Course Material

Chromatographic Techniques

Index
Sr. No. Content
1. Gas Chromatography basics
2. ThermoFisher_GCMS presentation
3. Application Guidebook
4. Apple Extract_ION TRAP
5. Brochure_TRACE1300 series
6. Drinking water_GCMS
7. Rice_IONTRAP
8. TSQ8000_Brochure
9. URINE-SIX Opiates-ISQ
10. IC_Theory and Principles
11. HPLC_Theory and Principlesx
12. Bro_Carbohydrates_Food_Beverage
13. Bro_Environmental_IC
14. Bro-Chemicals-IC
15. Bro-Chromeleon-6
16. Bro-Chromeleon-7
17. Bro-Corona-ultraRS
18. Bro-UltiMate-3000-LC-Systems
19. Bro-UltiMate-3000-RSLC
20. Bro-Water-Analysis
21. DS-ICS-2100
22. DS-RFIC-EG
23. UltiMate-Standard_UHPLC
 Index
Sr. No. Content
1. Gas Chromatography basics
2. ThermoFisher_GCMS presentation
3. Application Guidebook
4. Apple Extract_ION TRAP
5. Brochure_TRACE1300 series
6. Drinking water_GCMS
7. Rice_IONTRAP
8. TSQ8000_Brochure
9. URINE-SIX Opiates-ISQ
10. IC_Theory and Principles
11. HPLC_Theory and Principlesx
12. Bro_Carbohydrates_Food_Beverage
13. Bro_Environmental_IC
14. Bro-Chemicals-IC
15. Bro-Chromeleon-6
16. Bro-Chromeleon-7
17. Bro-Corona-ultraRS
18. Bro-UltiMate-3000-LC-Systems
19. Bro-UltiMate-3000-RSLC
20. Bro-Water-Analysis
21. DS-ICS-2100
22. DS-RFIC-EG
23. UltiMate-Standard_UHPLC
Basic Concepts of
Gas Chromatography

Ms.Aarti Karkhanis
Asst.Manager (Applications GCMS group)
ThermoFisher Scientific Mumbai
 What is GC?

The technique

• GC stands for Gas Chromatography, an analytical technique which

allows separation organic compounds in a sample

2
 Why we need GC?

The information

• By using GC we can perform:

• Qualitative analysis
• Quantitative analysis

3
 Samples for GC

• Strictly speaking, samples for GC are organic compounds with

1. sufficient volatility- Either in vapor form or can be turned to vapor at 400oC

or less)
2. sufficient thermal stability (does not decompose on heating)

• Some compounds which do not fulfill the criteria

• To fulfill the criteria by means of chemical reaction
(‘derivatization’),

4
5
6
7
8
9
10
11
12
13
 Typical applications of GC:

• Pesticide residues and pollutants in water, agricultural products, foodstuff

• Organic solvents in packaging materials, ink, etc.
• Drugs of abuse in urine, blood, tablets
• Fatty acid contents in edible oils, fat, etc.
• Essential oil Food Agriculture
• etc… industry industry
Environmental Forensic
field field
Biotechnology Chemical Fragrance
field industry industry

14
 Flow chart of GC

15
 What is chromatography?

1. Chromatography is a dynamic separation process based on the

selective distribution of the different components between two
phases.

2. Stationary phase, is held immobilised inside the column while the

the mobile phase travels through the column i.e. Carrier gas, flowing
through the column.

3. Compounds that exhibit a higher affinity for the stationary phase will
travel more slowly. The compounds exhibiting a lower affinity travels
faster

16
GC Columns
 GC Column

PACKED COLUMN

Packed column Glass or Metal

Length: 0.5-20 m
ID (Inner Diameter): 2-4 mm

Packing Material:
-Adsorbent (molecular sieve, activated alumina,
Stationary
silica gel
phase (thin
Solid support -Solid support coated with thin film of
film)
material stationary phase (refer to the picture)

18 18
 Selecting GC Column

• Packed or capillary?
• What stationary phase?
• What stationary phase film thickness?
• Column inner diameter?
• Column length?

19 19
Reference: http://www.restekcorp.com/colsel/colsel.htm
 Packed or Capillary Column?

20 20
 Which Stationary Phase?

• Gas Liquid Chromatography:

• Stationary phase polarity and analytes polarity
• For compounds with similar volatility
• Solutes with polarities similar to the polarity of the stationary
phase are more retained (“like attracts like”)
• In other words, polar compounds are more strongly retained by a
polar stationary phase than by a less polar stationary phase

• Stationary phase polarity is determined by the polarity of

the functional groups on the stationary phase material

21 21
 GC Column

Stationary Polyimide
CAPILLARY COLUMN phase resin

Fused silica
tubing

Capillary column

Length: 10 - ~100 m
ID (inner diameter): 0.1 mm – 0.53 mm
Stationary phase film thickness: 0.1-5 um

22
22
 Selection of column

ability of stationary phase to

differentiate (‘recognize’)
solutes by means of differences
in solutes’ chemical or physical
properties, such as

chemical composition

chemical structure

boiling points

polarity

23
 Column selection in method development
a) Stationary phase:-
1. Non-Polar-100% dimethyl polysiloxane,5% phenyl polysilphenylene siloxane,
5% Phenyl polycarborane siloxane.
(Used for applications of hydrocarbons, solvents, pesticides, phenols, amines)
2. Polar-100% Polyethylene glycol
(Used for applications of esters, alcohols,ketones,glycols,aromatic isomers)
3. Mid-polar- 35% Phenyl polysilphenylene-siloxane,14% Cynopropylphenyl polysiloxane
(Used for applications of pesticides, PCB’s, PAH’s, organic acids, Drugs, steroids)

b) Inner diameter:- With larger ID column sample capacity increases, but resolution may
decrease. Smaller ID can improve resolution

c) Film Thickness:- A higher film thickness increases sample capacity of the column. A
thicker film thickness also reduces the potential of overloading the column and improves
resolution

d) Length :- A longer column will provide greater efficiency & resolution. Resolution is
proportional to column square root of length.

24
 The different sample components travel at different speed through the column and
eluting from the column at different times.

• Migration rate of compounds in column depend on:

• Compound chemical structure

• Stationary phase chemical structure

• Column temperature

25
 Migration rates of compounds in column

• Different migration rates of compounds can be achieved if these

compounds have different interaction strengths with the
stationary phase

Stronger interaction Weaker interaction

Stationary phase

26
 Inner Diameter (I.D.)
• Efficiency of a column
increases with column inner
diameter decrease.

• The higher the efficiency, the

narrower the peak at a given
retention time.

• Column capacity is bigger as

the column diameter gets
larger.

• Smaller column diameter

requires higher pressure and
vice versa.

27
 Column length

• Column efficiency proportional to column

length
• Increasing column length will increase the N
and hence double the column efficiency.

• Drawbacks of increasing column length:

• Increase of analysis time
• Increase in cost

28
 Column Parameters

HETP (H)

Resolution

Number of theoretical plates

29
 Theoretical plate
• Theoretical plate is a term coined by Martin and Synge.

• Theoretical plates (N) is a measure of how efficiently a column can separate a mixture into its
components. This efficiency is based on retention time of the components and the width of the peaks.

• Theoretical plates is a
concept to check column
efficiency
• Theoretical plates
numbers are an indirect
measure of peak width
for a peak at a specific
retention time
• Columns with high N are
considered to be more
efficient than those with
lower N
• A column with a high N
will have a narrower peak
at a given retention time

30
 Resolution

• A measure of overlap between two peaks; the higher the resolution, the
less the overlap
• Separation (α) is only the distance between two peak maxima;
resolution takes both α and the width of the peaks into account
• Baseline resolution usually occurs at R = 1.50

31
 Height equivalent to a theoretical plate (HETP or H)

• Another measure of column efficiency

• Small plate heights indicate higher efficiency

L
H=
N
L = column length (mm)
N = theoretical plates number

32 32
Effect of column length

1 2
1
30 meters 60 meters

5
7
4 6 8
11
12 11
13 4
3 10 12 13
8 10
3 6 7
9
9

4 8 12 16 20 24 28 32 36
4 8 12 16

1. phenol 4. p-cresol 7. 2,4-xylenol 10. p-ethylphenol 13. 3,4-xylenol

2. o-cresol 5. m-cresol 8. 2,5-xylenol 11. m-ethylphenol

33
3. 2,6-xylenol 6. o-ethylphenol 9. 2,3-xylenol 12. 3,5-xylenol 33
 General capillary column maintenance
• Do not heat column above the
upper temperature limit
• Conditioned the column before
using
• Make sure carrier gas is flowing
before heating the column/oven
• Avoid unnecessary bending and
contact of column with rough
surfaces
• Seal column ends with GC septa
• Follow instructions from column
manufacturer

34
34
 Sample Introduction Technique
• Liquid and gas samples can be introduced to gas chromatograph.

• Most common technique:

1.Liquid injection- Sample is diluted in solvent and injected to GC

Injection Port by a syringe (volume of injection = normally 1-2 µL).

2. Introduction of gas sample :-

a. Gas tight syringe
b. Gas sampling valve

3.Headspace
4.SPME (Solid Phase Microextraction)
5. Pyrolysis

35
 Why HeadSpace?

• Keeps injectors and columns clean

• Matrix conditions can be modified to fit the

analysis

• Solid samples can be analyzed directly

• High-concentration samples can be evaluated

• Increased Sensitivity

36
 Headspace

Parameters effectively used to extract the VOC’s

Agitation temperature,
Syringe temp,
incubation time
37
 SPME

38
 Gas Chromatographic Equipment
Gas supply and flow/pressure control Detector
The mobile phase (the carrier gas in GC) is Injector Following elution from the column, the individual sample
supplied by either gas cylinders or by Samples to be components are detected as they flow through the
generators designed for the purpose. This analysed are detector. The output from the detector is supplied to an
supply must be controlled precisely and introduced onto the amplifier, followed by a data system or recorder. The
accurately by pressure or flow controllers column by means of analog monitoring of the detector output versus time is
the injection system. called a chromatogram. It consists of a series of peaks
2 Type-SSL/PTV corresponding to the components.

Column Data system

.
The central and most important Qualitative identification
Oven
component of a gas is based on the time
Programmed temperature
chromatograph is the column. that the peaks elute
/Isothermal column oven
The column is the heart of the from the column and is
programming required
system where the separation subsequently detected.
depending on the
occurs. Quantitative analysis is
requirement
Selection of column most based on peak size or
important factor peak area.

39
Inlet systems in Gas
Chromatography
 Split / splitless injection

Split injection: Transferred Splitless injection:

amount related to the split ratio. Used for low concentration
• Used for high concentration samples. samples.

Split flow is CLOSED during

• The sample splitted with respect to split injection
flow and column flow.
• Only a small flow enters the column to After a determined period of
prevent column overloading. time, the split flow is opened
to remove solvent residue
• Split Ratio= (Split Flow)/(Column Flow) from the injector.

41
 a) Split / splitless inlet-working

• Most common Vaporizing inlet

Split Injection
• In Split injection system, only
portion of sample injected into the injection port .
• Remaining sample is sent to the split line .

• This technique is used for high sample concentration.

Splitless Injection
• Before sampling time the split vent is closed
so that all the components should go inside the column
• After sampling time the split vent is opened to
vent out the stray vapors in the inlet as per the set split ratio.

• Used to analyze samples of low concentration/trace detection

42
 Cool on column injection
• The sample solvent is directly injected directly into column using a
small diameter needle

• This technique is used for

thermally labile compounds.

• The sample solvent is

directly injected onto column
using a small diameter
needle. (does not use flash
vaporization).

• After injection the oven

temperature raised as the
given programming.

43
 PTV injection

• First describe by VOGT in 1979.

• Closely resemble to split/splitless inlet. 2 main differences:
• inlet is kept cool during injection- allowing the analyte to condence
inside the liner, while solvent is vented via split.
• Inlet has very low thermal mass, allow rapid heating for analyte
transfer
Large volume may be injected at control speed into the inlet allowing
the injection of very large volume.

44
 Headspace Injection technique

45
 Applications-HS

• Volatile impurities
Packaging films
-Food wrapers
-Sun control window films

-Flavor & Fragrance compounds in matrix

-OVI-water, sea water, sludge, environmental
hazards,inks,paint,resins,alcohol content in blood samples

46
 Applications-HS

Typical Static Headspace Applications Include:

• Pharmaceuticals –
• 1. OVI analysis –as per monograph <467>
• 2.Pesticide analysis- as per monograph
{Articles of Botonical Origin<561> } General methods for pesticide
residues analysis
3. Genotoxic Impurities
• Environmental Samples
• Forensics (Blood Alcohols)
• Polymers
• Food/Flavors
• Coatings

47
General Overview of
Detector Systems
 Characteristics of GC Detector

• Sensitivity – Detector efficiency to convert a sample into an electrical signal

• Minimum detectability - S/N must be ≥ 3

• Linearity- A range of concentrations at which reliable quantitative measures can be

made.

• Selectivity- The ratio of the detector sensitivities of a given compound over a

potentially interfering compound

• Limit of detection: The lowest concentration level that can be determined by a

particular detector. The S/N ratio must be atleast 3.

• Limit of Quantification: Level above which quantitative results maybe obtained.

The S/N ratio must be 10 or above.

49
 Most commonly used detectors

 TCD –Thermal Conductivity Detector

 FID – Flame Ionisation Detector

 ECD – Electron Capture Detector

 FTD – Flame Thermionic Detector

 FPD – Flame Photometric Detector

50
 Thermal Conductivity Detector

• Detects almost all compounds except

the carrier gas.
• The TCD filament is heated by applying
a current
• When carrier gas + sample gas passes
over the filament, the temperature of the
filament increases, because the thermal
conductivity of the sample compounds
is less than the carrier gas.
• The changes in filament W or Ru
temperature affect its resistance
• The resistance change is measured
and produces the signal

51
 Thermal Conductivity Detector

Application fields

 Petroleum industry
 Chemicals (gas analysis)
 Semiconductor industry

Sensitivity in the ppm - % range

52
 Flame Ionization Detector
For organic compounds analysis Hydrogen and air
are needed to create the flame.

Sample is brought to hydrogen flame and converted

into ions. Current will be generated.

The current is proportional to the amount of the

organic compound present.

Ion quantity generated is almost proportional to the

number of carbon atoms in a compound, but
Carbon atoms in C=O bonds do not generate a
signal Presence of halogens in the compound
decreases sensitivity

53
 Flame Ionization Detector

Application fields

 Petroleum industry
 Enviromental
 Pharmaceuticals
 Food and flavors

Sensitivity in the ppm - % range

54
 Electron capture detector
• Non- destructive & Selective detector
• Measure electrical conductivity of the effluent after exposure to
ionizing radiation.
• Sensitive to ‘electron capturing species’- halogens.

Working:
• Radioactive 63Ni foil emit low energy electron (beta particle)
63Ni β-
• These β particle collide with carrier to produce high energy
electron
β- + N2 2e- + N2+

• Electronegative analyte elute from the column and ‘capture’

some of the electrons

• As a result there is a decrease in standing current and this

decrease is proportional to the concentration of the analytes.

55
 Applications

• ECD
• Chloro pesticides in Drinking water (EPA 508.1)
• Trihalomethanes in Drinking water (EPA 501)
• Chlorinated Acids (EPA 515.1)
• Acrylamide in food (EPA 8032)
• Chlorinated disinfectant by-product (EPA 551)
• Halogenated acetic acids in drinking water (EPA 552)
• Polychlorophenols

- analysis of PCBs (polychlorinated biphenyls)

- analysis of PBBs (polybrominated biphenyls)
- analysis of PBDEs (polybrominated diphenyl ethers)
- analysis of organochlorine pesticides (DDT, BHC, etc.)

56
 Flame Thermionic Detector
• Highly sensitive towards organo-nitrogen and organo-phosphorus compounds

• Working:
 Rb or Ce silicate bead when heated
emits thermionic electrons which
migrate to the collector electrode
forming background current.

 H2 mixed with the column eluant

undergoes partial combustion at the
surface of the bead.

 The N & P in the sample after

combustion get adsorbed on the bead surface.

 The work function of the bead decreases and the

current in the detector increases.

 This increase in current is proportional to the concentration of the analyte in the sample.

57
 Application

• Examples of analyses:
- analysis of nitrosamines
- analysis of organophosphorous and nitrogen-containing
pesticides

58
 Flame Photometric Detector

Highly sensitive towards compounds containing
Sulphur and Phosphorus.

Working:
• Detects compounds that contains P(phosphorous),
S(sulfur), and Sn (tin)
• Lights of different wavelengths will be produced when
samples are brought to the flame.
• Filter is placed inside FPD. Only certain wavelengths
can pass through the filter.
• Example of analysis:

• - analysis of organophosphorous pesticides

(malathion, dichlorvos, chlorpyrifos, etc.)

59
 Applications

• FPD
• Butyl tin compounds
• Organophosphorous pesticides
• Organosulphar pesticides

60
 Gas analysis

61
 Analysis of Freon GC analysis

62
 Aromatic compounds in Tea

63
 Adulteration in wine

64
 Analysis of Light oil & kerosene by GC

65
 4.5.Use of GC detectors in different application fields
Environmental Food Safety
• ECD • ECD
• EDB and DBCP in wastewater and soil (EPA 504, EPA • Chloro pesticides in Drinking water (EPA 508.1)
8011) • Trihalomethanes in Drinking water (EPA 501)
• Purgeables Halocarbon (EPA 502.1) • Chlorinated Acids (EPA 515.1)
• Halogenated pesticides in wastewater and soil (EPA • Acrylamide in food (EPA 8032)
505/508, EPA 515, EPA 8081) • Chlorinated disinfectant by-product (EPA 551)
• Herbicides (EPA 515.4) • Halogenated acetic acids in drinking water (EPA
• Chlorinated pesticides and PCBs (EPA 8080, EPA 8081A, 552)
EPA 8082) • Polychlorophenols
• NPD • PID
• Herbicides, Insecticides, Pesticides (EPA 507, EPA 8141) • Phthalates in drinking water (EPA 506)
• FPD • Purgeable Aromatics (EPA 503.1)
• Butyl tin compounds
• Organophosphorous pesticides
• PID
• PAHs , Purgeables Aromatic Organics
Petrochemical
• FID
• FID
• Simdist
•TPH in water and soil
• Oxygenated in Fuel
• Benzene in Gasoline
• DHA
Toxicology and forensic • TCD
• NPD • RGA, NGA
• Drugs • PDD
• FID • High Purity Gases
• Ethanol and methanol in blood • PFPD
• Arson accelerant in fire debris • Sulfur in Diesel
• Sulfur in LPG

66
 Thermo GC 1300 Series- with Instant connect module

Exchange modules
....set a new era in GC technology
• They allow users to re-think the way to use a gas chromatograph:
• Satisfy incremental business needs
• Enhance laboratory productivity at any time
• Upgrading a GC from single to multiple channels
• Change instrument configuration, using the proper injector/detector for the samples you run today
• Postpone routine maintenance for continuous operations when the laboratory schedule allows
• Remove contaminated injector/detector bodies, replace them with clean ones in a few minutes

67
 TRACE 1300 series – A Completely New Platform
Versatility: SSL compatible
Versatility: new modular concept with multi-vendor
Build for stock instead for order consumables and methods.
New injectors with higher
robustness.
Performance: new
detectors with fast
response and
auto-ranging Reliability/Up time:
capabilities High quality, smart
New fast oven maintenance,
routine grade
reliability
Ease of Use:
new touch
screen UI
Incorporate all new parts
for next 10 years product
life cycle
Versatility: New chassis with
smaller footprint: 2/3 of
standard dual column GC

68
 TRACE 1300 Series – Two flavors available
TRACE 1310: Touch screen TRACE 1300: Local built-in ultra-
interface provides instant access simplified user interface – two
for ease of use and local control buttons and four LEDs.

Modules available:
INLETS: SSL - SSL backflush - PTV - PTV backflush
DETECTORS: FID - TCD - ECD - NPD - MS*
OTHER OPTIONS: Oven Cryo - Aux carrier
Software drivers: Chromeleon, Xcalibur, ChromQuest, ChromCard

* Supported MS: ISQ; ITQ; TSQ Quantum XLS; DFS; Delta V

69
 TRACE 1300 GC

TRACE 1300 GC is ideal for industry routine labs:

• For budget-conscious investments

• Dedicated to less experienced lab-operators
• Minimal instrument interaction desired
• Simplified user interface
• Single button start/stop and preventive maintenance
• Maintains all system features and capabilities

70
 TRACE 1310 GC

TRACE 1310 GC is dedicated to larger production and routine laboratories

• Complete touch screen for direct instrument control

• Method development and status controls
• Multi language capabilities
• Maintenance, run log and video tutorials
• Ideal front-end to MS systems

71
 Thermo Scientific+Dionex

72
Basic Concepts of
Gas Chromatography

Ms.Aarti Karkhanis
Asst.Manager (Applications GCMS group)
ThermoFisher Scientific Mumbai
 What is GC?

The technique

• GC stands for Gas Chromatography, an analytical technique which

allows separation organic compounds in a sample

2
 Why we need GC?

The information

• By using GC we can perform:

• Qualitative analysis
• Quantitative analysis

3
 Samples for GC

• Strictly speaking, samples for GC are organic compounds with

1. sufficient volatility- Either in vapor form or can be turned to vapor at 400oC

or less)
2. sufficient thermal stability (does not decompose on heating)

• Some compounds which do not fulfill the criteria

• To fulfill the criteria by means of chemical reaction
(‘derivatization’),

4
5
6
7
8
9
10
11
12
13
 Typical applications of GC:

• Pesticide residues and pollutants in water, agricultural products, foodstuff

• Organic solvents in packaging materials, ink, etc.
• Drugs of abuse in urine, blood, tablets
• Fatty acid contents in edible oils, fat, etc.
• Essential oil Food Agriculture
• etc… industry industry
Environmental Forensic
field field
Biotechnology Chemical Fragrance
field industry industry

14
 Flow chart of GC

15
 What is chromatography?

1. Chromatography is a dynamic separation process based on the

selective distribution of the different components between two
phases.

2. Stationary phase, is held immobilised inside the column while the

the mobile phase travels through the column i.e. Carrier gas, flowing
through the column.

3. Compounds that exhibit a higher affinity for the stationary phase will
travel more slowly. The compounds exhibiting a lower affinity travels
faster

16
GC Columns
 GC Column

PACKED COLUMN

Packed column Glass or Metal

Length: 0.5-20 m
ID (Inner Diameter): 2-4 mm

Packing Material:
-Adsorbent (molecular sieve, activated alumina,
Stationary
silica gel
phase (thin
Solid support -Solid support coated with thin film of
film)
material stationary phase (refer to the picture)

18 18
 Selecting GC Column

• Packed or capillary?
• What stationary phase?
• What stationary phase film thickness?
• Column inner diameter?
• Column length?

19 19
Reference: http://www.restekcorp.com/colsel/colsel.htm
 Packed or Capillary Column?

20 20
 Which Stationary Phase?

• Gas Liquid Chromatography:

• Stationary phase polarity and analytes polarity
• For compounds with similar volatility
• Solutes with polarities similar to the polarity of the stationary
phase are more retained (“like attracts like”)
• In other words, polar compounds are more strongly retained by a
polar stationary phase than by a less polar stationary phase

• Stationary phase polarity is determined by the polarity of

the functional groups on the stationary phase material

21 21
 GC Column

Stationary Polyimide
CAPILLARY COLUMN phase resin

Fused silica
tubing

Capillary column

Length: 10 - ~100 m
ID (inner diameter): 0.1 mm – 0.53 mm
Stationary phase film thickness: 0.1-5 um

22
22
 Selection of column

ability of stationary phase to

differentiate (‘recognize’)
solutes by means of differences
in solutes’ chemical or physical
properties, such as

chemical composition

chemical structure

boiling points

polarity

23
 Column selection in method development
a) Stationary phase:-
1. Non-Polar-100% dimethyl polysiloxane,5% phenyl polysilphenylene siloxane,
5% Phenyl polycarborane siloxane.
(Used for applications of hydrocarbons, solvents, pesticides, phenols, amines)
2. Polar-100% Polyethylene glycol
(Used for applications of esters, alcohols,ketones,glycols,aromatic isomers)
3. Mid-polar- 35% Phenyl polysilphenylene-siloxane,14% Cynopropylphenyl polysiloxane
(Used for applications of pesticides, PCB’s, PAH’s, organic acids, Drugs, steroids)

b) Inner diameter:- With larger ID column sample capacity increases, but resolution may
decrease. Smaller ID can improve resolution

c) Film Thickness:- A higher film thickness increases sample capacity of the column. A
thicker film thickness also reduces the potential of overloading the column and improves
resolution

d) Length :- A longer column will provide greater efficiency & resolution. Resolution is
proportional to column square root of length.

24
 The different sample components travel at different speed through the column and
eluting from the column at different times.

• Migration rate of compounds in column depend on:

• Compound chemical structure

• Stationary phase chemical structure

• Column temperature

25
 Migration rates of compounds in column

• Different migration rates of compounds can be achieved if these

compounds have different interaction strengths with the
stationary phase

Stronger interaction Weaker interaction

Stationary phase

26
 Inner Diameter (I.D.)
• Efficiency of a column
increases with column inner
diameter decrease.

• The higher the efficiency, the

narrower the peak at a given
retention time.

• Column capacity is bigger as

the column diameter gets
larger.

• Smaller column diameter

requires higher pressure and
vice versa.

27
 Column length

• Column efficiency proportional to column

length
• Increasing column length will increase the N
and hence double the column efficiency.

• Drawbacks of increasing column length:

• Increase of analysis time
• Increase in cost

28
 Column Parameters

HETP (H)

Resolution

Number of theoretical plates

29
 Theoretical plate
• Theoretical plate is a term coined by Martin and Synge.

• Theoretical plates (N) is a measure of how efficiently a column can separate a mixture into its
components. This efficiency is based on retention time of the components and the width of the peaks.

• Theoretical plates is a
concept to check column
efficiency
• Theoretical plates
numbers are an indirect
measure of peak width
for a peak at a specific
retention time
• Columns with high N are
considered to be more
efficient than those with
lower N
• A column with a high N
will have a narrower peak
at a given retention time

30
 Resolution

• A measure of overlap between two peaks; the higher the resolution, the
less the overlap
• Separation (α) is only the distance between two peak maxima;
resolution takes both α and the width of the peaks into account
• Baseline resolution usually occurs at R = 1.50

31
 Height equivalent to a theoretical plate (HETP or H)

• Another measure of column efficiency

• Small plate heights indicate higher efficiency

L
H=
N
L = column length (mm)
N = theoretical plates number

32 32
Effect of column length

1 2
1
30 meters 60 meters

5
7
4 6 8
11
12 11
13 4
3 10 12 13
8 10
3 6 7
9
9

4 8 12 16 20 24 28 32 36
4 8 12 16

1. phenol 4. p-cresol 7. 2,4-xylenol 10. p-ethylphenol 13. 3,4-xylenol

2. o-cresol 5. m-cresol 8. 2,5-xylenol 11. m-ethylphenol

33
3. 2,6-xylenol 6. o-ethylphenol 9. 2,3-xylenol 12. 3,5-xylenol 33
 General capillary column maintenance
• Do not heat column above the
upper temperature limit
• Conditioned the column before
using
• Make sure carrier gas is flowing
before heating the column/oven
• Avoid unnecessary bending and
contact of column with rough
surfaces
• Seal column ends with GC septa
• Follow instructions from column
manufacturer

34
34
 Sample Introduction Technique
• Liquid and gas samples can be introduced to gas chromatograph.

• Most common technique:

1.Liquid injection- Sample is diluted in solvent and injected to GC

Injection Port by a syringe (volume of injection = normally 1-2 µL).

2. Introduction of gas sample :-

a. Gas tight syringe
b. Gas sampling valve

3.Headspace
4.SPME (Solid Phase Microextraction)
5. Pyrolysis

35
 Why HeadSpace?

• Keeps injectors and columns clean

• Matrix conditions can be modified to fit the

analysis

• Solid samples can be analyzed directly

• High-concentration samples can be evaluated

• Increased Sensitivity

36
 Headspace

Parameters effectively used to extract the VOC’s

Agitation temperature,
Syringe temp,
incubation time
37
 SPME

38
 Gas Chromatographic Equipment
Gas supply and flow/pressure control Detector
The mobile phase (the carrier gas in GC) is Injector Following elution from the column, the individual sample
supplied by either gas cylinders or by Samples to be components are detected as they flow through the
generators designed for the purpose. This analysed are detector. The output from the detector is supplied to an
supply must be controlled precisely and introduced onto the amplifier, followed by a data system or recorder. The
accurately by pressure or flow controllers column by means of analog monitoring of the detector output versus time is
the injection system. called a chromatogram. It consists of a series of peaks
2 Type-SSL/PTV corresponding to the components.

Column Data system

.
The central and most important Qualitative identification
Oven
component of a gas is based on the time
Programmed temperature
chromatograph is the column. that the peaks elute
/Isothermal column oven
The column is the heart of the from the column and is
programming required
system where the separation subsequently detected.
depending on the
occurs. Quantitative analysis is
requirement
Selection of column most based on peak size or
important factor peak area.

39
Inlet systems in Gas
Chromatography
 Split / splitless injection

Split injection: Transferred Splitless injection:

amount related to the split ratio. Used for low concentration
• Used for high concentration samples. samples.

Split flow is CLOSED during

• The sample splitted with respect to split injection
flow and column flow.
• Only a small flow enters the column to After a determined period of
prevent column overloading. time, the split flow is opened
to remove solvent residue
• Split Ratio= (Split Flow)/(Column Flow) from the injector.

41
 a) Split / splitless inlet-working

• Most common Vaporizing inlet

Split Injection
• In Split injection system, only
portion of sample injected into the injection port .
• Remaining sample is sent to the split line .

• This technique is used for high sample concentration.

Splitless Injection
• Before sampling time the split vent is closed
so that all the components should go inside the column
• After sampling time the split vent is opened to
vent out the stray vapors in the inlet as per the set split ratio.

• Used to analyze samples of low concentration/trace detection

42
 Cool on column injection
• The sample solvent is directly injected directly into column using a
small diameter needle

• This technique is used for

thermally labile compounds.

• The sample solvent is

directly injected onto column
using a small diameter
needle. (does not use flash
vaporization).

• After injection the oven

temperature raised as the
given programming.

43
 PTV injection

• First describe by VOGT in 1979.

• Closely resemble to split/splitless inlet. 2 main differences:
• inlet is kept cool during injection- allowing the analyte to condence
inside the liner, while solvent is vented via split.
• Inlet has very low thermal mass, allow rapid heating for analyte
transfer
Large volume may be injected at control speed into the inlet allowing
the injection of very large volume.

44
 Headspace Injection technique

45
 Applications-HS

• Volatile impurities
Packaging films
-Food wrapers
-Sun control window films

-Flavor & Fragrance compounds in matrix

-OVI-water, sea water, sludge, environmental
hazards,inks,paint,resins,alcohol content in blood samples

46
 Applications-HS

Typical Static Headspace Applications Include:

• Pharmaceuticals –
• 1. OVI analysis –as per monograph <467>
• 2.Pesticide analysis- as per monograph
{Articles of Botonical Origin<561> } General methods for pesticide
residues analysis
3. Genotoxic Impurities
• Environmental Samples
• Forensics (Blood Alcohols)
• Polymers
• Food/Flavors
• Coatings

47
General Overview of
Detector Systems
 Characteristics of GC Detector

• Sensitivity – Detector efficiency to convert a sample into an electrical signal

• Minimum detectability - S/N must be ≥ 3

• Linearity- A range of concentrations at which reliable quantitative measures can be

made.

• Selectivity- The ratio of the detector sensitivities of a given compound over a

potentially interfering compound

• Limit of detection: The lowest concentration level that can be determined by a

particular detector. The S/N ratio must be atleast 3.

• Limit of Quantification: Level above which quantitative results maybe obtained.

The S/N ratio must be 10 or above.

49
 Most commonly used detectors

 TCD –Thermal Conductivity Detector

 FID – Flame Ionisation Detector

 ECD – Electron Capture Detector

 FTD – Flame Thermionic Detector

 FPD – Flame Photometric Detector

50
 Thermal Conductivity Detector

• Detects almost all compounds except

the carrier gas.
• The TCD filament is heated by applying
a current
• When carrier gas + sample gas passes
over the filament, the temperature of the
filament increases, because the thermal
conductivity of the sample compounds
is less than the carrier gas.
• The changes in filament W or Ru
temperature affect its resistance
• The resistance change is measured
and produces the signal

51
 Thermal Conductivity Detector

Application fields

 Petroleum industry
 Chemicals (gas analysis)
 Semiconductor industry

Sensitivity in the ppm - % range

52
 Flame Ionization Detector
For organic compounds analysis Hydrogen and air
are needed to create the flame.

Sample is brought to hydrogen flame and converted

into ions. Current will be generated.

The current is proportional to the amount of the

organic compound present.

Ion quantity generated is almost proportional to the

number of carbon atoms in a compound, but
Carbon atoms in C=O bonds do not generate a
signal Presence of halogens in the compound
decreases sensitivity

53
 Flame Ionization Detector

Application fields

 Petroleum industry
 Enviromental
 Pharmaceuticals
 Food and flavors

Sensitivity in the ppm - % range

54
 Electron capture detector
• Non- destructive & Selective detector
• Measure electrical conductivity of the effluent after exposure to
ionizing radiation.
• Sensitive to ‘electron capturing species’- halogens.

Working:
• Radioactive 63Ni foil emit low energy electron (beta particle)
63Ni β-
• These β particle collide with carrier to produce high energy
electron
β- + N2 2e- + N2+

• Electronegative analyte elute from the column and ‘capture’

some of the electrons

• As a result there is a decrease in standing current and this

decrease is proportional to the concentration of the analytes.

55
 Applications

• ECD
• Chloro pesticides in Drinking water (EPA 508.1)
• Trihalomethanes in Drinking water (EPA 501)
• Chlorinated Acids (EPA 515.1)
• Acrylamide in food (EPA 8032)
• Chlorinated disinfectant by-product (EPA 551)
• Halogenated acetic acids in drinking water (EPA 552)
• Polychlorophenols

- analysis of PCBs (polychlorinated biphenyls)

- analysis of PBBs (polybrominated biphenyls)
- analysis of PBDEs (polybrominated diphenyl ethers)
- analysis of organochlorine pesticides (DDT, BHC, etc.)

56
 Flame Thermionic Detector
• Highly sensitive towards organo-nitrogen and organo-phosphorus compounds

• Working:
 Rb or Ce silicate bead when heated
emits thermionic electrons which
migrate to the collector electrode
forming background current.

 H2 mixed with the column eluant

undergoes partial combustion at the
surface of the bead.

 The N & P in the sample after

combustion get adsorbed on the bead surface.

 The work function of the bead decreases and the

current in the detector increases.

 This increase in current is proportional to the concentration of the analyte in the sample.

57
 Application

• Examples of analyses:
- analysis of nitrosamines
- analysis of organophosphorous and nitrogen-containing
pesticides

58
 Flame Photometric Detector

Highly sensitive towards compounds containing
Sulphur and Phosphorus.

Working:
• Detects compounds that contains P(phosphorous),
S(sulfur), and Sn (tin)
• Lights of different wavelengths will be produced when
samples are brought to the flame.
• Filter is placed inside FPD. Only certain wavelengths
can pass through the filter.
• Example of analysis:

• - analysis of organophosphorous pesticides

(malathion, dichlorvos, chlorpyrifos, etc.)

59
 Applications

• FPD
• Butyl tin compounds
• Organophosphorous pesticides
• Organosulphar pesticides

60
 Gas analysis

61
 Analysis of Freon GC analysis

62
 Aromatic compounds in Tea

63
 Adulteration in wine

64
 Analysis of Light oil & kerosene by GC

65
 4.5.Use of GC detectors in different application fields
Environmental Food Safety
• ECD • ECD
• EDB and DBCP in wastewater and soil (EPA 504, EPA • Chloro pesticides in Drinking water (EPA 508.1)
8011) • Trihalomethanes in Drinking water (EPA 501)
• Purgeables Halocarbon (EPA 502.1) • Chlorinated Acids (EPA 515.1)
• Halogenated pesticides in wastewater and soil (EPA • Acrylamide in food (EPA 8032)
505/508, EPA 515, EPA 8081) • Chlorinated disinfectant by-product (EPA 551)
• Herbicides (EPA 515.4) • Halogenated acetic acids in drinking water (EPA
• Chlorinated pesticides and PCBs (EPA 8080, EPA 8081A, 552)
EPA 8082) • Polychlorophenols
• NPD • PID
• Herbicides, Insecticides, Pesticides (EPA 507, EPA 8141) • Phthalates in drinking water (EPA 506)
• FPD • Purgeable Aromatics (EPA 503.1)
• Butyl tin compounds
• Organophosphorous pesticides
• PID
• PAHs , Purgeables Aromatic Organics
Petrochemical
• FID
• FID
• Simdist
•TPH in water and soil
• Oxygenated in Fuel
• Benzene in Gasoline
• DHA
Toxicology and forensic • TCD
• NPD • RGA, NGA
• Drugs • PDD
• FID • High Purity Gases
• Ethanol and methanol in blood • PFPD
• Arson accelerant in fire debris • Sulfur in Diesel
• Sulfur in LPG

66
 Thermo GC 1300 Series- with Instant connect module

Exchange modules
....set a new era in GC technology
• They allow users to re-think the way to use a gas chromatograph:
• Satisfy incremental business needs
• Enhance laboratory productivity at any time
• Upgrading a GC from single to multiple channels
• Change instrument configuration, using the proper injector/detector for the samples you run today
• Postpone routine maintenance for continuous operations when the laboratory schedule allows
• Remove contaminated injector/detector bodies, replace them with clean ones in a few minutes

67
 TRACE 1300 series – A Completely New Platform
Versatility: SSL compatible
Versatility: new modular concept with multi-vendor
Build for stock instead for order consumables and methods.
New injectors with higher
robustness.
Performance: new
detectors with fast
response and
auto-ranging Reliability/Up time:
capabilities High quality, smart
New fast oven maintenance,
routine grade
reliability
Ease of Use:
new touch
screen UI
Incorporate all new parts
for next 10 years product
life cycle
Versatility: New chassis with
smaller footprint: 2/3 of
standard dual column GC

68
 TRACE 1300 Series – Two flavors available
TRACE 1310: Touch screen TRACE 1300: Local built-in ultra-
interface provides instant access simplified user interface – two
for ease of use and local control buttons and four LEDs.

Modules available:
INLETS: SSL - SSL backflush - PTV - PTV backflush
DETECTORS: FID - TCD - ECD - NPD - MS*
OTHER OPTIONS: Oven Cryo - Aux carrier
Software drivers: Chromeleon, Xcalibur, ChromQuest, ChromCard

* Supported MS: ISQ; ITQ; TSQ Quantum XLS; DFS; Delta V

69
 TRACE 1300 GC

TRACE 1300 GC is ideal for industry routine labs:

• For budget-conscious investments

• Dedicated to less experienced lab-operators
• Minimal instrument interaction desired
• Simplified user interface
• Single button start/stop and preventive maintenance
• Maintains all system features and capabilities

70
 TRACE 1310 GC

TRACE 1310 GC is dedicated to larger production and routine laboratories

• Complete touch screen for direct instrument control

• Method development and status controls
• Multi language capabilities
• Maintenance, run log and video tutorials
• Ideal front-end to MS systems

71
 Thermo Scientific+Dionex

72
Basics of GCMS

Ms.Aarti Karkhanis
Asst.Manager (Applications GCMS group)
ThermoFisher Scientific Mumbai
 Historical Development of GC/MS

• 1910- J.J. Thompson developed the first MS

• 1910- M.S.Tswett –published his book
• 1918- Dempster used EI for first time
• 1941- Martin & Synge- Published a paper on GLC
• 1946- Stephens proposed a TOF
• 1947- NBS started collection of MS spectra
• 1950- Gohlke-First time published coupling of GC with MS
• 1950-Martin & Synge-Nobel prize
• 1955- Desty -First Commercial GC
• 1958- Paul published information on quadrupole mass filter
• 1958- Ken Shoulders manufactured the first Quadrupole MS
• 1958- Golay –Open Tubular columns
• 1964- First commercial quadrupole MS( Quad RGA) –Bob Finnigan

2
 Historical Development of GC/MS

• 1966- Munson & Field – CI

• 1968-First commercial quadrupole GCMS – Finnigan Instruments
• 1978- Fused Silica Capillary columns- Dandenau
• 1978- Triple quadrupole technology – Yost & Enke
• 1982- Finnigan –first patents on Ion trap technology
• 1989- Prof Wolfgang Paul – Nobel prize
• 2000-A. Makarov – New Type of mass analyzer concept -Orbitrap
• 2005- Thermo – Introduced Orbitrap

3
 Analytical Challenges

• Complex Samples (Tissues, water,soil, sediments, food and beverages,

etc…)

• Multiple components of various character (multi residue analysis)

• Metabolites , product degradations and new contaminants

• Method selectivity in order to determine the components of interest only

• Low levels of detection

• Higher quality results: results which are reliable

4
 How to identify in GC ?

By Injecting Standards!

5
 What is Mass Spectrometry?

“The basis of MS (mass spectrometry) is the production of

ions that are subsequently separated or filtered according to
their mass-to-charge (m/z) ratio and detected. The
resulting mass spectrum is a plot of the (relative) abundance of
the produced ions as a function of the m/z ratio.”

6
 Mass Spectrometer

…Introduction of mass ions in a

separation field…

+
_

Ion Source Mass Analyzer Detector

7
 Relative abundance

m/z

8
Components of a Mass Spectrometer

Atmosphere Vacuum
System

Sample Ionisation Mass Data

Detector
Inlet Method Analyser System

9
10
 Ion source

11
 Quadrupole mass spectrometer

• Voltages applied to four cylindrical quadrupole rods generate a quadrupole field in the center
axis of the mass spectrometer
• Ions entering the field will oscillate; the oscillation characteristics depend on the mass-to-
charge ratio of the ions and the voltage applied
• Voltage applied to the quadrupole can be varied/selected so that only ions with specific m/z will
have stable oscillations in the center axis, and will reach the ion detector

12
 Electron Multiplier

• Electron multiplier
• A conversion dynode is used to convert either negative or positive ions
into electrons.
• These electrons are amplified by a cascade effect in a horn shape
device, to produce a current.
• This device, also called channeltron, is widely used in quadrupole and
ion trap instruments.

13
 Vacuum System

• All MS systems require high vacuum of the order of 10-5 to 10-6 torr. It is
because of free movement of ions in the field and to reduce the
background

14
15
 PFTBA EI+ Spectrum

16
Ionization Methods

Electron Impact Ionisation

Positive Chemical Ionisation
Negative Chemical Ionisation
 Electron Impact Ionization
• Most common method of ionization for GC-MS
• Used as both -qualitative and quantitative tool
• Produces mass spectra of molecules
• Fragmentation fingerprints
• Combine with retention time for positive identification

• Extensive fragmentation of sample molecules gives rise to “fingerprint” of

compound, useful for identification purpose

• m/z value of the molecular ion (M+) gives information about the molecular weight of
the compound, also useful for identification purpose

• In some cases, M+ ion does not survive fragmentation due to the high energy
involved in the process. Two approaches can be taken:
• Use single ion for quantitation with one or more ions for verification
• Electron impact ionization-EI-where analytes are directly ionized through collision with
a bombarding electron stream resulting in the removal of an electron to form a
fragments.

18
 EI

Filament (Rh) Ion Lens

M: “inlet system”

M:

Repeller

Collector

1000 V 800 V 0 V

Vacuum system

19
 - +

Ion Lens
-

EI

Repeller

Collector
+
1000 V 800 V 0 V

Vacuum system

20
 - +

M: + e- * → M.* + 2e-
Ion Lens
-

M:

Repeller

Collector +

1000 V 800 V 0 V

Vacuum system

21
 + -

Ion Lens
-

+
+
+ + ++
++ +
+
+ -
Repeller

Collector

1000 V 800 V 0 V

+ -
Vacuum system

22
 Fragmentation Process

BC + + D

A + + BCD• A • + BCD+ A • + BD+

ABCD + e- ABCD+• + 2eq ADB+•

(~70 eV ; 6700 kJ/mol)
AB • + CD+ CD • + AB+ A + + BD•

C+D+ C ++ D A ++ B A+B+
FRAGMENTATION
REARRANGEMENTS
23
 Fragmentation process

Detected BC +
Abundance
%
ADB+•
A+ BCD+ BC+
A+ AB + BC +
ABCD+• ADB+•

CD AB AB +
B C+ D+
+
BCD+ ABCD+•
+
CD+
+

D+ C+ A+ B+
m/z

24
 Fragmentation Process

“Base Peak”
Relative
Abundance Fragmentation ABC+•
% (“product Ions”) “Molecular
A+ AB + BC + Ion”
(“Precursor”)

B+ C+ D+ CD+ BCD+ ABCD+•

m/z
Spectrum is a normalized line plot
25
 NIST Library Result

• NIST Library Search Result for Acenaphthylene

152
63 76 86 98 126
63 76 86 98 125
152
60 80 100 120 140 160 180 200 220 240 260
semivol_10000scan_01#4653 RT: 13.59 AV: 1 NL: 3.61E6Acenaphthylene
26
 NIST Result

27
 Electron Energy

Yield (%) 70eV

50 100 150 200 250

Electron Energy
10
ABC = M M+ 20eV
20eV
A+
Abundance

100

A+ M+ 40eV
40eV
B+
1000
C+ B+ A+
M+ 70eV
70eV
(Compound Dependent)

28
 PFTBA EI+ Spectrum

29
PICI Summary

Molecular weight information

Increased selectivity for many compounds

Selectivity affected by reagent gas

High pressure CI produces true CI spectra

(including adduct ions for confirmation)

30
 Positive Ion Chemical Ionization

• Reagent gas reacts with electrons to form reactive species

• Result is “softer” ionization which results in primary formation of
molecular ions.
• Main use is molecular weight confirmation

31
 Positive Ion Chemical Ionization

Reagent gas reactions (methane)

CH 4 + e − → CH 4+ , CH 3+ , CH 2+ m/z 16, 15, 14

+ +
CH + CH 4 → CH + CH 3
4 5
m/z 17

CH 3+ + CH 4 → C 2 H 5+ + H 2 m/z 29

CH 2+ + CH 4 → C 2 H 4+ + H 2 m/z 28
+
→ C2 H + H 2 + H
3
m/z 27

C 2 H 3+ + CH 4 → C 3 H 5+ + H 2 m/z 41

32
 Positive Ion Chemical Ionization

Proton transfer
M + CH → [M + H ] + CH 4
+ +
5
[M+1]+
M + C 2 H → [M + H ] + C 2 H 4
+ +
5

Hydride abstraction
M + C 2 H 5+ → [M − H ] + C 2 H 6
+
[M-1]+

Adduct formation
M + C 2 H 5+ → [M + C 2 H 5 ]
+
[M+29]+

M + C 3 H → [M + C 3 H 5 ]
+ +
5 [M+41]+

33
 Chemical Ionisation

• Chemical ionization mass spectrometry (CI-MS) begins with ionization

of methane, ammonia or another gas, creating a radical cation (e.g.
CH4•+ or NH3•+).
• This in turn will impact the sample molecule M to produce MH•+
molecular ions.
• Some of MH•+ fragments into smaller daughter ions and neutral
fragments. Both positive and negative ions are formed but only
positively charged species will be detected.
• Less fragmentation occurs with CI than with EI
• CI gives less information about the detailed structure of a molecule, but
does yield the molecular ion; sometimes the molecular ion cannot be
detected by the EI method, hence the two methods are complementary.

34
 100 pg of Benzophenone PCI – Scan Mode

M+1
M+29 M+41

35
 Negative Ion Chemical Ionization
• Reagent gas reacts with electrons to form “plasma” of thermal electrons
• Ionization is favored by molecules which have a high electron affinity –
electron capture
• Useful for selective analysis in heavy matrices, i.e. pesticide in food or
waste matrix.
• Compound is ionized through interaction between molecules and low
energy electrons
• Main reactions/interactions are resonance electron capture, forming
negative ions
• Ionization is selective for compounds whose molecules can undergo
electron capture process
• Fragmentation is not as extensive as EI  limited structural
information, but simple mass spectrum

36
 Data Acquisition Modes

• There are 2 methods of acquiring data in MS mode.

• Full Scan Mode

• Record all ions in the chosen mass range.

• Selected Ion Mode

• Look only at selected mass, jumping from one to the next.

37
 Improving Sensitivity

Single Ion Monitoring

• SIM
• Set quadrupole to pass a single characteristic ion during a retention time
window in the chromatogram
• Increases sensitivity
• More sensitivity than
obtained with full scan
• Typically get 5 - 50 times
better sensitivity compared
to full scan (depends on #
of ions monitored in SIM &
matrix interference)

38
 Thermo ISQ-Single Quadrupole
Designing in Selectivity (and Sensitivity)
• S-Shaped Ion Guide
• Dramatic reduction in neutral
noise
• Excited but neutral helium
follows a linear path from the
ion source, and therefore
misses the mass analyzer and
detector
• Maintain ion ratio performance,
even at very low concentrations

39
 Designing in Robustness

• ExtractaBrite Ion Source

• Repeller, ion volume, lenses and “RF lens”
• Contained in an easy-to-work-with cartridge sleeve
• No tools required for assembly or disassembly
• High efficiency ion focusing optics
• Repeller works with the RF Lens for a ‘push / pull’ approach to ion
extraction

40
 Ion Volume Technology

Each analyte has right ion

volume
• Ionvolume EI/PCI/NCI

• Vacuum lock technology

EI/CI

CI
EI

41
 Designing in Robustness

• ExtractaBrite Ion Source

• Highly inert ion volume material
• High temperature ion optical path
• Open geometry flow pathway
• Independent ion source and ion optical
path heating
• Select source temperature for method
performance; system will automatically run
ion optics as hot as possible

42
 Designing in Unique Innovation

• Full Source Removal

• Extraction of the cartridge
containing the repeller, source
and lenses while still under
vacuum

Cartridge Source

Source block

43
 Designing in Unique Innovation

Step 2. Remove source

Step 1. Insert removal tool

Step 4. Push source out of tool

Step 3. Hot source is held in tool

44
Type of Mass Analyzer
 Ion Trap
Ionization occurs in the Source, Mass Sorting occurs in the
Trap, followed by Detection via a conversion Dynode and
Electron Multiplier.
Additional capabilities include MSn.

46
47
 Helium Buffer Gas
1. The kinetic energy that allowed the ions to enter the ion trap also allows them to
subsequently escape.
2. To remove kinetic energy from the ions, different flow of helium buffer gas or damping gas
is present in the ion trap. Collisions remove kinetic energy and allow ions to be trapped.

Without Helium With Helium

+ + + He
collision He +
He

+ + He

+ He +

48
 How we improve Sensitivity

S/N= Sensitivity

Ion Trap Full scan mode most sensitive

Ion trap uses MS/MS to MSn

49
 Real World -Matrix Interference
• Background interferences confuse interpretation
• Useful characteristic ion information is hidden

EI –Full Scan spectrum

Standard

Body Tissue Extract Standard Full Scan Spectrum

Interference in Peak
Interference in
Spectrum

50
 Matrix Elimination Using MS/MS

• Enhanced Selectivity
• MS/MS spectrum for standards and samples are same

Standard Sample

Spectrum from Standard Spectrum from Body Tissue Extract

The Perfect
Separation
51
 MS/MS in an Ion Trap

1. Inject 3. Fragment

2. Isolate 4. Detect

52
Full Scan – 10pg/ul Dioxin in 25g Cow’s milk

RT: 0.00 - 40.04

7.92 NL:
100 7.75 2.93E7
95 TIC MS
CS3-MILK-
90 7.26 PCDD-
PCDF-FS-
85 402
80

75
8.26
70

65 27.28
Relative Abundance

60
6.99
55
26.78
50 8.42
25.34 27.46
45
29.00
40
25.08
35 29.31
30 6.46 24.84
21.60 29.72 37.36
25 23.38
29.96
20 8.73 20.09 20.84 30.40
15 19.85
18.13 35.13
8.99 17.49 31.28
10 12.23 12.78 34.51 36.12 38.00
6.08 9.76
5 5.69
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Time (min)

53
Dioxins: Full Scan vs. MS/MS – 10pg/ul

54
 Table of Contrasts

Ion Traps Single Quadrupoles

Mass Separation in time Mass Separation in space

High sensitivity Full Scan Lower sensitivity Full Scan

Selectivity of MS/MS High sensitivity SIM

55
Triple stage quadrupole -TSQ
 “To make the productivity advantages
of high performance GC-MS/MS easy
to achieve and available routinely;
especially for high-throughput
laboratories.”
Design philosophy

57
58
 Unstoppable productivity starts with
TRACE™ 1300 Series GC
•Instant Connect (iC) injector and detector modules
•User switching in minutes
•For maintenance or flexibility
•Low thermal mass design throughout
•Fast heating and cooling for higher sample
throughput

•TriPlus™
TriPlus™ RSH autosampler
•Automation of sample
preparation steps
•Dilutions
•Addition of standards

59
 Not just MS/MS performance

Full
Scan

SIM

•High full scan spectral quality

•Searchable with NIST etc.
•High sensitivity SIM & Full Scan
•Virtually indistinguishable from a
Lindane isomers 40 ppb in cucumber in
simultaneous full scan/SIM
single quadrupole instrument
•Dual acquisition modes
•Quan & qual in the same run

60
 Routinely achieve regulatory detection limits with confidence

Dichlorvos in mixed herbal

extract at 5 ppb

Full
Scan

SIM

•Cut through chemical background

•High selectivity in matrix compared with
SRM single stage MS
•Selected Reaction Monitoring (SRM)
•S-shaped curved ion guide lens
•High precision at low concentrations
• Femtogram level detection
• Confirming ions

61
 Triple Stage Quadrupole

• The analyzer of a “triple quadrupole” instrument consists of two

quadrupoles, separated by a collision cell. Such a configuration is
often referred as a "tandem in space" arrangement.

• Precursor ions and product ions are created and analyzed in different
physical spaces.

• Ions must be moved from the ion-source to the analyzer (different

physical regions) where different functions take place.

62
 Selected Reaction Monitoring
Purpose of SRM: Quantitation on a single product ion, no spectrum
The second quadrupole fragments the precursor This multiple
The first quadrupole,
ion using argon gas. Argon is chosen due to its number of SRMs
Q1, isolates one
low cost and high efficiency. Only small
mass. This is the is termed MRM.
amounts of Ar are used
precursor ion. This
is the same process “Monitor a Transition”
that happens in a from Precursor in Q1 to Product Ion in Q3
single quad in SIM

Argon
Collision Gas
Q1 on precursor ion Q3 on product ion
m/z 272 SRM on m/z 237

63
 Quinoxyfen in Hops using SIM and MS/MS
RT: 23.76
100

Single Quad
Relative Abundance

24.01
80 237
60 23.55 24.11 24.55 25.35 25.52
23.28
40

20
SIM Mode 20.27 20.40
21.09 21.30
21.67
22.17
22.52 22.97 25.65
25.87 26.45
26.78

0
25.37
100
Relative Abundance

80
23.53 RT: 23.76 272
60
27.35
40 22.93
20.19 23.43 26.62
21.25 24.55 24.67 25.48
20 19.61 20.57 22.17 22.41 24.02 26.26

0
RT: 23.76
100
Relative Abundance

80

60
24.59 307
23.13 24.17 25.18 25.34
22.93 26.31 27.09
40 23.37 26.46
21.67 21.89 22.52
20 19.65 20.15 20.57 21.15
0
0 RT:
RT: 23.77
23.77
100
Relative Abundance

80
307>207
60

40

20

0
RT: 23.77
100
Relative Abundance

80

60 Triple Quad MS/MS 307 >237

40

20 MRM Mode
0
18 19 20 21 22 23 24 25 26 27
Time (min)

64
 Scan Modes Benefits

• Full Scan
• Best for obtaining much spectral information
• Ideal for unknowns
• Selected Ion Monitoring (SIM)
• Best when more sensitivity is needed than with full
scan
• Works best when sample matrix is not too complicated
• Selective Reaction Monitoring (SRM) & Multiple
Reaction Monitoring (MRM)
• Ideal for improving sensitivity with samples containing
dirty or complex matrix
• Useful tool for structure elucidation

65
65
 PTV Backflush of Pear Extracted with AcEt
Area of high boiling matrix
RT: 5.07 - 24.68
Octadecanoic Acid 14.68
100 Extract
Sitosterol (area)

80 BKF at 20 min (No BKF) 14.19

• No BKF
Vitamine E 21.15
13.39

Relative Abundance
7.64 17.70 19.90
60
21.47
18.30
11.76
40 8.15 9.73 16.52
12.64 22.03
7.42 16.86
8.72 15.38 22.17
20 19.45
5.81 11.23 22.51 23.47
6.74

• With BKF 0
100
Extract
Octadecanoic Acid
14.20

BKF at 10 min Sitosterol (area)

• BKF ON 10 min 80
13.78
Vitamine E

Relative Abundance
13.04
60 7.65 19.08

• After injection 40
7.42
9.38 9.73
11.61

12.38
15.93
17.00

8.72
20 5.83 6.88 11.15 15.27 17.59
19.24 19.98 23.05 23.69
20.84
0
14.88
100 Standard Mix
Pesticides

80
12.95 15.97
Relative Abundance

BKF at 10 min

• Pesticide standard 60
12.32
13.72

14.51
16.78

40 17.07

• BKF ON 10 min 20
7.29 7.80
9.08 10.41 11.53
11.29
17.82
18.44

18.65
6.45 7.18 8.39 9.26 20.79 21.30 21.44 23.65
0
6 8 10 12 14 16 18 20 22 24
Time (min)
Full scan data acquisition – Trace GC w PTV-BKF – 30 m TR-Pesticides, 5 m pre-column 0.53 mm ID

66
GC/MS Scan Modes
 Scan Modes – SRM

MS Mode Q1 Q2 Q3
Selected reaction
monitoring (SRM)

Selected reaction
monitoring (SRM)

= scanned; = selected m/z; = CID

Selected reaction monitoring (SRM) mode is a MS/MS technique that
allows the first RF/DC quadrupole filter (Q1) to be set at one RF/DC
settings to filter for a specific m/z (the precursor ion). A collision
induced dissociation (CID) is performed in the collision cell (Q2). The
second RF/DC quadrupole filter (Q3) to be set at one (or more) RF/DC
settings to filter for a specific m/z value(s) (the product ion) or can be
scanned for many product ions. Selecting Q3 for 1 or 2 ions will yield
better sensitivities than scanning Q3.
68
68
 MS/MS Simplicity

• Automatic SRM Development

• True Timed-SRM
• Links between SRM Instrument method and TraceFinder
• Compound based SRM method creation through CDS

69
 ExtractoBrite Ion Source

• Source cartridge containing:

• Ion reseller
• Ion volume
• Lens stack
• No wires!

70
 ExtractaBrite Ion Source

Patented RF Lens

• Patented RF lens protects ion guide and quads

• RF lens is removed with source cartridge without venting
• RF lens kept hot to keep clean

71
 ExtractaBrite Ion Source

Patented RF Lens

Repeller

• Patented RF lens protects ion guide and quads

• RF lens is removed with source cartridge without venting
• RF lens kept hot to keep clean
• Repeller designed to overcome ion burn once it forms

72
 MS/MS Simplicity - AutoSRM

Step 1: Select Step 2: Select Step 3: Optimize

your precursor your product ions SRM collision
ions from from product ion energies
fullscan scans

SRM Development Workflow

73
 Highlights of AutoSRM

• Automates the following for transition selection and

optimization:
• Creation of fullscan, product ion scan and SRM methods
• Creation of sample sequences
• Creation of data layouts for analyzing results
• Selection of precursor, product and collision energies

End result showing optimized transition

74
 Automated Update of Transition Windows

75
 Associate Raw File to Update Scan Filters

76
 Your next competitive advantage

77
 Thermo Scientific+Dionex

78
Basics of GCMS

Ms.Aarti Karkhanis
Asst.Manager (Applications GCMS group)
ThermoFisher Scientific Mumbai
 Historical Development of GC/MS

• 1910- J.J. Thompson developed the first MS

• 1910- M.S.Tswett –published his book
• 1918- Dempster used EI for first time
• 1941- Martin & Synge- Published a paper on GLC
• 1946- Stephens proposed a TOF
• 1947- NBS started collection of MS spectra
• 1950- Gohlke-First time published coupling of GC with MS
• 1950-Martin & Synge-Nobel prize
• 1955- Desty -First Commercial GC
• 1958- Paul published information on quadrupole mass filter
• 1958- Ken Shoulders manufactured the first Quadrupole MS
• 1958- Golay –Open Tubular columns
• 1964- First commercial quadrupole MS( Quad RGA) –Bob Finnigan

2
 Historical Development of GC/MS

• 1966- Munson & Field – CI

• 1968-First commercial quadrupole GCMS – Finnigan Instruments
• 1978- Fused Silica Capillary columns- Dandenau
• 1978- Triple quadrupole technology – Yost & Enke
• 1982- Finnigan –first patents on Ion trap technology
• 1989- Prof Wolfgang Paul – Nobel prize
• 2000-A. Makarov – New Type of mass analyzer concept -Orbitrap
• 2005- Thermo – Introduced Orbitrap

3
 Analytical Challenges

• Complex Samples (Tissues, water,soil, sediments, food and beverages,

etc…)

• Multiple components of various character (multi residue analysis)

• Metabolites , product degradations and new contaminants

• Method selectivity in order to determine the components of interest only

• Low levels of detection

• Higher quality results: results which are reliable

4
 How to identify in GC ?

By Injecting Standards!

5
 What is Mass Spectrometry?

“The basis of MS (mass spectrometry) is the production of

ions that are subsequently separated or filtered according to
their mass-to-charge (m/z) ratio and detected. The
resulting mass spectrum is a plot of the (relative) abundance of
the produced ions as a function of the m/z ratio.”

6
 Mass Spectrometer

…Introduction of mass ions in a

separation field…

+
_

Ion Source Mass Analyzer Detector

7
 Relative abundance

m/z

8
Components of a Mass Spectrometer

Atmosphere Vacuum
System

Sample Ionisation Mass Data

Detector
Inlet Method Analyser System

9
10
 Ion source

11
 Quadrupole mass spectrometer

• Voltages applied to four cylindrical quadrupole rods generate a quadrupole field in the center
axis of the mass spectrometer
• Ions entering the field will oscillate; the oscillation characteristics depend on the mass-to-
charge ratio of the ions and the voltage applied
• Voltage applied to the quadrupole can be varied/selected so that only ions with specific m/z will
have stable oscillations in the center axis, and will reach the ion detector

12
 Electron Multiplier

• Electron multiplier
• A conversion dynode is used to convert either negative or positive ions
into electrons.
• These electrons are amplified by a cascade effect in a horn shape
device, to produce a current.
• This device, also called channeltron, is widely used in quadrupole and
ion trap instruments.

13
 Vacuum System

• All MS systems require high vacuum of the order of 10-5 to 10-6 torr. It is
because of free movement of ions in the field and to reduce the
background

14
15
 PFTBA EI+ Spectrum

16
Ionization Methods

Electron Impact Ionisation

Positive Chemical Ionisation
Negative Chemical Ionisation
 Electron Impact Ionization
• Most common method of ionization for GC-MS
• Used as both -qualitative and quantitative tool
• Produces mass spectra of molecules
• Fragmentation fingerprints
• Combine with retention time for positive identification

• Extensive fragmentation of sample molecules gives rise to “fingerprint” of

compound, useful for identification purpose

• m/z value of the molecular ion (M+) gives information about the molecular weight of
the compound, also useful for identification purpose

• In some cases, M+ ion does not survive fragmentation due to the high energy
involved in the process. Two approaches can be taken:
• Use single ion for quantitation with one or more ions for verification
• Electron impact ionization-EI-where analytes are directly ionized through collision with
a bombarding electron stream resulting in the removal of an electron to form a
fragments.

18
 EI

Filament (Rh) Ion Lens

M: “inlet system”

M:

Repeller

Collector

1000 V 800 V 0 V

Vacuum system

19
 - +

Ion Lens
-

EI

Repeller

Collector
+
1000 V 800 V 0 V

Vacuum system

20
 - +

M: + e- * → M.* + 2e-
Ion Lens
-

M:

Repeller

Collector +

1000 V 800 V 0 V

Vacuum system

21
 + -

Ion Lens
-

+
+
+ + ++
++ +
+
+ -
Repeller

Collector

1000 V 800 V 0 V

+ -
Vacuum system

22
 Fragmentation Process

BC + + D

A + + BCD• A • + BCD+ A • + BD+

ABCD + e- ABCD+• + 2eq ADB+•

(~70 eV ; 6700 kJ/mol)
AB • + CD+ CD • + AB+ A + + BD•

C+D+ C ++ D A ++ B A+B+
FRAGMENTATION
REARRANGEMENTS
23
 Fragmentation process

Detected BC +
Abundance
%
ADB+•
A+ BCD+ BC+
A+ AB + BC +
ABCD+• ADB+•

CD AB AB +
B C+ D+
+
BCD+ ABCD+•
+
CD+
+

D+ C+ A+ B+
m/z

24
 Fragmentation Process

“Base Peak”
Relative
Abundance Fragmentation ABC+•
% (“product Ions”) “Molecular
A+ AB + BC + Ion”
(“Precursor”)

B+ C+ D+ CD+ BCD+ ABCD+•

m/z
Spectrum is a normalized line plot
25
 NIST Library Result

• NIST Library Search Result for Acenaphthylene

152
63 76 86 98 126
63 76 86 98 125
152
60 80 100 120 140 160 180 200 220 240 260
semivol_10000scan_01#4653 RT: 13.59 AV: 1 NL: 3.61E6Acenaphthylene
26
 NIST Result

27
 Electron Energy

Yield (%) 70eV

50 100 150 200 250

Electron Energy
10
ABC = M M+ 20eV
20eV
A+
Abundance

100

A+ M+ 40eV
40eV
B+
1000
C+ B+ A+
M+ 70eV
70eV
(Compound Dependent)

28
 PFTBA EI+ Spectrum

29
PICI Summary

Molecular weight information

Increased selectivity for many compounds

Selectivity affected by reagent gas

High pressure CI produces true CI spectra

(including adduct ions for confirmation)

30
 Positive Ion Chemical Ionization

• Reagent gas reacts with electrons to form reactive species

• Result is “softer” ionization which results in primary formation of
molecular ions.
• Main use is molecular weight confirmation

31
 Positive Ion Chemical Ionization

Reagent gas reactions (methane)

CH 4 + e − → CH 4+ , CH 3+ , CH 2+ m/z 16, 15, 14

+ +
CH + CH 4 → CH + CH 3
4 5
m/z 17

CH 3+ + CH 4 → C 2 H 5+ + H 2 m/z 29

CH 2+ + CH 4 → C 2 H 4+ + H 2 m/z 28
+
→ C2 H + H 2 + H
3
m/z 27

C 2 H 3+ + CH 4 → C 3 H 5+ + H 2 m/z 41

32
 Positive Ion Chemical Ionization

Proton transfer
M + CH → [M + H ] + CH 4
+ +
5
[M+1]+
M + C 2 H → [M + H ] + C 2 H 4
+ +
5

Hydride abstraction
M + C 2 H 5+ → [M − H ] + C 2 H 6
+
[M-1]+

Adduct formation
M + C 2 H 5+ → [M + C 2 H 5 ]
+
[M+29]+

M + C 3 H → [M + C 3 H 5 ]
+ +
5 [M+41]+

33
 Chemical Ionisation

• Chemical ionization mass spectrometry (CI-MS) begins with ionization

of methane, ammonia or another gas, creating a radical cation (e.g.
CH4•+ or NH3•+).
• This in turn will impact the sample molecule M to produce MH•+
molecular ions.
• Some of MH•+ fragments into smaller daughter ions and neutral
fragments. Both positive and negative ions are formed but only
positively charged species will be detected.
• Less fragmentation occurs with CI than with EI
• CI gives less information about the detailed structure of a molecule, but
does yield the molecular ion; sometimes the molecular ion cannot be
detected by the EI method, hence the two methods are complementary.

34
 100 pg of Benzophenone PCI – Scan Mode

M+1
M+29 M+41

35
 Negative Ion Chemical Ionization
• Reagent gas reacts with electrons to form “plasma” of thermal electrons
• Ionization is favored by molecules which have a high electron affinity –
electron capture
• Useful for selective analysis in heavy matrices, i.e. pesticide in food or
waste matrix.
• Compound is ionized through interaction between molecules and low
energy electrons
• Main reactions/interactions are resonance electron capture, forming
negative ions
• Ionization is selective for compounds whose molecules can undergo
electron capture process
• Fragmentation is not as extensive as EI  limited structural
information, but simple mass spectrum

36
 Data Acquisition Modes

• There are 2 methods of acquiring data in MS mode.

• Full Scan Mode

• Record all ions in the chosen mass range.

• Selected Ion Mode

• Look only at selected mass, jumping from one to the next.

37
 Improving Sensitivity

Single Ion Monitoring

• SIM
• Set quadrupole to pass a single characteristic ion during a retention time
window in the chromatogram
• Increases sensitivity
• More sensitivity than
obtained with full scan
• Typically get 5 - 50 times
better sensitivity compared
to full scan (depends on #
of ions monitored in SIM &
matrix interference)

38
 Thermo ISQ-Single Quadrupole
Designing in Selectivity (and Sensitivity)
• S-Shaped Ion Guide
• Dramatic reduction in neutral
noise
• Excited but neutral helium
follows a linear path from the
ion source, and therefore
misses the mass analyzer and
detector
• Maintain ion ratio performance,
even at very low concentrations

39
 Designing in Robustness

• ExtractaBrite Ion Source

• Repeller, ion volume, lenses and “RF lens”
• Contained in an easy-to-work-with cartridge sleeve
• No tools required for assembly or disassembly
• High efficiency ion focusing optics
• Repeller works with the RF Lens for a ‘push / pull’ approach to ion
extraction

40
 Ion Volume Technology

Each analyte has right ion

volume
• Ionvolume EI/PCI/NCI

• Vacuum lock technology

EI/CI

CI
EI

41
 Designing in Robustness

• ExtractaBrite Ion Source

• Highly inert ion volume material
• High temperature ion optical path
• Open geometry flow pathway
• Independent ion source and ion optical
path heating
• Select source temperature for method
performance; system will automatically run
ion optics as hot as possible

42
 Designing in Unique Innovation

• Full Source Removal

• Extraction of the cartridge
containing the repeller, source
and lenses while still under
vacuum

Cartridge Source

Source block

43
 Designing in Unique Innovation

Step 2. Remove source

Step 1. Insert removal tool

Step 4. Push source out of tool

Step 3. Hot source is held in tool

44
Type of Mass Analyzer
 Ion Trap
Ionization occurs in the Source, Mass Sorting occurs in the
Trap, followed by Detection via a conversion Dynode and
Electron Multiplier.
Additional capabilities include MSn.

46
47
 Helium Buffer Gas
1. The kinetic energy that allowed the ions to enter the ion trap also allows them to
subsequently escape.
2. To remove kinetic energy from the ions, different flow of helium buffer gas or damping gas
is present in the ion trap. Collisions remove kinetic energy and allow ions to be trapped.

Without Helium With Helium

+ + + He
collision He +
He

+ + He

+ He +

48
 How we improve Sensitivity

S/N= Sensitivity

Ion Trap Full scan mode most sensitive

Ion trap uses MS/MS to MSn

49
 Real World -Matrix Interference
• Background interferences confuse interpretation
• Useful characteristic ion information is hidden

EI –Full Scan spectrum

Standard

Body Tissue Extract Standard Full Scan Spectrum

Interference in Peak
Interference in
Spectrum

50
 Matrix Elimination Using MS/MS

• Enhanced Selectivity
• MS/MS spectrum for standards and samples are same

Standard Sample

Spectrum from Standard Spectrum from Body Tissue Extract

The Perfect
Separation
51
 MS/MS in an Ion Trap

1. Inject 3. Fragment

2. Isolate 4. Detect

52
Full Scan – 10pg/ul Dioxin in 25g Cow’s milk

RT: 0.00 - 40.04

7.92 NL:
100 7.75 2.93E7
95 TIC MS
CS3-MILK-
90 7.26 PCDD-
PCDF-FS-
85 402
80

75
8.26
70

65 27.28
Relative Abundance

60
6.99
55
26.78
50 8.42
25.34 27.46
45
29.00
40
25.08
35 29.31
30 6.46 24.84
21.60 29.72 37.36
25 23.38
29.96
20 8.73 20.09 20.84 30.40
15 19.85
18.13 35.13
8.99 17.49 31.28
10 12.23 12.78 34.51 36.12 38.00
6.08 9.76
5 5.69
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Time (min)

53
Dioxins: Full Scan vs. MS/MS – 10pg/ul

54
 Table of Contrasts

Ion Traps Single Quadrupoles

Mass Separation in time Mass Separation in space

High sensitivity Full Scan Lower sensitivity Full Scan

Selectivity of MS/MS High sensitivity SIM

55
Triple stage quadrupole -TSQ
 “To make the productivity advantages
of high performance GC-MS/MS easy
to achieve and available routinely;
especially for high-throughput
laboratories.”
Design philosophy

57
58
 Unstoppable productivity starts with
TRACE™ 1300 Series GC
•Instant Connect (iC) injector and detector modules
•User switching in minutes
•For maintenance or flexibility
•Low thermal mass design throughout
•Fast heating and cooling for higher sample
throughput

•TriPlus™
TriPlus™ RSH autosampler
•Automation of sample
preparation steps
•Dilutions
•Addition of standards

59
 Not just MS/MS performance

Full
Scan

SIM

•High full scan spectral quality

•Searchable with NIST etc.
•High sensitivity SIM & Full Scan
•Virtually indistinguishable from a
Lindane isomers 40 ppb in cucumber in
simultaneous full scan/SIM
single quadrupole instrument
•Dual acquisition modes
•Quan & qual in the same run

60
 Routinely achieve regulatory detection limits with confidence

Dichlorvos in mixed herbal

extract at 5 ppb

Full
Scan

SIM

•Cut through chemical background

•High selectivity in matrix compared with
SRM single stage MS
•Selected Reaction Monitoring (SRM)
•S-shaped curved ion guide lens
•High precision at low concentrations
• Femtogram level detection
• Confirming ions

61
 Triple Stage Quadrupole

• The analyzer of a “triple quadrupole” instrument consists of two

quadrupoles, separated by a collision cell. Such a configuration is
often referred as a "tandem in space" arrangement.

• Precursor ions and product ions are created and analyzed in different
physical spaces.

• Ions must be moved from the ion-source to the analyzer (different

physical regions) where different functions take place.

62
 Selected Reaction Monitoring
Purpose of SRM: Quantitation on a single product ion, no spectrum
The second quadrupole fragments the precursor This multiple
The first quadrupole,
ion using argon gas. Argon is chosen due to its number of SRMs
Q1, isolates one
low cost and high efficiency. Only small
mass. This is the is termed MRM.
amounts of Ar are used
precursor ion. This
is the same process “Monitor a Transition”
that happens in a from Precursor in Q1 to Product Ion in Q3
single quad in SIM

Argon
Collision Gas
Q1 on precursor ion Q3 on product ion
m/z 272 SRM on m/z 237

63
 Quinoxyfen in Hops using SIM and MS/MS
RT: 23.76
100

Single Quad
Relative Abundance

24.01
80 237
60 23.55 24.11 24.55 25.35 25.52
23.28
40

20
SIM Mode 20.27 20.40
21.09 21.30
21.67
22.17
22.52 22.97 25.65
25.87 26.45
26.78

0
25.37
100
Relative Abundance

80
23.53 RT: 23.76 272
60
27.35
40 22.93
20.19 23.43 26.62
21.25 24.55 24.67 25.48
20 19.61 20.57 22.17 22.41 24.02 26.26

0
RT: 23.76
100
Relative Abundance

80

60
24.59 307
23.13 24.17 25.18 25.34
22.93 26.31 27.09
40 23.37 26.46
21.67 21.89 22.52
20 19.65 20.15 20.57 21.15
0
0 RT:
RT: 23.77
23.77
100
Relative Abundance

80
307>207
60

40

20

0
RT: 23.77
100
Relative Abundance

80

60 Triple Quad MS/MS 307 >237

40

20 MRM Mode
0
18 19 20 21 22 23 24 25 26 27
Time (min)

64
 Scan Modes Benefits

• Full Scan
• Best for obtaining much spectral information
• Ideal for unknowns
• Selected Ion Monitoring (SIM)
• Best when more sensitivity is needed than with full
scan
• Works best when sample matrix is not too complicated
• Selective Reaction Monitoring (SRM) & Multiple
Reaction Monitoring (MRM)
• Ideal for improving sensitivity with samples containing
dirty or complex matrix
• Useful tool for structure elucidation

65
65
 PTV Backflush of Pear Extracted with AcEt
Area of high boiling matrix
RT: 5.07 - 24.68
Octadecanoic Acid 14.68
100 Extract
Sitosterol (area)

80 BKF at 20 min (No BKF) 14.19

• No BKF
Vitamine E 21.15
13.39

Relative Abundance
7.64 17.70 19.90
60
21.47
18.30
11.76
40 8.15 9.73 16.52
12.64 22.03
7.42 16.86
8.72 15.38 22.17
20 19.45
5.81 11.23 22.51 23.47
6.74

• With BKF 0
100
Extract
Octadecanoic Acid
14.20

BKF at 10 min Sitosterol (area)

• BKF ON 10 min 80
13.78
Vitamine E

Relative Abundance
13.04
60 7.65 19.08

• After injection 40
7.42
9.38 9.73
11.61

12.38
15.93
17.00

8.72
20 5.83 6.88 11.15 15.27 17.59
19.24 19.98 23.05 23.69
20.84
0
14.88
100 Standard Mix
Pesticides

80
12.95 15.97
Relative Abundance

BKF at 10 min

• Pesticide standard 60
12.32
13.72

14.51
16.78

40 17.07

• BKF ON 10 min 20
7.29 7.80
9.08 10.41 11.53
11.29
17.82
18.44

18.65
6.45 7.18 8.39 9.26 20.79 21.30 21.44 23.65
0
6 8 10 12 14 16 18 20 22 24
Time (min)
Full scan data acquisition – Trace GC w PTV-BKF – 30 m TR-Pesticides, 5 m pre-column 0.53 mm ID

66
GC/MS Scan Modes
 Scan Modes – SRM

MS Mode Q1 Q2 Q3
Selected reaction
monitoring (SRM)

Selected reaction
monitoring (SRM)

= scanned; = selected m/z; = CID

Selected reaction monitoring (SRM) mode is a MS/MS technique that
allows the first RF/DC quadrupole filter (Q1) to be set at one RF/DC
settings to filter for a specific m/z (the precursor ion). A collision
induced dissociation (CID) is performed in the collision cell (Q2). The
second RF/DC quadrupole filter (Q3) to be set at one (or more) RF/DC
settings to filter for a specific m/z value(s) (the product ion) or can be
scanned for many product ions. Selecting Q3 for 1 or 2 ions will yield
better sensitivities than scanning Q3.
68
68
 MS/MS Simplicity

• Automatic SRM Development

• True Timed-SRM
• Links between SRM Instrument method and TraceFinder
• Compound based SRM method creation through CDS

69
 ExtractoBrite Ion Source

• Source cartridge containing:

• Ion reseller
• Ion volume
• Lens stack
• No wires!

70
 ExtractaBrite Ion Source

Patented RF Lens

• Patented RF lens protects ion guide and quads

• RF lens is removed with source cartridge without venting
• RF lens kept hot to keep clean

71
 ExtractaBrite Ion Source

Patented RF Lens

Repeller

• Patented RF lens protects ion guide and quads

• RF lens is removed with source cartridge without venting
• RF lens kept hot to keep clean
• Repeller designed to overcome ion burn once it forms

72
 MS/MS Simplicity - AutoSRM

Step 1: Select Step 2: Select Step 3: Optimize

your precursor your product ions SRM collision
ions from from product ion energies
fullscan scans

SRM Development Workflow

73
 Highlights of AutoSRM

• Automates the following for transition selection and

optimization:
• Creation of fullscan, product ion scan and SRM methods
• Creation of sample sequences
• Creation of data layouts for analyzing results
• Selection of precursor, product and collision energies

End result showing optimized transition

74
 Automated Update of Transition Windows

75
 Associate Raw File to Update Scan Filters

76
 Your next competitive advantage

77
 Thermo Scientific+Dionex

78
 Applications Notebook
Edition 1, February 2009

Chromatography &
Mass Spectrometry

Environmental/Food Safety

Drug Discovery

Clinical Research/Toxicology

Metabolism/Metabolomics

Proteomics

Part of Thermo Fisher Scientific

 Welcome to the Thermo Scientific Application
Notebook, a compilation of application note
abstracts that give you a glimpse of our
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LC, LC-MS and ICP-MS.

We offer complete laboratory solutions for all

analysis needs, whether your area of interest is
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Table of Contents
Environmental / Food Safety
LC-MS/MS Analysis of Herbicides in Drinking Water at Femtogram Levels
Using 20 mL EQuan Direct Injection Techniques...................................................... 4
Analysis of Triazine Pesticides in Drinking Water Using LC-MS/MS
(EPA Method 536.0)............................................................................................... 5
GC Single Quadrupole MS Analysis of Pesticides and Flame Retardants

Table of Contents
in Drinking Water According to US EPA Method 527 ............................................... 6
Speciation of Bromine in Water Samples Using HPLC Coupled to the
XSERIES 2 ICP-MS................................................................................................ 7
Rapid Screening of Pesticides Using U-HPLC/MS........................................................ 8
Analysis of Melamine and Cyanuric Acid in Food Matrices by LC–MS/MS .................... 9
LC-MS/MS Analysis of Malachite Green, Leucomalachite Green, Ciprofloxacin,
and Tetracycline in Food Samples Using a TurboFlow Method......................................10
Identification of Lysergic Acid Diethylamide (LSD) in Candy by U-HPLC/MS ..................12

Drug Discovery
Enhanced Reproducibility Performance of the TSQ Vantage LC-MS/MS:
Bioanalysis of Paroxetine in Rat Plasma.................................................................13
Comparing Manual Compound Optimizations Against Automated
Optimizations Obtained by QuickQuan on the TSQ Quantum Access LC-MS/MS........14

Clinical Research / Toxicology

Quantitation of Fentanyl and Norfentanyl from Urine Using On-line
High-Throughput System.......................................................................................16
High-Throughput LC-MS/MS Method for Quantitative Analysis
of Four Immunosuppressant Drugs in Whole Blood.................................................17

Metabolism / Metabolomics
Determining Distribution of Erlotinib in Pancreatic Tumors by MALDI
Using the LTQ XL Linear Ion Trap ...........................................................................18
Fast, Sensitive, and Accurate Metabolite Identification Using
Multiple Mass Defect Filters and Higher Energy Collisional Dissociation ..................19
Identification of GSH Conjugates Using Accurate Mass Data and
MetWorks Software .............................................................................................20
Determination of Occupational Exposure to Aromatic Solvents
Through Metabolite Monitoring Using Isocratic HPLC .............................................21
Analysis of Green and Black Tea Extracts Using an Accela High-Speed U-HPLC
Coupled to an LTQ Orbitrap XL Hybrid Linear Ion Trap Mass Spectrometer ...............22

Proteomics
Using Mass List Preprocessing to Increase Confidence of Protein Identification
from ETD and ECD Spectra....................................................................................23
A New Methodology for Targeted Peptide Quantitation in Complex Mixtures
Using a High-Resolution Triple Quadrupole Mass Spectrometer...............................24
MALDI Produced Ions Examined with a Linear Ion Trap – Orbitrap Mass Analyzer ........26

Legal Notices
©2009 Thermo Fisher Scientific Inc. All rights reserved. All trademarks not specifically referenced are the property of Thermo Fisher Scientific Inc. and its
subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these
products in any manners that might infringe the intellectual property rights of others. Not all products are available in all countries. Please consult your local sales
representative for details. 3
 LC-MS/MS Analysis of Herbicides in Drinking Water
at Femtogram Levels Using 20 mL EQuan Direct
Injection Techniques
Jonathan R. Beck, Charles Yang, Thermo Fisher Scientific, San Jose, CA, USA

Introduction integration is not shown in the chromatogram. Injections at

As concerns grow over the toxic effects of herbicides and higher volumes show superior signal-to-noise ratios and
Environmental/

other chemicals in our environment, the need to accurately intensity, which allow for analysis of very low concentration
Food Safety

monitor these substances in drinking water and foods becomes samples (pg/mL and fg/mL). The results of this study are
even more critical. LC-MS/MS is routinely used by the shown in Table 1.
environmental and beverage industries to identify and quantify
Conclusion
pesticide and herbicide residues. However, this method
typically requires extensive offline sample preconcentration Using a preconcentration column in tandem with an analytical
methods, which can be expensive and time-consuming, to HPLC column allows for the quantitation of a triazine herb-
meet the stringent requirements and low limits of detection icide mixture over the concentration range 0.1 – 10.0 pg/mL.
set forth by federal and international regulatory authorities. The large injection volume capabilities of the EQuan™
The Thermo Scientific EQuan system provides online sample system eliminated the need for laborious and expensive offline
preconcentration and analysis of sample volumes up to 20 mL. preconcentration using solid phase extraction. Injection
volumes ranging from 1 mL to 20 mL are possible using
Experimental Conditions this configuration, thus offering flexibility for laboratories
based on their sensitivity and reporting requirements.
Sample Preparation
Drinking water containing 0.1% formic acid was spiked
with a mixture of herbicides (see Table 1). The concentrations
of the herbicides in the spiked water ranged from 0.1 pg/mL
to 10 pg/mL. Calibration standards were prepared at the
following concentrations: 0.1, 0.5, 1.0, 5.0, and 10.0 pg/mL.

HPLC
Spiked water samples and blank water samples (1 mL, 5 mL, or
20 mL) were injected directly onto a loading column (Thermo
Scientific Hypersil GOLD 20 mm x 2.1 mm ID, 12 µm)
using an HTC PAL autosampler (CTC Analytics, Zwingen,
Switzerland). After preconcentration, the herbicides were
back flushed onto the analytical column (Hypersil GOLD™
50 mm x 2.1 mm ID, 3 µm), where the compounds were
separated prior to introduction into the mass spectrometer.

MS
MS analysis was carried out on a Thermo Scientific TSQ
Quantum Access triple stage quadrupole mass spectrometer
with an electrospray ionization (ESI) source.

Results
Chromatograms of the herbicide simazine at three different Figure 1: Chromatograms showing the injection of simazine with 1, 5, and 20 mL
injection volumes are shown in Figure 1. A very small peak injection volumes. The concentration of simazine is 1 pg/mL for all three injections.
can be seen for the 1 mL injection volume; however, the
Factor Factor %RSD
Compound Area, 1 mL Area, 5 mL Area, 20 mL 1 mL to 5 mL 5 mL to 20 mL (n = 8)
Atraton ND 1.16E+07 5.42E+07 N/A 4.69 11.15
Simetryn ND 4.27E+06 1.94E+07 N/A 4.56 8.93
Prometon/Secbumeton 3.26E+06 1.07E+07 4.80E+07 3.30 4.47 9.89
Ametryn 4.34E+06 1.42E+07 5.99E+07 3.27 4.22 11.59
Simazine ND 1.28E+06 5.70E+06 N/A 4.44 5.32
Prometryn/Terbutryn 6.19E+06 1.89E+07 7.61E+07 3.05 4.02 3.99
Atrazine 1.26E+06 4.45E+06 1.55E+07 3.53 3.49 4.97
Table 1: Peak areas and reproducibility for 20 mL injections (n = 8) at a 1 pg/mL concentration level, without an internal standard.

4 To download the full text of this application note, visit www.thermo.com/msnotebook

Analysis of Triazine Pesticides in Drinking Water Using
LC-MS/MS (EPA Method 536.0)
Jonathan Beck, Thermo Fisher Scientific, San Jose, CA, USA

Introduction
The United States Environmental Protection Agency (EPA)
has recently issued a draft form of a proposed method for
the analysis of triazine compounds in drinking water.1 This

Environmental/
Food Safety
method uses a simple method to directly analyze triazine
compounds using LC-MS/MS without requiring any solid
phase extraction (SPE) or other lengthy sample preparation
steps. This application note demonstrates the analysis of
these compounds over the concentration range 0.25 –
5.0 ng/mL (ppb) using the Thermo Scientific TSQ Quantum
Access triple stage quadrupole mass spectrometer and the
Thermo Scientific Accela HPLC system.

Experimental Conditions
Sample Preparation
While no SPE was required for this method, samples were
treated as per the EPA’s draft method. The method calls for the
addition of ammonium acetate at 20 mM for pH adjustment
and dechlorination and sodium omadine at 64 mg/L to prevent
microbial degradation, both purchased from Sigma-Aldrich,
St. Louis, MO. All samples were prepared in reagent water.
All samples were spiked with the internal standard solution,
resulting in a final concentration of 5 ng/mL (ppb) for each
internal standard. Calibration standards were prepared at
the following levels: 0.25, 0.5, 1, 2, 2.5 and 5 ng/mL.

HPLC Conditions
Column: Thermo Scientific Hypersil GOLD 100 x 2.1 mm, 3 µm
Solvent A: 5 mM Ammonium Acetate Figure 1: Chromatogram of the triazine compounds at 2 ppb, and their
Solvent B: Methanol internal standards
Flow Rate: 400 µL/min
Injection Volume: 100 µL
internal standards is shown in Figure 1. All peaks are
HPLC Gradient: Time %A %B
chromatographically resolved from one another. Calibration
0:00 98 2
curves were generated for each compound over the range
10:00 98 2
0.25 – 5.0 ppb. All calibration curves exhibited excellent
20:00 10 90
linearity, ranging from 0.9964 for Atrazine-desethyl to
25:00 10 90
25:06 98 2
0.9982 for Atrazine. The other compounds exhibit similar
30:00 98 2 linearity, and are not shown in this application note.

Mass Spectrometer Conditions Conclusion

Ionization Source: Positive Electrospray
The TSQ Quantum Access™ LC-MS/MS is an excellent
Sheath Gas: 30 arbitrary units
choice for the analysis of triazine compounds and their
Auxiliary Gas: 10 arbitrary units
degradates. Linearity over the entire calibration range of
ESI Voltage: 3.5 kV
0.25 to 5.0 ppb is observed. Separation of all the analytes is
Ion Transfer Tube Temperature: 350 °C
Collision Gas: 1.5 mTorr
achieved with the Hypersil GOLD™ column allowing for
Q1/Q3 Peak Resolution: 0.7 Da unambiguous identification and quantitation of all of the
Scan Width: 0.01 Da compounds in this application note.

Results References
1. Smith, G.A., Pepich, B.V., Munch, D.J. “Determination of Triazine Pesticides
The triazine compounds eluted from the LC column in and their Degradates in Drinking Water by Liquid Chromatography
20 minutes. A chromatogram of each compound and the Electrospray Ionization Mass Spectrometry (LC/ESI/MS)” Draft 5.0, April 2007.

To download the full text of this application note, visit www.thermo.com/msnotebook 5

 GC Single Quadrupole MS Analysis of Pesticides
and Flame Retardants in Drinking Water According
to US EPA Method 527
Jessie Butler, Eric Phillips, Thermo Fisher Scientific, Austin, TX, USA

Introduction
The United States Environmental Protection Agency (EPA)
Environmental/

released Method 527 to address the analysis of flame

Food Safety

retardants, pesticides, and synthetic pyrethroids in drinking

water.1 Flame retardants are a broad group of brominated
organic compounds used in plastics, electronics, textiles,
and clothing.2 Method 527 covers compounds with broad
mass ranges and activities, and requires low detection limits,
making this method challenging to optimize. This application
describes the optimization of EPA Method 527 using the
Thermo Scientific DSQ II single quadrupole mass spectrometer,
and the Thermo Scientific FOCUS GC gas chromatograph
using alternative injection, separation and detection settings
a
from those cited in the EPA method.

Experimental Conditions Tetrabromodiphenyl ether

This study did not evaluate sample preparation. Samples were
analyzed using a 1 µL surge splitless injection, which minimized
band broadening, provided narrow GC peaks, and reduced Pentabromodiphenyl
breakdown and activity in the injection port. Column flow was
set at 3 mL/min, shortening the time that the compounds
spent in the stationary phase. A 35% diphenyl/65% dimethyl
polysiloxane phase minimized adverse chemical interactions Hexabromodiphenyl
that occurred with the phase recommended in the EPA Method.
Detection was performed using the DSQ II single
quadrupole MS operated in full-scan electron ionization (EI) Hexabromodiphenyl ether
mode. To ensure that the system met Method 527 tune
requirements for DFTPP and also delivered low detection b
limits for the high-mass brominated flame retardants, two
Figure 1a. Total ion chromatogram for the 10 µg/L standard.
separate tune files and two different mass ranges were used.
1b. Extracted ion chromatograms for selected brominated flame retardants.
DFTPP criteria were achieved with a low-mass tune, while
high-mass sensitivity was preserved using a high-mass tune
file. Two mass ranges were used, with a 5 scan/s scan rate: EPA Method, which had an average IDL of 0.061 µg/L. To
(a) Segment 1: 45-460 amu (b) Segment 2: 120-670 amu. further test the precision of the method, replicate injections
Method linearity, accuracy, and sensitivity were evaluated were made at 1.0 µg/L. An average relative standard deviation
across the working range of the calibration curve (0.25 to (% RSD) of 3.8% and average percent difference of -6.5%
10 µg/L). Instrument detection limits (IDLs) were determined from the theoretical value demonstrate good accuracy.
by running replicate injections at 0.25 µg/L. Accuracy of Conclusions
quantitation was tested by determining the percent recovery
The analysis of flame retardants and pesticides in drinking
of each calibration level.
water using the Thermo Scientific DSQ II mass spectrometer
Results and FOCUS GC resulted in an average IDL of 0.034 µg/L.
The final analytical method resulted in GC run times of less The FOCUS GC and DSQ II system met the quality control
than 30 minutes, with excellent separation (Figure 1a). Peaks criteria of the EPA method over a calibration range from
were Gaussian, even for the high-mass flame retardants 0.25 to 10 µg/L. Tune criteria for DFTPP were easily met
(Figure 1b). The method met the tuning criteria for DFTPP with a low-mass tune and the sensitivity for brominated
and calibration curve criteria, and low IDLs were achieved flame retardants were enhanced with a high-mass tune.
for all target compounds. Using two tune files allowed for References
meeting the broad range of requirements in a single injection. 1. EPA Method 527 Determination of Selected Pesticides and Flame Retardants
All compounds at calibration levels 2, 3, 4, 5, and 6 met the in Drinking Water by Solid Phase Extraction and Capillary Column Gas
Chromatography/Mass Spectrometry (GC/MS), Revision 1.0 April 2005,
70 – 130% recovery criteria for the method, while the Level 1 USEPA Technical Support Center Office of Ground Water and Drinking
calibration standard met the criteria of 50 – 150% recovery, Water U. S. Environmental Protection Agency.
with an average recovery of 102%. The average IDL was 2. Development of U.S. EPA Method 527 for the Analysis of Selected Pesticides
and Flame Retardants in UCMR Survey, Environmental Science &
0.036 µg/L. These detection limits compare favorably to the Technology, Vol 39, NO. 13, pg 4996.

6 To download the full text of this application note, visit www.thermo.com/msnotebook

Speciation of Bromine in Water Samples Using HPLC
Coupled to the XSERIES 2 ICP-MS
Aleksandra Polatajko, Shona McSheehy, Julian Wills, Meike Hamester, Thermo Fisher Scientific, Bremen, Germany

Goal HPLC Parameters (Accela)

Develop a comprehensive HPLC-ICP-MS method for the Column: Thermo Scientific BioBasic AX (100 x 4.6 mm, 5 µm)
with BioBasic AX guard column (10 x 4.6 mm, 5 µm)
determination of bromide and bromate in under ten minutes Injection Volume: 100 µL
with excellent limits of detection.

Environmental/
Flow Rate: 1 mL min -1

Food Safety
Isocratic Elution: 6 mM HN03 and 10 mM NH4OH (pH 7.5)
Introduction
ICP-MS Parameters (XSERIES 2)
Bromine is naturally present in the earth’s crust and seawater
Forward Power: 1400 W
in a range of chemical forms. In natural waters (including
Nebulizer Gas Flow: 0.9 L min -1
drinking water), it is present as the bromide ion (Br-). During Data Acquisition Mode: PlasmaLab transient time resolved analysis (TRA)
the ozonation process, the most commonly used method for Isotopes and Dwell Time: 79Br (200 ms), 81Br (200 ms)
the purification of drinking water, the bromide may be con- Transient Acquisition Time: 600 s
verted into bromate (BrO3-) which is recognized as a highly Nebulizer: Glass concentric
carcinogenic compound.1 Thus, monitoring total bromine Spray Chamber: Glass impact bead
concentration in drinking water does not fully indicate the real Cones: Xt
risk to human health; only through speciation analysis and Table 1: HPLC-ICP-MS parameters
determination of the bromate ion can this evaluation be made.
Various national and international agencies have introduced a
limits on bromate. United States’ Environmental Protection
Agency (EPA) Disinfectants and Disinfection Byproducts Rule
(Stage 2 DBP rule), the European Drinking Water Directive
(98/83EC)2, as well as the German Drinking Water Regulation
(TVO) stipulate a maximum contaminant level (MCL) of 10 µg
L-1 bromate in drinking waters. The EPA also requires a mini-
mum reporting level (MRL) of 1 µg bromate L-1. EPA Method
321.8 describes the determination of bromate in drinking
water by ion chromatography in combination with inductively
coupled plasma mass spectrometry (ICP-MS). Based on this b
method, an HPLC-ICP-MS instrument package from Thermo
Fisher Scientific was used for the highly sensitive and selective
analysis of bromide and bromate in drinking and tap water.

Experimental Conditions
The Thermo Scientific Accela High Speed LC system coupled
to the Thermo Scientific XSERIES 2 quadrupole ICP-MS
via an HPLC-ICP-MS coupling kit was used for separation
and detection of bromine species. Thermo Scientific PlasmaLab Figure 1: Chromatograms of (a) commercially available BrO3- and Br - standards
and Xcalibur software packages were used to provide a at a concentration of 10 µg L-1 (b) tap water
completely automated HPLC-ICP-MS device. The HPLC- BrO3- Br-
ICP-MS conditions used are shown in Table 1.
LOD (µg L-1) 0.23 0.19
LOQ (µg L-1) 0.75 0.63
Results
Table 2: Figures of merit for the HPLC-ICP-MS analytical method
Anion exchange chromatography coupled to ICP-MS allowed
for the successful separation of bromine standards in less than
Conclusions
ten minutes (Figure 1a). Only one bromine species, bromide
(Br-) was found in tap water (Figure 1b) and a commercially The HPLC-ICP-MS method described provides an analytical
available mineral water (chromatogram not shown). No solution for bromine speciation in water samples. Isocratic
bromate (BrO3-) was detected at concentrations above the elution allows for a total run time of ten minutes. The low
detection and quantification limits obtained with this method
detection limit in either water sample investigated.
facilitate the determination of bromate at concentrations
Quantification of Br- and BrO3- species in the tap and
conforming to EU and US legislation.
mineral waters was achieved using external calibration curves.
The concentrations of Br- in the tap and mineral water samples References
analyzed were 43.3 µg L-1 and 82.3 µg L-1 respectively. 1. Chipman J. K., Davies, J. E., Parsons, J. L., Nair, J., O’Neill, G. and Fawell,
Detection (3σ) and quantification (10σ) limits were J. K (1998) Toxicology, 126, 93-102
2. Council Directive 1999/83/EC The Drinking Water Directive on the quality
determined from the measurement of (n=5) blanks (Table 2). of water intended for human consumption

To download the full text of this application note, visit www.thermo.com/msnotebook 7

 Rapid Screening of Pesticides Using U-HPLC/MS
Markus Kellmann, Andreas Wieghaus, Helmut Muenster, Thermo Fisher Scientific, Bremen, Germany
Lester Taylor, Dipankar Ghosh, Thermo Fisher Scientific, San Jose, CA, USA

Introduction Results
The screening of pesticides, mycotoxins and veterinary drugs Comparison of mass measurements at medium and high
is of great importance in regulated environments such as resolution settings clearly indicates the need for high
food or feed analysis. Due to some of the limitations of resolution for screening of compounds in complex matrix
traditional triple quadrupole approaches (e.g., targeted samples. Since in many cases interferences cannot be resolved
Environmental/

analyte detection, limited number of compounds, and from the analytes, medium resolution results in poor mass
Food Safety

unidentified unknowns compounds), there is currently a accuracies (>10 ppm). In contrast, high resolution yields
trend towards use of full-scan MS data for the analysis of very high mass accuracies (<2 ppm) for the vast majority
residue samples. Current screening approaches mainly rely of measured compounds. Consequently, selectivity and
on the use of ToF instruments coupled to U-HPLC delivering also sensitivity is increased. In addition, the linearity of
mass accuracy (~5 ppm) at a maximum resolution of quantitation benefits from the use of high resolution, since
<15,000. This can produce inaccurate mass measurements analytes are not integrated together with interfering matrix
due the presence of unresolved background matrix background. A minimum resolution of 50,000 was required
interferences. In this work we show a full-scan MS to ensure matrix interference-free mass accuracy. Limits
screening approach the Thermo Scientific Exactive mass of detection of 2 to 10 ppb were observed in matrix
spectrometer, a novel single-stage Orbitrap™ MS capable of containing samples.
providing precise mass accuracies at resolutions of up to
100,000 without the need for internal mass calibration. Conclusion
This work shows that a combination of U-HPLC and high
Experimental Conditions resolution mass spectrometry is an excellent approach for
An Exactive™ mass spectrometer coupled to a U-HPLC residue screening in the field of food safety because it
chromatography system was used to evaluate a highly overcomes the limitations associated with the use of
complex mixture of ~150 pesticides, mycotoxins and plant medium resolving instruments.
toxins in different concentrations. A 12 min water/acetonitrile
gradient was used with a 50 x 2 mm RP C18 column (1.9
µm particles). Sensitivity, selectivity and linearity of
quantification were evaluated in standard solutions and
extracts from animal feed. Orbitrap detection was carried
out using automatic control of the number of ions entering
the detector. Mass measurements were performed at
different resolution settings to enable comparisons to data
generated using ToF instruments and to demonstrate the
advantages of ultra high resolution.

Display of the mass spectrum generated

at retention time 4.89 minutes showing
the pesticide Sulcotrion (m/z 329.024675).
This is masked by background ions at a
resolution of 15,000 (top). However it
is easily detected at 50,000 resolution
(bottom). The inset shows the mass
chromatogram for Sulcotrion at two
different resolutions resulting in a
false negative detection at 15,000
resolution and correct positive
detection at 50,000 resolution.

8 To download the full text of this application note, visit www.thermo.com/msnotebook

Analysis of Melamine and Cyanuric Acid in Food Matrices
by LC–MS/MS
Peter Varelis, National Center for Food Safety and Technology, Chicago, IL, USA
Jonathan Beck, Kefei Wang, Dipankar Ghosh, Thermo Fisher Scientific, San Jose, CA, USA

Introduction
In March 2007, several North American manufacturers of
pet food voluntarily issued nationwide recall notices for

Environmental/
some of their products that were reportedly associated with

Food Safety
renal failure in pets. The raw material wheat gluten, used to
manufacture the pet food, was imported from China and
was identified as the source of contamination.
Although initial reports suggested that contamination
was confined to pet food, further investigations revealed
that melamine-tainted fodder may have been used to feed
animals intended for human consumption.1, 2 In particular, it
was discovered that melamine-contaminated ingredients had
been used to prepare feed for chickens, swine and catfish.1, 2
Consequently, the US Food and Drug Administration
(FDA)1 and the US Department of Agriculture (USDA)2
have developed methods for the analysis of melamine
residues in animal tissue. This note describes a method for
the detection and analysis of melamine and cyanuric acid in Figure 1: Chromatogram of cyanuric acid and melamine spiked into catfish
food matrices by LC–MS/MS using the Thermo Scientific matrix, at a level of 50 ppb for cyanuric acid and 10 ppb for melamine.
TSQ Quantum Ultra mass spectrometry system.

Experimental Conditions Results

Sample Preparation Calibration curves ranged from 1–1000 ppb for melamine
and cyanuric acid, respectively. Melamine and cyanuric acid
Solid samples were homogenized using an Ultra-Turrax®
were spiked into a matrix of catfish and processed as
(IKA-Werke GmbH & Co. KG, Staufen, Germany)
described in this note. A chromatogram of this sample,
homogenizer. After extraction into aqueous 1:1 Water:MeOH
spiked at 10 ppb for melamine and 50 ppb for cyanuric
and addition of the internal standards, the samples were
acid, is shown in Figure 1. Very low noise is observed,
prepared by off-line ion exchange chromatography using
emphasizing the effectiveness of the clean-up procedure
SPE cartridges.
for a complicated matrix.
Liquid Chromatography
Conclusion
Aliquots (10–25 µL) of the above extract were separated
using a Thermo Scientific BioBasic AX analytical column A simple, sensitive, and specific method for the detection
(2.1 x 150 mm, 5 µm), which was kept at 30 °C in an oven. and quantification of melamine and cyanuric acid in food
matrices has been demonstrated. The method is robust and
MS Conditions allows for the analysis of a large number of samples,
MS: TSQ Quantum Ultra™ without degradation in column performance.
Source: Heated Electrospray (H-ESI)
Ionization: Positive ESI References
1. Weise, E. and Schmit, J. “Melamine in pet food may not be accidental.”
USA Today. Available at www.usatoday.com/money/industries/2007-04-24-
SRM Transitions fda-pet-food-probe_N.htm.
Melamine: Melamine 13C3 (Internal Standard): 2. “Fish on US fish farms fed melamine-contaminated feed; FDA discovers
m/z 127 → 68 @ 32 eV m/z 130 → 70 @ 32 eV contaminated food products from China mislabeled.” American Veterinary
m/z 127 → 85 @ 18 eV m/z 130 → 87 @ 18 eV Medical Association. Available at www.avma.org/press/releases/070508_
petfoodrecall.asp. Accessed 10 December 2007.
Cyanuric acid: Cyanuric acid 13C3 :
m/z 128 → 42 @ 17 eV m/z 131 → 43 @ 17 eV
Ultra-Turrax is a registered trademark of IKA-Werke GmbH & Co. KG.
m/z 128 → 85 @ 11 eV m/z 131 → 87 @ 11 eV

To download the full text of this application note, visit www.thermo.com/msnotebook 9

 LC-MS/MS Analysis of Malachite Green,
Leucomalachite Green, Ciprofloxacin, and Tetracycline
in Food Samples Using a TurboFlow Method
Charles Yang, Dipankar Ghosh, Thermo Fisher Scientific, San Jose, CA, USA

Introduction Experimental Conditions

The analysis of chemical residues in food requires
Environmental/

TurboFlow Conditions
techniques sensitive enough to detect and quantify
Food Safety

Loading Column: TurboFlow XL C18 column

contaminants at or below the maximum residue limit
(Thermo Fisher Scientific, Franklin, MA)
(MRL) of the compound in a given sample matrix. Because
Analytical Column: 50 × 3 mm, 5 µm Hypersil GOLD™ column
of increased food safety regulations and the growing (Thermo Fisher Scientific, Bellefonte, PA)
numbers of samples to be analyzed, it is critical that the
Mobile Phase A: 0.1% formic acid in water
analytical techniques provide high sample throughput.
Mobile Phase B: 0.1% formic acid in acetonitrile
In this study, ciprofloxacin, tetracycline, and malachite
Mobile Phase D: 20% acetone/40% methanol/40% acetonitrile
green/leucomalachite green (Figures 1-3) were examined in
Autosampler Injection Size: 10 µL
shrimp, fish, and pork liver. We show a solution that combines
Sample Extraction Solution: 50:50 (A/B)
the Thermo Scientific Aria TLX system utilizing TurboFlow™
technology with a Thermo Scientific TSQ Quantum Access
mass spectrometer. Compared to traditional offline extraction MS Conditions
methods, this solution provides fast and reliable sample MS analysis was carried out on a TSQ Quantum Access™
analysis of chemical residues in food by online sample triple stage quadrupole mass spectrometer. The MS
extraction followed by LC-MS/MS. The Aria™ TLX system conditions were as follows:
uses TurboFlow technology to retain small molecules and filter
out proteins and larger materials by diffusion, size exclusion, Ion Source Polarity: Positive ion mode
and column chemistry. This enables users to directly inject Spray Voltage: 3500 V
samples into the LC-MS system for analysis, greatly Vaporizer Temperature: 472 °C
simplifying sample preparation and increasing throughput. Sheath Gas Pressure (N2): 40 arbitrary unit
Auxiliary Gas Pressure (N2): 50 arbitrary unit
Ion Transfer Tube Temperature: 270 °C
Skimmer Offset: 5V
Collision Gas (Ar): 1.5 mTorr
Q1/Q3 Peak Resolution: 0.7 u (unit mass resolution)
Scan Mode: Selected Reaction Monitoring

Figure 1: Structures of malachite green and leucomalachite green

Figure 2: Structure of Tetracycline

Figure 3: Structure of Ciprofloxacin Figure 4: Chromatogram comparison of tetracycline at 500 ng/kg in pig liver matrix in standard HPLC and
TurboFlow method

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 Fish
Standard HPLC TurboFlow Method
LOD %RSD LOQ %RSD R2 LOD %RSD LOQ %RSD R2
(µg/kg) n=3 (µg/kg) n=3 (µg/kg) n=3 (µg/kg) n=3
Ciprofloxacin 0.5 32.0 1.0 8.0 0.9875 0.1 15.9 0.5 7.1 0.9968
MG 0.1 23.0 0.5 1.4 0.9988 0.1 6.8 0.1 8.2 0.9984
LMG 0.5 7.4 0.5 7.4 0.9990 0.1 12.0 0.1 12.0 0.9983
Shrimp
Ciprofloxacin 5.0 10.5 5.0 10.5 0.9580 0.5 16.0 1.0 2.0 0.9906
MG 0.1 21.4 0.5 7.2 0.9991 0.05 7.9 0.05 7.9 0.9990

Environmental/
LMG 0.1 20.5 0.5 11.0 0.9975 0.05 13.5 0.1 11.2 0.9988

Food Safety
Pig Liver
Ciprofloxacin 0.5 38.8 1.0 10.4 0.9707 0.1 29.0 0.5 8.6 0.9969
Tetracycline 0.5 14.8 1.0 10.5 0.9932 0.1 11.5 0.5 11.3 0.9953
Table 1: Data summary showing the improvement in the TurboFlow method results over those of the standard HPLC results. Note that results are only shown for a
compound in the matrix in which it would be found; for example, MG and LMG would be found in fish but not in pig liver.

Results
The results of the assay and a comparison between standard
HPLC method without any sample cleanup versus TurboFlow
LC-MS method with online sample cleanup are shown in
Table 1. An example chromatogram comparing tetracycline
at 500 ng/kg in pig liver analyzed with standard HPLC
versus TurboFlow method is shown in Figure 4. The
calibration curves for ciprofloxacin in pig liver on standard
HPLC versus TurboFlow method are shown in Figure 5.
Because the TurboFlow method removed matrix
interferences, lower limits of detection (LOD) and lower
limits of quantitation (LOQ) were obtained than in the
standard HPLC method (see Table 1). The corresponding
percent relative standard deviation values (%RSD) were
also improved, providing more confidence in the results.
The limits of detection and limits of quantitation were well
below the maximum residue limits for all of the substances.

Conclusion
A rapid, sensitive and reliable method for the quantitation
of veterinary drugs in food matrices was developed using
the Aria TLX-1 system with the TSQ Quantum Access mass
spectrometer. Minimal sample preparation was required
because the TurboFlow method allows direct injection of
samples into the system. The overall processing time for
analysis was significantly shortened compared to methods
using offline sample preparation. The Aria TLX system
reduced ion suppression and matrix effects compared to
standard HPLC runs.
Figure 5: Ciprofloxacin calibration 1/x on standard HPLC vs. TurboFlow method
in pig liver matrix

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 Identification of Lysergic Acid Diethylamide (LSD)
in Candy by U-HPLC/MS
Jason R. Stenzel, Washington State Patrol – Crime Laboratory Division, Cheney, WA, USA
Guifeng Jiang, Thermo Fisher Scientific, San Jose, CA, USA

Goal The candy hearts and sugar cubes were treated with
Positively identify trace amounts of lysergic acid 3-5 drops of this LSD solution and allowed to stand for
diethylamide (LSD) in sugar candy quickly, with minimal 24 hours prior to use. Ten (10) mg scrapings from the sugar
Environmental/

sample preparation and no chemical derivatization. cube or candy heart were added to 2 mL methanol. This
Food Safety

mixture was vortexed for 30 sec, and the supernatant was

Introduction filtered through a cotton-plugged Pasteur pipette.
Lysergic acid diethylamide (LSD) is a controlled substance Chromatographic and Mass Spectrometer Conditions
in forensic chemistry that is notorious for being difficult to
Chromatographic analyses were performed using the
identify. Most forensic laboratories confirm the presence of
LSD by using gas chromatography with mass spectrometry Thermo Scientific Accela U-HPLC system. MS analysis was
(GC/MS). The disadvantages of GC/MS are its requirements carried out on a Thermo Scientific MSQ Plus™ single
for extensive sample preparation, including chemical quadrupole LC-MS detector. The chromatographic and MS
derivatization of LSD to a more volatile form, and its impaired conditions were as follows:
performance with analytes that are polar, thermally labile,
or nonvolatile. Column: Thermo Scientific Hypersil GOLD PFP (perfluorinated
An alternative method to positively identify LSD in phenyl) 1.9 µm, 100 x 2.1 mm
complex food matrices is to use ultra-high performance liquid Flow Rate: 1 mL/min
chromatography with mass spectrometric detection (U-HPLC/MS). Mobile Phase: A: Water with 0.06 % acetic acid
U-HPLC/MS offers a threefold benefit compared to GC/MS; B: Acetonitrile (ACN) with 0.06% acetic acid
C: Methanol with 0.06% acetic acid
simpler sample preparation, no derivatization, and less time
Gradient: T (min) A% B% C%
wasted baking out or cleaning the instrument.
0.00 95.0 0.0 5.0
1.00 95.0 0.0 5.0
Experimental Conditions
1.50 90.0 5.0 5.0
Standard and Sample Preparation 2.70 70.0 10.0 20.0
3.00 5.0 15.0 80.0
A 1000 mg/L solution of LSD in methanol was purchased 7.00 5.0 0.0 95.0
from Alltech (State College, PA, USA) and diluted to about 7.10 95.0 0.0 5.0
5 mg/L with methanol. 8.00 95.0 0.0 5.0
Ionization: Positive Electrospray
Probe Temperature: 500 °C
Cone Voltage: 90 V
ESI Voltage: 3.5 kV
Scan Mode: Full scan with mass range of m/z = 125-425 amu

Results
LSD elutes at 4.49 min and is detected by using full scans
(m/z 125 – 425) of the single quadrupole mass spectrometer.
The extracted ion chromatograms from m/z 324 ± 0.5 are
displayed in Figure 1A.
The methanol extracts from the candy hearts and sugar
cubes doped with LSD were analyzed using the same
U-HPLC/MS method as for the standard LSD (Figure 1B,
1C). Positive confirmation of LSD in the samples is assured
both by retention time matching and MS spectra matching
of the samples with the LSD standard.

Conclusion
U-HPLC/MS can positively identify trace amounts of lysergic
acid diethylamide (LSD) in sugar candy in 8 min, after a
Figure 1: Extracted ion chromatograms at m/z = 324 ± 0.5 amu obtained by simple 10 min sample prep involving no chemical derivatization.
U-HPLC/MS on a Hypersil GOLD™ PFP column: (A) LSD standard; (B) methanol
extract of LSD-doped candy heart; (C) methanol extract of LSD-doped sugar
cube. See text for details.

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Enhanced Reproducibility Performance of the TSQ Vantage
LC-MS/MS: Bioanalysis of Paroxetine in Rat Plasma
Jonathan McNally, Rohan A. Thakur, Thermo Fisher Scientific, San Jose, CA, USA

Goal Sample Analysis

The aim of this study was to demonstrate the robustness and HPLC Pump: Thermo Scientific Accela pump
reproducibility of the Thermo Scientific TSQ Vantage for Auto-sampler: CTC PAL (CTC Analytics, Basel, Switzerland)
highly sensitive bioanalysis of paroxetine spiked in rat plasma. Column: Thermo Scientific Hypersil GOLD C18 50 mm × 2.1 mm
(3 µm) column
HPLC Method: 10% Solvent B (acetonitrile containing 0.1% formic acid) to
Introduction
95% B over five minutes. Solvent A was water containing
In May 2001, the FDA promulgated its first official guidance 0.1% formic acid. The flow rate was 1 mL/min. Injection

Drug Discovery
for bioanalytical methods.1 This document is titled Guidance volumes of 10 µL were used.
for Industry, Bioanalytical Method Validation, and serves as
MS Conditions
a guide for bioanalytical methods supporting clinical and
Mass Spectrometer: TSQ Vantage™
non-clinical studies. Recently, the reproducibility and
Ionization Mode: HESI-II in positive ion mode
accuracy of the concentration determined in incurred
Vaporizer Temperature: 400 °C
samples was subject of significant debate, outlined in the
Ion Sweep Gas: 5 arbitrary units
American Association of Pharmaceutical Scientists (AAPS)
Ion Transfer Tube Temp: 300 °C
journal paper titled Workshop/Conference Report –
Sheath Gas: 60 arbitrary units
Quantitative Bioanalytical Methods Validation and
Auxiliary Gas: 30 arbitrary units
Implementation: Best Practices for Chromatographic
Resolution: 0.7 Da (FWHM) on Q1 and Q3
and Ligand Binding Assays.2 Recommendations were put
Scan Time: 0.2 s
forth to randomly re-assay a subset of previously assayed
incurred samples and compare them to the original value to Selected Reaction Monitoring
ensure reproducibility and accuracy. Significant deviation Paroxetine m/z 330.20 → 192.1 Da
(>15-20%) from the original measurement would then Alprazolam m/z 309.1 → 281.0 Da
require re-evaluation of the bioanalytical method. Collision Energy: 22 V
Collision Gas Pressure: 1.5 mTorr
Experimental Conditions
Sample Preparation Results
Aliquots (5 mL) of rat plasma were spiked with paroxetine Reproducibility is an important parameter for a bioanalytical
in the concentration range of 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, method. The TSQ Vantage was designed to perform
100.0, 250.0, and 500.0 pg/mL for the calibration standards reproducibly at high levels of sensitivity after repeated
and at levels of 2.5, 25.0 and 250.0 pg/mL for quality control exposure to samples containing biological matrix, as shown by
samples. Alprazolam (analogue internal standard) was spiked Figure 1. This figure shows 5 standard curves (1-500 pg/mL)
at a concentration of 50.0 pg/mL. Blank rat plasma and plasma run once a day during the analysis of 1000 rat plasma samples
containing internal standard were also prepared. Eight replicate over a 5-day period. The 5 curves are overlaid to demonstrate
plasma aliquots of 100 µL were taken from each spiked level the robustness of the ion source.
and protein precipitated using 500 µL of acetonitrile (5:1,
v/v protein precipitate), centrifuged at 13,000 rpm for 10 Conclusion
minutes and the supernatants decanted. The extracts were Reproducibility is important because it predicates reliability
evaporated under a stream of nitrogen gas and reconstituted or the ability of the method to yield similar concentration
in 100 µL of water/methanol/acetic acid (80/20/0.1 v/v/v). for a sample when measured on different occasions.3 This is
of particular importance to the summary guidance issued at
the recent AAPS/FDA Crystal City meeting which requires
demonstrating reproducibility of incurred samples. The data
shown clearly demonstrates the superior reproducibility
performance of the TSQ Vantage for high sensitivity LC-MS/MS.

References
1. Guidance for Industry, Bioanalytical Method Validation. Drug Information
Branch (HFD-210), Center for Drug Evaluation and Research (CDER),
5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573,
http://www.fda.gov/cder/guidance/index.htm
2. Workshop/Conference Report – Quantitative Bioanalytical Methods Validation
and Implementation: Best Practices for Chromatographic and Ligand Binding
Assays. The AAPS Journal 2007; 9 (1) Article 4 (http://www.aapsj.org).
Figure 1: An overlay of 5 standard curves in spiked rat plasma, sampled during 3. Key Elements of Bioanalytical Method Validation for Small Molecules.
the analysis of 1000 rat plasma samples over a 5-day period. The AAPS Journal 2007; 9 (1) Article 11, http://www.aapsj.org.

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 Comparing Manual Compound Optimizations Against
Automated Optimizations Obtained by QuickQuan on
the TSQ Quantum Access LC-MS/MS
Nicholas Duczak, Jim Koers, Thermo Fisher Scientific, San Jose, CA, USA

Goal
To determine the sensitivity difference in methods created
by Thermo Scientific QuickQuan high-throughput screening
(HTS) software versus methods created from manual
optimization procedures.
Drug Discovery

Introduction
One of the rate limiting steps in LC-MS/MS sample analysis
is the time required to create accurate and sensitive instrument
methods. To ensure the best possible performance, operators
may spend an extended period of time optimizing the mass
spectrometer for compounds of interest. In laboratories where
higher throughput is required, the allotted time for instrument
optimization is minimal. Automated optimization packages
are frequently utilized to increase throughput, but these
solutions compromise assay sensitivity for the sake of speed.

Experimental Conditions
Thermo Scientific TSQ Quantum Access Optimization
Figure 1: Diagram of the QuickQuan Total Solution package. The QuickQuan
For the experiment, four compounds (Minoxidil, Imipramine, method utilized the autosampler and a dedicated injection valve to infuse
Paroxetine and Nefazodone) were optimized using the compounds for MS optimization. During the manual and automated
automated infusion optimization procedure available in optimizations, mobile phase composition and flow were kept identical;
QuickQuan™ (Figure 1). The QuickQuan method utilized however, the manual optimization required the infusion of compound via the
syringe pump on the front of the TSQ Quantum Access MS.
the autosampler and a dedicated injection valve to infuse
compounds for MS optimization. During the manual and
automated optimizations, mobile phase composition and
flow were kept identical; however, the manual optimization
required the infusion of compound via the syringe pump on
the front of the TSQ Quantum Access™ mass spectrometer.
Optimal gas flow rates and temperatures were determined
through the TSQ Quantum EZ-Tune interface (Figure 2). A
5 ng/mL neat standard was prepared in 50/50 methanol to
water and samples were analyzed through QuickQuan software.
After results were obtained, the instrument parameters
(gas flows, temperatures, and product ion energies) were
optimized manually for the four compounds. The same sample
was then injected using the manually optimized parameters
under identical LC conditions and ion source positioning to
compare intensity results between the two techniques.

Sample Analysis
Mobile Phase A was water with 0.1% formic acid and
mobile phase B was methanol with 0.1% formic acid.
Solvent was pumped at 400 µL/min and chromatographic
separations were achieved using a Thermo Scientific Figure 2: For QuickQuan auto-optimization, the appropriate gas and temperature
Hypersil GOLD (50 x 2; 5 µM) column. Compounds were settings were determined using the TSQ Quantum EZ-Tune interface. EZ-Tune
eluted using a linear gradient starting at 10% B to 95% B automatically calculated the source parameters when given the LC flow rate
in 1 minute, allowing a hold at 95% B for 1 minute. MS of the experiment.
analysis was performed on a TSQ Quantum Access
instrument equipped with heated electrospray (H-ESI).

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MS Conditions
For both QuickQuan and manual optimizations:

Instrument: TSQ Quantum Access

Ionization Mode: Positive
Scan Type: Selective Reaction Monitoring (SRM)
Scan Time: 0.020 seconds

Results
Figure 3 shows the extracted ion chromatograms for the
manual (left) and QuickQuan (right) methods. In both cases,
the most intense product ion determined for each compound
was identical (Table 1). The percentage of change in intensity

Drug Discovery
is displayed in Table 2. Manual optimization of the four
compounds required approximately 30 minutes; QuickQuan
software performed the procedure in approximately 5 minutes.

QuickQuan Results Manual Optimization Results

Most Intense Collision Most Intense Collision
SRM Transition Energy SRM Transition Energy
210 → 193 14 210 → 193 15
281 → 86 17 281 → 86 15
330 → 192 20 330 → 192 20
470 → 274 28 470 → 274 25

Table 1: Table comparing optimization results. In both cases, the most intense
product ion for each compound was identical.

SRM QuickQuan Manual Percent

Transition Optimizations Optimizations Difference
210 → 193 5.63E+05 6.85E+05 21.67%
281 → 86 1.07E+06 1.94E+06 81.31%
330 → 192 8.58E+04 1.10E+05 28.21%
470 → 274 3.88E+05 3.53E+05 -9.02%

Table 2: Table comparing intensities obtained from the QuickQuan-generated

method against a manually created method. All LC and autosampler conditions
were kept identical.

Conclusion
QuickQuan software provided an equivalent optimization
result to the manual procedure in one-sixth the time. Manual
optimization only showed a slight improvement in on-column
response for one of the four compounds tested. In order to
achieve this increase in signal, an extra 25 minutes of manual
tuning was required. Also in comparison, the user did not Figure 3: Comparative chromatograms from on column injection using manually
determined method (left) and QuickQuan generated method (right)
need to tend to the TSQ Quantum Access MS during the
QuickQuan optimization procedure. In high-throughput
environments where a large number of compounds are
analyzed daily, the time required for manual optimization is
extremely inefficient. QuickQuan software proves to be an
excellent solution for compound optimization when sample
throughput is a high priority.

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 Quantitation of Fentanyl and Norfentanyl from Urine
Using On-line High-Throughput System
Francois A. Espourteille, Ph. D., Thermo Fisher Scientific, San Jose, CA, USA

Introduction and increases the efficiency of the TSQ Quantum Access™

The use of Thermo Scientific Aria TLX-4 system with Thermo mass spectrometer. The TLX-4 and TSQ Quantum Access
Scientific TurboFlow methods for automated on-line sample triple quadrupole system can run over 1000 sample analyses
cleanup of a biological sample is well documented in the per day with a 92.9% sample completion rate with 7.1%
literature.1 The Aria™ TLX-4 system enhances the sensitivity, re-injection.2 The method run time was 5.5 minutes with a
specificity, and precision for mass spectrometric detection of sample injection every 82 seconds. This system provides a
fentanyl and norfentanyl. Increasing demand in clinical reliable, high-thoughput method for fentanyl and norfentanyl.
research laboratories for higher sample throughput has put
the emphasis on automated methods and platforms that have
the ability to quickly ramp up throughput to meet demand.
The Aria TLX-4 system extracts both fentanyl and its
Clinical Research /

metabolite, norfentanyl, from the many interferences found in

urine. In addition, the system chromatographically separates
Toxicology

them from each other, before sending them to the mass

spectrometer. TurboFlow™ extraction methods exclude both
high molecular weight species and salts while the stationary
phase coating retains the analyte(s) through reverse phase
column chemistry. This results in fast, efficient, on-line
separation of fentanyl and its metabolite prior to introduction
into the mass spectrometer.

Experimental Conditions Y = 0.018469X - 0.00275

R 2 = 0.99635 (0.01% CV)
Method Information
Figure 1: Calibration curves of fentanyl from 4 channels of the Aria TLX-4 System.
This method describes the analysis for the determination of
Data courtesy of Dennis Crouch, Ameritox, LTD.
fentanyl and its metabolite, norfentanyl, from a human
urine sample. An LOQ of 0.5 ng/mL was observed in human
urine, with an LOD below 0.1 ng/mL. A
Instrumentation: Aria TLX-4 with Thermo Scientific TSQ Quantum Access
triple quadrupole mass spectrometer
Extraction Column: TurboFlow XL C18 P 0.5 x 50 mm
Analytical Column: Thermo Scientific Hypersil GOLD aQ 3 x 50, 5 µm

Sample Preparation B
A working solution containing fentanyl and norfentanyl at
1000 ng/mL was made. Subsequent dilutions yielded a curve
from 200 ng/mL to 0.5 ng/mL. An internal standard solution
containing both fentanyl-D5 and norfentanyl-D5 was added
to all standards. Samples were vortexed, centrifuged at
10,000 RCF for 5 minutes and analyzed immediately.
Figure 2: Excellent signal/noise at LOQ for (A) norfentanyl and (B) fentanyl at
Results 0.5 ng/mL calibration.
The data in Figure 1 shows linear regression from 0.5 ng/mL Data courtesy of Dennis Crouch Ameritox, LTD.
to 200 ng/mL, with 1/x weighing. Figure 2 demonstrates the
limit of quantitation with excellent signal to noise ratio. Mass Spectrometers are general purpose laboratory instruments. They have not
been cleared or approved by the United States Food and Drug Administration,
the European IVD Directive or any other agency for diagnostic, clinical or
Conclusion other medical use.

The Aria TLX-4 system powered by TurboFlow technology References

provides a fast, efficient, and automated on-line separation
1. Sauvage et al. 2006. Therapeutic Drug Monitoring 28(1), pp. 123-130.
technology for the extraction and analysis of fentanyl and
2. Crouch, Dennis. “The Analysis of Fentanyl and Norfentanyl using
its metabolite, norfentanyl. The ability to run 5.5 minute TurboFlow Column Analyte Isolation and Multiplex-HPLC/MS/MS”.
methods on four channels further decreases analysis time Oral presentation, AAFS, Washington DC February 17-20, 2008.

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High-Throughput LC-MS/MS Method for Quantitative
Analysis of Four Immunosuppressant Drugs in Whole Blood
Marta Kozak, Thermo Fisher Scientific, San Jose, CA, USA
Raimund Ruzicka, Thermo Fisher Scientific, Fremont, CA, USA

Introduction Conclusion
Immunosuppressants are drugs used to treat transplant A fast 2-minute sensitive and reliable method for quantification
organ recipients in order to prevent organ rejection. of tacrolimus, sirolimus, everolimus and cyclosporin A was
Current immunosuppressants on the market are sirolimus, developed. (See chromatograms on Figure 1.) The following
cyclosporin A, tacrolimus, everolimus and mycophenolic dynamic ranges were established:
acid. LC-MS is a widely used technique for fast, accurate
and precise determination of immunosuppressant drug Analyte Dynamic Range (ng/mL)
concentrations in blood samples for use by researchers Tacrolimus 1 - 50
and pharmacologists. Sirolimus 1 - 50
Everolimus 1 - 50

Clinical Research /
Experimental Conditions
Cyclosporin A 10 - 2000

Toxicology
Sample Preparation
To 100 µL of blood, add protein precipitation solution The inter-assay and intra-assay variabilities were
prepared by mixing 0.1 M ZnSO4 and MeOH containing respectively 6.9 and 6.8 for tacrolimus; <7.0 and <6.7 for
two internal standards (concentrations of 20 ng/mL sirolimus; <9.7and <8.6 for everolimus; and <4.2 and <5.5
ascomycin and 250 ng/mL cyclosporin D) in 1:2 ratio. for cyclosporin A. Data obtained from proficiency sample
Vortex, centrifuge and inject 50 µL into the LC-MS system. analysis showed good correlation with peer group mean
values which demonstrated good method accuracy.
LC Method Interferences tested negatively.
A two-minute gradient method was implemented on a We proved a lower limit of quantification with
Thermo Scientific Accela liquid chromatography system. concentrations of 0.25 ng/mL for tacrolimus, sirolimus and
everolimus and 1 ng/mL for cyclosporin A with a % CV
Solvent A: 10 mM ammonium formate in 0.1% formic acid below 20% for the analysis of five replicates.
Solvent B: MeOH containing 10 mM ammonium formate and This method is also available in a high-throughput version
0.1% formic acid with an acquisition time of 1 minute.
Solvent C: ACN/IPA/acetone = 70/20/10 v/v/v
Analytical Column: Thermo Scientific Javelin GOLD C18 guard column,
10 x 2.1 mm

MS Conditions
Samples were analyzed using a Thermo Scientific TSQ
Quantum Ultra triple quadrupole mass spectrometer
equipped with an APCI Ion Max™ source in SRM data
acquisition mode.

Method Validation
Whole blood calibration standards in a concentration range
of 1-50 ng/mL for tacrolimus, sirolimus and everolimus and
10-2000 ng/mL for cyclosporin A and QC samples with
concentrations across the calibration range were prepared.
The method was validated by processing and analyzing five
replicates of each QC sample in three different batches.
Proficiency samples from Analytical Services International
Proficiency Testing Scheme were analyzed for method
accuracy. The method was tested for interferences as required
by “Class II Special Controls Guidance Document” and
Figure 1: Chromatograms of the lowest calibration standard: 1 ng/mL tacrolimus,
“NCCLS Interference Testing in Clinical Chemistry; Approval sirolimus and everolimus and 10 ng/mL cyclosporin A.
Guideline EP7-A”. Additionally, lower than currently
required limits of quantification were investigated for future Mass Spectrometers are general purpose laboratory instruments. They have not
been cleared or approved by the United States Food and Drug Administration,
research and pharmacology applications.
the European IVD Directive or any other agency for diagnostic, clinical or
other medical use.

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 Determining Distribution of Erlotinib in Pancreatic
Tumors by MALDI Using the LTQ XL Linear Ion Trap
M.C. Prieto Conaway, H. Bui, Thermo Fisher Scientific, San Jose, CA, USA
S. Beachy, R. Pitoniak, B. Hylander, P. Wang, K. Marlar, E. Repasky, L. Kazim, Roswell Park Cancer Institute, Buffalo, NY, USA

Goal Erlotinib-treated pancreas tumor Erlotinib-treated pancreas tumor

Use imaging mass spectrometry to determine the biodistribution 2D map of m/z 336 fragment 2D map of m/z 278 fragment
of erlotinib (Tarceva®) in pancreatic tumors from treated
and untreated mice.

Introduction
Imaging mass spectrometry (IMS) using matrix-assisted
laser desorption ionization (MALDI) is being investigated
using a linear ion trap with the overall goal of analyzing the
distribution of various targeted therapies within patients’
tumors. The drug chosen for evaluation is erlotinib which Untreated pancreas tumor Untreated pancreas tumor
2D map of m/z 336 fragment 2D map of m/z 278 fragment
targets the epidermal growth factor receptor (EGFR), a type I
receptor tyrosine kinase (TK) involved in cellular differentiation
and proliferation, by binding to the ATP pocket and inhibiting
the autophosphorylation of the receptor. Erlotinib has
Metabolomics
Metabolism/

demonstrated clinical activity in non-small cell lung cancer,

head and neck cancer, and ovarian cancer in Phase II studies.
The sensitivity and MS/MS capabilities of the Thermo Scientific
LTQ XL linear ion trap mass spectrometers are exploited
for the unambiguous determination of the distribution of
this drug within human pancreatic tumors.

Experimental Conditions Figure 1: 2-Dimensional maps of erlotinib characteristic fragments (MS/MS 380),
ATT matrix in treated (top) vs. untreated (bottom) pancreas tumors.
Methods
Solution Studies: A series of matrices were tested with Conclusion
erlotinib and erlotinib/Captisol®, looking for best ionization The LTQ XL™ with a MALDI source makes an outstanding
of the analyte of interest. platform for the study of drug distribution and its metabolites
Tissue Studies: SCID mice bearing patients’ pancreas directly off tissue. The sensitivity of the linear ion trap, in
tumors grown as xenografts were dosed once with 2.5 mg particular, allows for an MS/MS spectrum with less than
erlotinib in 6% Captisol, Captisol alone, or left untreated. 10 laser shots at each location. Thermo Scientific ImageQuest
software was used for the visualization of images after
Mass Spectrometry: Thermo Scientific Tune Plus software
MS acquisition.
with tissue imaging was used to acquire data in a raster mode.
MALDI as conducted in this study, without the use of
The laser spacing was set to 100 µm. Data was acquired
isotopic labels or internal standards, is not considered a
with Automatic Gain Control (AGC) on, a standard feature
quantitative technique. However, data processing for both
of all LTQ™ mass spectrometers, to automatically determine
treated and untreated tissues were kept consistent and
the optimal number of laser shots for a particular crystal.
normalized to the same intensity scale. A relative
The tissue area for MS imaging was selected with the
comparison and visualization of the different distribution
Free-Draw feature of the software.
and amounts of measurable drug between the two was
therefore possible.
Results
Selected reaction monitoring (SRM) experiments were used Acknowledgements
to monitor the m/z 278 fragment characteristic of both 1. Zhao, M, He, P. Rudek, M. A., Hidalgo, M., Baker, S. D. 2003. Specific
erlotinib (m/z 394) and its metabolite (m/z 380). Both method for determination of OSI-774 and its metabolite OSI-420 in human
parent drug and metabolite appeared to be distributed plasma by using liquid chromatography-tandem mass spectrometry.
J Chromatography B, 793, 413-420.
evenly over the tumor. Untreated specimens were used as
controls and did not show distribution of either drug or Tarceva is a registered trademark of OSI Pharmaceuticals, Inc.
metabolite when compared with the erlotinib-treated ones. Captisol is a registered trademark of CyDex Pharmaceuticals, Inc.

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Fast, Sensitive, and Accurate Metabolite Identification
Using Multiple Mass Defect Filters and Higher Energy
Collisional Dissociation
Yingying Huang, Patrick M. Jeanville, Thermo Fisher Scientific, San Jose, CA, USA
Shirley Liu, Shichang Miao, ChemoCentryx, Mountain View, CA, USA

Goal from using only a single mass defect filter (MDF), results
Uncover phase I and phase II metabolites specifically and from MMDFs are more distinct and specific, allowing users to
concurrently with the use of multiple mass defect filters do faster, more sensitive and more accurate analyses (Figure 1).
(MMDF) and higher-energy collisional dissociation (HCD) HCD provides complementary fragmentation pathways,
with Thermo Scientific LTQ Orbitrap XL data for in addition to the collision induced dissociation (CID)
structural elucidation. available in the ion trap, and produces low mass diagnostic
ions in MS-MS spectra that are very useful for metabolite
Introduction structural elucidation.
An integral part of drug discovery and development is the
identification of drug metabolites formed through phase I
and phase II metabolic reactions. LC-MS has become the
cornerstone in drug metabolite identification because of its
sensitivity and ability to analyze complex mixtures. In

Metabolomics
Metabolism\
particular, LC-MSn employing linear ion trap (LIT) technology
has become widely used because of its speed, sensitivity and
robustness in generating rich structure information.
Coupling an Orbitrap mass analyzer to the LIT greatly
facilitates the task of metabolite identification because it
enables parallel data acquisition, high mass accuracy and
resolution, and post- LIT ion manipulations. In this study,
we utilize MMDF, a feature in Thermo Scientific MetWorks
Software, and HCD on an LTQ Orbitrap™ to study the
biotransformations of Irinotecan in hepatocyte incubation
and identify its metabolites.

Experimental Conditions
Samples
Incubation was carried out using rat hepatocytes pooled
from 1 male and 1 female with a cell density of 0.5 million/mL
and 10 M of Irinotecan in the final 1 mL incubation solution.
The solution was shaken overnight and quenched by cooling
down on dry ice, followed by the addition of 200 µL chilled
AcN. The solution was then vortexed and centrifuged. The
Figure 1: Base peak chromatograms of 10 mM Irinotecan rat hepatocyte
supernatant (~1 mL) was taken out, and 10 µL from such incubation: (a) Original; (b) After single mass defect filter; (c) After multiple
solution was directly injected for each LC-MS/MS run. mass defect filters (MMDF).

Methods
HPLC: Thermo Scientific Accela High Speed LC Conclusion
Column: Thermo Scientific Hypersil GOLD, With the help of MMDF and the combination of HCD and
C18, 1 x 100 mm, 1.9 µm particle size. CID MS/MS, 13 Irinotecan metabolites whose peak areas
Solvents were water and acetonitrile, both with 0.1% were less than 1% of that of the parent were identified. This
formic acid. A shallow gradient was used to raise the organic report demonstrates that MMDF is more effective than a
composition from 15% to 25% over 12 minutes. The total single MDF to uncover phase I and II metabolites specifically
run time was 20 minutes including wash and equilibration. and concurrently. MMDF also enables the use of different
MDFs for N/O-dealkylation products, which often have mass
Results defects that are significantly different from the parent drug.
MMDFs were able to filter out the vast majority of the
background ions in the full scan spectra while preserving those
related to the parent drug. Compared with the results gathered

To download the full text of this application note, visit www.thermo.com/msnotebook 19

 Identification of GSH Conjugates Using Accurate Mass
Data and MetWorks Software
Amber Kohl, Thermo Fisher Scientific, West Palm Beach, FL, USA, Yingying Huang, Thermo Fisher Scientific, San Jose, CA, USA
Heng-Keang Lim, Johnson and Johnson Pharmaceutical Research Institute, Raritan, NJ, USA

Goal processed using MetWorks™ software. For full scan accurate

Comprehensive glutathione conjugates screening via isotope MS data, the Multiple Mass Defect Filters (MMDF) option
pattern dependent acquisition and identification using the in MetWorks software (see Figure 1) can be utilized to filter
multiple mass defect filtering (MMDF) and isotopic search out many background ions that are unrelated to the parent
functions in Thermo Scientific MetWorks software. drug and improve the capabilities of finding unexpected
modifications of an analyte.
Introduction
In this report, a cofactor-fortified human liver microsomal
incubation of 10 µM of nefazodone was analyzed using the
Thermo Scientific LTQ Orbitrap. A workflow was developed
to identify expected and unexpected glutathione conjugates.

Experimental Conditions
Human Liver Microsomal Incubation
Metabolomics
Metabolism/

All human liver microsomal incubations (1 mL) consisted of

2 mM EDTA, 5 mM MgCl2, 1 mg/mL microsomes, 10 µM
nefazodone, 5 mM glutathione and 1 mM NADPH. All
reagents were prepared in 0.1 M phosphate buffer (pH 7.4),
except for nefazodone, which was spiked as 10 µL of 1 mM
acetonitrile: H2O (1:1) stock solution to keep the final
acetonitrile content to less than 0.1%. After incubation Figure 1: Multiple Mass Defect Filter dialogue box in Thermo Scientific MetWorks 1.2.
for one hour at 37 °C in an open tube, the incubation was This dialogue box can be accessed by clicking on “Tools” →“Multiple Mass
terminated by the addition of 3 mL of ice-cold acetonitrile, Defect Filters”.
and the tube was vortexed prior to centrifugation at 1,203g
for 10 minutes at 5 °C. The supernatant was then transferred Conclusion
to a clean tube and dried at room temperature under a gentle
An approach for identifying GSH conjugates from incubations
stream of nitrogen. The residue was reconstituted in 200 µL
of water:acetonitrile (9:1), hand-vortexed and then passed of compounds has been developed. The isotopic data
through a 0.45 µM nylon filter using centrifugation at 7,200g dependence in the method setup allows for selective triggering
for 10 minutes at room temperature. The filtrate was analyzed of MSn data for metabolites of interest. Compared to other
by LC-MS and MS-MS using the methods described below: methods for post-processing of data, multiple mass defect
filters allow the user to identify known as well as unknown or
HPLC
unexpected metabolites of interest. Followed by the accurate
LC Instrument: Thermo Scientific Surveyor Plus HPLC System mass isotope search, this combination is well-suited for taking
Column: Thermo Scientific HyPURITY Aquastar column advantage of accurate mass data for selectively identifying
(100 x 2.1 mm ID, 3 m)
GSH conjugates that have distinct isotope signature patterns.
Mobile Phase: A: Water with 0.2% formic acid
B: Acetonitrile with 0.2% formic acid The availability of both accurate mass full scan and product
Flow Rate: 400 µL/min. ion mass spectra in the data allows structure elucidation of
Injection Volume: 20 µL the conjugates from a single LC-MSn experiment.

Mass Spectrometer References

1. Kola I and Landis J. Nature Rev. Drug Discov. 2004, 3, 711.
The LTQ Orbitrap mass spectrometer was operated in positive
™
2. Peters TS. Toxicol. Pathol. 2005, 33, 146.
electrospray ionization mode with an electrospray voltage of 3. Evans DC, Watt AP, Nicoll-Griffith DA and Baillie TA. Chem. Res. Toxicol.
5 kV. The sample was sprayed into the mass spectrometer 2004, 17, 3.
4. Baillie TA and Davis MR. Biol Mass Spectrom. 1993, 32, 319.
with the sheath, auxiliary and sweep gas set at 70, 20, and
5. Yan ZY and Caldwell QW. Anal. Chem. 2004, 76, 6836.
5 arbitrary units, respectively, and desolvation was further
6. Castro-Perez J, Plumb R, Liang L and Yang E. Rapid Commun. Mass
aided by an ion transfer tube temperature of 300 ºC. Spectrom. 2005, 19, 798.
7. Kalgutkar AS, Vaz AND, Lame ME, Henne KR, Soglia J, Zhao SX,
Abramov YA, Laombardo F, Collin C, Hendsch ZS and Hop CECA. Drug
Results Metab Dispos 2005, 33, 243.
The high resolution accurate mass data from the analysis 8. Lim H-K, Chen J, Cook K, Sensenhauser C, Silva J, Evans DC. Rapid
Commun. Mass Spectrom. 2008, 22, 1295.
of human liver microsomal incubation of nefazodone was

20 To download the full text of this application note, visit www.thermo.com/msnotebook

Determination of Occupational Exposure to
Aromatic Solvents Through Metabolite Monitoring
Using Isocratic HPLC
Stephen Aspey, Luisa Pereira, Daffyd Milton, Thermo Fisher Scientific, Runcorn, UK

Introduction
Exposure to styrene, toluene or xylene has a detrimental
effect upon the central nervous system. At low concentrations,
symptoms of dizziness, headache, nausea and vomiting are
experienced together with respiratory tract irritation. Higher
level exposure can cause in coordination and mental confusion
and, as the central nervous system becomes further depressed,
can result in unconsciousness and death. The monitoring and
evaluation of the biological effects upon health of workers
exposed to such solvents is, therefore, vitally important.
Traditionally, solvent exposure has been evaluated through
measurement of solvent concentration in ambient air,
however, it has been found in recent years that biological Figure 1: Metabolite separation
monitoring (in blood or urine) affords a far more accurate

Metabolomics
1. Mandelic acid 5. p -Hydroxybenzoic acid (internal standard)

Metabolism/
estimate of exposure. 2. 2-Methylhippuric acid 6. 3-Methylhippuric acid
This application note highlights the use of Hypercarb 3. Hippuric acid 7. 4-Methylhippuric acid
columns for the effective separation of the associated 4. Phenylglyoxylic acid
compounds of styrene, toluene and xylene metabolism.
Using a simple two component isocratic mobile phase all
major metabolites (and an internal standard) are separated The separation achieved for the biological metabolites
in under 11 minutes. using the conditions described above is illustrated in Figure 1.
The analysis is achieved in less than eleven minutes with all
Experimental Conditions analytes possessing good peak shape (greatest asymmetry at
10% value measured for mandelic acid at 1.18).
Column: Thermo Scientific Hypercarb, 100 x 4.6 mm, 3 µm The suitability of the method for quantitative analysis
Part Number: 35003-104630 was investigated through linearity and reproducibility
Mobile Phase: (H2O/MeOH, 50/50) + 0.1% TFA assessment. All analytes were observed to generate a linear
Flow Rate: 1.0 mL/min response (R2 ≥ 0.9978) over the concentration range of
Temperature: 25 °C 1–100 µg/mL. With respect to reproducibility of area, ratio
Injection Volume: 10 µL of analyte area to internal standard (p-hydroxybenzoic acid)
Detection: UV @ 240 nm and repeatability of retention times, the percentage relative
standard deviation values recorded over twenty injections
(equating to 240 column volumes) were low (≤ 1.15%).
Results
Porous graphitic carbon provides unique retention and Conclusion
separation of highly polar compounds. The retention
A simple, isocratic HPLC method, using a porous graphitic
mechanisms are understood to be a combination of
carbon column, has been developed for the determination of
dispersive interactions between the analyte-mobile phase
aromatic solvent metabolites. These polar metabolites are
and analyte-graphitic surface together with charge induced
well retained on the column and good separation and peak
interactions of polar analytes with the polarizable surface of
shape is observed. The method shows excellent linearity of
the graphite. Analyte interaction with the stationary phase
calibration and reproducibility, both in terms of peak area and
is dependent upon the type and positioning of functional
retention time, making it suitable for quantitative analysis.
groups together with the molecular area in contact with the
planar graphite surface.
The unique separation mechanism provides enhanced
selectivity of diastereoisomers, isomers and structurally related
compounds. These properties indicate that a Hypercarb
column should be suitable for the separation of these
structurally similar metabolites.

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 Analysis of Green and Black Tea Extracts Using an Accela
High-Speed U-HPLC Coupled to an LTQ Orbitrap XL
Hybrid Linear Ion Trap Mass Spectrometer
Donna L. Wilson, Charles Yang, Thermo Fisher Scientific, San Jose, CA, USA

Goal A
Develop an untargeted metabolomic workflow, from data
acquisition through metabolite ID, for differential and
structural characterization of polyphenolic catechin
(flavan-3-ol) derivatives and theaflavin components of
green tea and black tea.

Introduction
B
The analysis of tea represents a significant analytical
challenge, as do comprehensive metabolomics investigations.
Tea contains a wide range of components including vitamins,
amino acids, and polyphenols many of which are structurally
similar and may differ only in the type and location of a
side chain. The use of high resolution chromatography is
Metabolomics
Metabolism/

essential for the analysis of such a complex mixture.

Acquisition of accurate mass data in both full scan and MSn
mode enables complete structural characterization.
This application note shows a complete analytical
metabolomic workflow highlighting the component differences
between green and black tea including: 1) data acquisition
utilizing a high-resolution accurate mass instrument coupled
to an high-pressure LC fitted with a higher-energy collisional
dissociation (HCD) cell; 2) metabolite differential abundance
analysis; 3) structural elucidation of relevant metabolites Figure 1: Differential metabolite abundance analysis with Thermo Scientific
using accurate mass and HCD fragmentation information. SIEVE software. A) Chromatographic alignment of the various LC sample
traces is the first step in the SIEVE process. It is easy to see the differences
Methods between the green tea samples (BLUE) and the black tea samples (RED). Accela
U-HPLC provided highly resolved chromatographic peaks and high signal-to-
Samples noise ratios. B) After alignment the corresponding peak intensities are
compared for green tea (BLUE) and black tea (RED). The relative abundances of
Green tea and black tea aqueous extracts without any several compounds of interest are shown with their ratios. These metabolites
pre-treatment. Each sample was repeated in quadruplicate. were identified using a combination of accurate mass database searching and
MSn spectra interpretation via Thermo Scientific Mass Frontier software.
Chromatography Conditions
Thermo Scientific Accela U-HPLC system
Results
Column: Thermo Scientific Hypersil GOLD C18,
100 x 2.1 mm, 1.9 µm particle size U-HPLC coupled with a small-particle column afforded a fast
Mobile Phase: analysis time while maintaining very high chromatographic
(A) water with 0.1% formic acid resolution (peak width 4 seconds at half height). The high-
(B) acetonitrile with 0.1% formic acid mass-accuracy data (mass difference less than 3 ppm with
external calibration) was used to confirm elemental
Flow Rate: 500 µL/min
composition and identification of compounds while also
Injection Volume: 10 µL enabling precise quantitation. The fast cycle time of the
Gradient: Linear gradient of 100-1% A over 20 minutes LTQ Orbitrap XL™ mass spectrometer enabled HCD
fragmentation at high resolution which provided detailed
Mass Spectrometry Settings
elucidation of fragmentation pathways, as demonstrated
Thermo Scientific LTQ Orbitrap XL mass spectrometer
with the example of epigallocatechin gallate (EGCG). The
Positive Electrospray Ion Source Voltage: 5.0 kV study also included a comparative analysis of green and black
All Methods: Full scan MS in Orbitrap with a mass tea using differential analysis software for identification of
resolution of 30,000. compositional variations between the two tea samples.
Data-dependent MS2 in the Orbitrap on the top 3 most
intense ions from full scan at a mass resolution of 7500.

22 To download the full text of this application note, visit www.thermo.com/msnotebook

Using Mass List Preprocessing to Increase Confidence
of Protein Identification from ETD and ECD Spectra
Martin Zeller, Torsten Ueckert, Bernard Delanghe, Thomas Moehring; Thermo Fisher Scientific, Bremen, Germany

Goal
Use mass list preprocessing to
remove all non-fragment ion peaks in
electron transfer dissociation (ETD)
and electron capture dissociation
(ECD) spectra and significantly
increase database search confidence.

Introduction
ETD and ECD fragment spectra
typically show true fragment ion
peaks and peaks that are related
to the precursor ions such as peaks
of the un-reacted precursor ions,
peaks of the charge reduced species
of the precursor ions and neutral
losses thereof.1 In addition, ECD
spectra often show peaks for the
overtones (harmonics) of the
Figure 1: Typical ECD spectrum of a BSA peptide
precursor as shown in Figure 1.

Proteomics
Non-fragment ion peaks can lead
to false positive identifications in database searches that For the complex Arabidopsis thaliana sample, 1107
use search algorithms that score experimentally generated peptides were identified with a 1% false-discovery rate
spectra versus calculated theoretical spectra. (FDR) and 1847 peptides with a 5% FDR without mass list
Instruments with a high resolving power such as the preprocessing. With mass list preprocessing, 1727 peptides
Thermo Scientific LTQ Orbitrap XL ETD and LTQ FT Ultra were identified with a 1% FDR and 2349 peptides with a
can unambiguously determine the charge state of precursor 5% FDR.
ions. With the knowledge of the charge states of the In almost every case where the score for the peptide
precursor ions, all non-fragment ion peaks can be calculated without spectrum preprocessing was higher, it was due to
and removed before database searches. In this work we un-reacted precursor ions or charge-reduced species being
evaluate the effect of the removal of the non-fragment ion included, leading to a dubious score or false-positive
peaks on the database search results. identification.
The average increase in the Xcorr value was 0.34. This
Experimental Conditions number included peptide pairs where the spectrum without
All spectra were acquired on an LTQ Orbitrap XL ETD™ or mass list preprocessing received a higher score due to the false
LTQ FT Ultra™ MS equipped with an ECD cathode. assignment of the precursor peak or charge-reduced species.
The Arabidopsis thaliana samples were separated using a
Thermo Scientific Surveyor LC equipped with a Thermo Conclusions
Scientific MicroAS autosampler and a 10 cm, 75 µm inner Mass list preprocessing can significantly increase confidence
diameter C18 column. in database searches of ETD and ECD data. Only instruments
Data analysis was done using Thermo Scientific Proteome with high resolving power such as the LTQ Orbitrap XL ETD
Discoverer software with a mass list preprocessor node. and LTQ FT Ultra mass spectrometers are able to determine
the charge state of the precursor ions and hence benefit
Results from removal of non-fragment ion peaks.
The mass list preprocessor tool has been implemented in the
Proteome Discoverer software. The Proteome Discoverer References
software includes multiple search engines for peptide and 1. Cooper, H. J., Hakansson, K., Marshall, A.G., Hudgins, R. R., Haselmann,
K. F., Kjeldsen, F., Budnik, B. A. Polfer, N. C., Zubarev, R. A., Letter: the
protein identification. It is designed to process complex data diagnostic value of amino acid side-chain losses in electron capture
sets with different search algorithms and/or dissociation dissociation of polypeptides. Comment on: “Can the (M(.)-X) region in
techniques in a single analysis (e.g. Data Dependent electron capture dissociation provide reliable information on amino acid
composition of polypeptides?”, Eur. J. Mass Spectrom. 8, 461-469 (2002).
decision tree).

To download the full text of this application note, visit www.thermo.com/msnotebook 23

 A New Methodology for Targeted Peptide Quantitation
in Complex Mixtures Using a High-Resolution Triple
Quadrupole Mass Spectrometer
Reiko Kiyonami, Scott Peterman, Rosa Viner, Amol Prakash, Vlad Zabrouskov, Thermo Fisher Scientific, San Jose, CA, USA

Goal monitoring (H-SRM) with high-resolution (0.2 FWHM)

Develop a highly sensitive and accurate method for the precursor ion selection. They can also monitor large
quantitative validation and verification of putative marker numbers of SRM transitions in a single run. This research
proteins in complex mixtures. sought to take advantage of those attributes to develop a
fast, robust H-SRM based workflow for accurate, highly
Introduction sensitive, quantitative validation and verification of targeted
proteins in complex mixtures. It was theorized that monitoring
A common endpoint for biomarker discovery experiments is
multiple b- or y-type fragment ions with the same retention
a list of putative marker proteins. A reasonable next step is
time would be sufficient to maintain high assay specificity.
targeted quantitative measurement of these proteins in an
It was also hoped that the shorter duty cycle of this approach
expanded patient population to affirm their validity as
(versus verification by full MS/MS scan) would allow peptide
markers. Selected reaction monitoring (SRM) by triple
confirmation and quantitation in a single run while
quadrupole mass spectrometry is commonly used for this
maintaining maximum sensitivity.
application due to its ability to measure low levels of
peptides in complex mixtures while maintaining excellent
Experimental Conditions
quantitative accuracy and precision.
Availability of protein standards is highly limited, so To test the proposed H-SRM workflow, both conventional
it is often necessary to confirm the identity of compounds (full MS/MS scan) and H-SRM workflows were run on a
detected during quantitative validation. Traditionally, this is TSQ Quantum Ultra™. The workflow is equally suitable for
Proteomics

done with an SRM-triggered full MS/MS scan and subsequent the TSQ Vantage™.
database search. However, most triple quadrupole mass An enzymatic digest of human serum was chosen as
spectrometers are limited to unit mass resolution (0.7 FWHM) the sample with which to compare sensitivity. Twenty four
for precursor ion selection. The quality of the MS/MS scan serum proteins with plasma concentrations ranging from
can be compromised by coeluting, isobaric background 3.40E5 pg/mL (vitronectin) to 1.25E9 pg/mL (haptoglobin)
compounds. The traditional SRM approach also suffers were selected. One to four proteotypic peptides were used
from reduced detection limits, and its relatively long cycle to monitor each targeted protein for a total of 50 peptides.
times make it difficult to quantify and confirm peptide The selected peptide sequences contained no Cys, Met, or
identity in a single LC run. other commonly modified residues and had masses in the
The Thermo Scientific TSQ Quantum Ultra and TSQ range of 800 – 2400 Da.
Vantage (Figure 1) are the only triple quadrupole mass spectro- For the conventional SRM workflow, two to three y-ions
meters that can employ high-resolution selected reaction from each peptide were monitored – a total of 152 SRM
transitions – and used to trigger the MS/MS
acquisition. For the H-SRM workflow, five to
seven y-ions from each peptide were monitored
for a total of 318 H-SRM transitions. Because
no full MS/MS scan was to be acquired in the
H-SRM workflow, the ions and precursor from
each targeted peptide were checked in advance
against the human SwissProt database using
Mascot™ Sequence Query to ensure the peptides
were unique and had high peptide scores.
A second goal was to evaluate the
quantitative reproducibility and accuracy of the
H-SRM assay. Thirteen serum proteins with
varying concentrations were targeted for this
experiment. A total of 61 SRM transitions,
chosen from 20 unique peptides representing
these proteins, were used to test the H-SRM
workflow. Dwell times of 5 ms, 10 ms, and
20 ms were compared.

Figure 1: TSQ Quantum Ultra Triple Quadrupole MS

24 To download the full text of this application note, visit www.thermo.com/msnotebook

Results Conclusion
The H-SRM workflow exhibited significant advantages over The H-SRM workflow takes advantage of the unique, high-
the conventional SRM workflow. resolution (0.2 FWHM) precursor ion selection available in
• The H-SRM assay provided significantly improved S/N ratios the TSQ Quantum Ultra and TSQ Vantage triple quadrupole
(see Figure 2) – greater than 10x for some peptides – mass spectrometers. It provided excellent sensitivity, analytical
by dramatically reducing non-specific interferences from assay precision, and quantitative accuracy for targeted
coeluting, isobaric species. protein quantitation in whole human serum by dramatically
• All 50 targeted peptides were unambiguously identified reducing non-specific interferences from serum background
using the H-SRM workflow. The traditional SRM ions. The H-SRM assay improved assay specificity and
workflow identified only 36 peptides. detection limits, thereby offering significant advantages
• Lower limits of detection and wider quantitative dynamic for biomarker verification and validation studies.
range (greater than four orders of magnitude) were both
observed with the H-SRM workflow. Mascot is a trademark of Matrix Science Ltd.

• The quantitative accuracy of the H-SRM assay was excellent.

The average relative quantitation error was ±4%.
• Excellent analytical precision
was obtained. Approximately
95% of the peptides gave CVs
<15% for all three dwell times.
No significant signal loss was
observed when using shorter
dwell times.
• The shorter duty cycle of the
H-SRM workflow (2.2 s vs. 3.9 s
for the conventional workflow)
allowed for efficient, simultaneous
targeted peptide identification and

Proteomics
quantitation in a single LC run.

Figure 2: The peptide LLDSLPSDTR representing complement component 1 inhibitor was not conclusively
identified by traditional SRM, but was identified by the H-SRM assay using time alignment of its multiple
fragment ions (inset). Compared with traditional SRM, the H-SRM assay significantly improved S/N.

To download the full text of this application note, visit www.thermo.com/msnotebook 25

 MALDI-Produced Ions Examined with a Linear Ion Trap –
Orbitrap Mass Analyzer
Kerstin Strupat, Huy Bui, Viatcheslav Kovtoun, Rosa Viner, Mari Prieto Conaway, Nick Izgarian, Oliver Lange, George Stafford,
Stevan Horning, Julian Phillips, Thomas Moehring, Thermo Fisher Scientific, Bremen, Germany and Thermo Fisher Scientific, San Jose, CA, USA

Introduction Results
Matrix-assisted laser desorption ionization (MALDI) has The Thermo Scientific MALDI LTQ Orbitrap system is
been used as ionization technique for a large variety of capable of providing reliable and accurate MALDI produced
thermo-labile (bio) molecules since the late 1980s, typically ions at high mass resolution. Ions or fragment ions produced
coupled to Time-of-Flight (ToF) instruments. Compared to can be analyzed with Orbitrap detection at a maximum mass
MALDI ToF and MALDI ToF-ToF, there are significantly resolution of > 100,000 @ m/z 400 with high mass accuracy.
fewer publications about MALDI coupled to mass analyzers This is shown in Figure 1: The full-scan information from
such as (q)QTof, ion trap or FTICR-based systems. While m/z 200 – 4000 shows various peptides acquired at mass
pulsed lasers are most straight forwardly coupled to ToF resolution 100,000 @ m/z 400. The insets display the isotope
systems, as the laser pulse can be used as start signal for pattern of mellitin (honey bee) @ m/z 2845 in raw data and
the ToF measure, the coupling to scanning mass analyzers simulation. Isotope pattern and mass measurement of the
is based upon different strategies.1 This application raw file perfectly agree with the proposed pattern and
demonstrates coupling a MALDI source to a hybrid theoretical mass, respectively. Mass accuracy of better than
linear ion trap – Orbitrap™ mass analyzer.2 2 ppm is achieved by internal mass calibration using lock-
mass functionality. Using external mass calibration, an
Experimental Conditions accuracy of better than 3 ppm is obtained routinely.
Peptide mass fingerprinting with accurate-mass support
The Thermo Scientific MALDI source is coupled to a state-
is available, routinely facilitating data interpretation. Data-
of-the-art linear ion trap – Orbitrap mass analyzer, the dependent analysis on single spots or on whole LC MALDI
Thermo Scientific LTQ Orbitrap. The source is equipped plates, followed by database searching makes it possible to
Proteomics

with a nitrogen laser (Lasertechnologie, Berlin, Germany) obtain complementary sequence and coverage information in
which operates at 337.1 nm wavelength, 3 ns pulse combination with LC-ESI-MS/MS experiments. For further
duration and 60 Hz repetition rate. The beam diameter is details, refer to application notes 30151 and 30161, and to
about 80 * 100 µm2 on the sample plate. Ions produced in the product brochure.
the source are collected in a quadrupole and subsequently
transferred to the linear ion trap and orbitrap analyzers, Conclusion
respectively. All linear ion trap as well as all orbitrap
MALDI-produced ions can be analyzed in MS and MS/MS
devices can be calibrated with the kit “MSCal4” provided
modes and detected with reliable mass accuracy and high
by Sigma-Aldrich. Analytical scans are achieved under the
mass resolution. The instrument combines the benefits of
conditions of Automatic Gain Control (AGC™). Various
MALDI technique with Orbitrap technology, and provides
matrices, such as alpha cyano 4-hydroxy cinnamic acid
fast, reliable, confident and complementary protein identification,
(HCCA) matrix as well as 2,5-di-hydoxybenzoic acid (DHB)
enabling multiple analytical approaches to localizing post-
matrix can be used for the analyses on the instrument. translational modifications.

References
1. Jun Qin and Brian T. Chait,
Anal. Chem. 1997, 69,
4002-4009 Identification
and Characterization of
Posttranslational Modifications
of Proteins by MALDI Ion
Trap Mass Spectrometry.
2. Michaela Scigelova and
Alexander Makarov,
Proteomics 2006 S 16-21
Orbitrap Mass Analyzer –
Overview and Application
in Proteomics.

Figure 1: Mellitin @ RP 50,000, ≤ 1 ppm mass accuracy. Sequence of mellitin from honey bee: GIGAVLKVLTTGGLPALISWIKRKRQQ,
Sum Formula: C131H230N39O31; Monisotopic Mass MH+: 2845.7614; N-Terminus: H; C-Terminus: amidation

26 To download the full text of this application note, visit www.thermo.com/msnotebook

© 2008 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc.
and its subsidiaries.

A streamlined solution for quantifying targeted proteins.

Our targeted protein quantitation solution provides a seamless

transition from discovery to verification of putative biomarkers.

• Application-specific SRM Workflow software leverages invaluable

information from discovery experiments to optimizing quantitation.
• The Thermo Scientific TSQ Vantage LC-MS/MS offers the highest
sensitivity and lowest noise for a virtually unlimited number of
targeted peptides. Its superior selectivity eliminates interferences
from complex matrices, lowering the limits of quantitation.
• Thermo Scientific HeavyPeptide stable isotope-labeled internal
standards can be customized to exactly match your target Application Notes
Let us send you our latest application
peptides for accurate absolute quantitation. notes demonstrating how we can
streamline this critical phase of
To streamline the critical transition from biomarker discovery to your research.
targeted quantitative verification, call 1-800-532-4752, or email
analyze@thermo.com

Moving science forward

©2008 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc.
and its subsidiaries.

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provides sub-ppm mass accuracies at resolutions up to 100,000. Introducing the Thermo Scientific
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Moving science forward

XX62978_E 02/09M
Application
Note: 10080
Quantitation and Confirmation of
Organophosphorus Pesticides in Apple Extract
in a Single Injection by GC-MS/MS
Thermo Fisher Scientific Inc., Austin, Texas, USA

Introduction
Key Words The analysis of organophosphorus pesticides is routinely
performed by injection on a selective GC detector, the
• PolarisQ ion trap
Flame Photometric Detector (FPD) or Nitrogen
GC/MSn
Phosphorus Detector (NPD). Both the FPD and NPD are
• GC-MS/MS very sensitive because of their selectivity and show good
linearity in the low picogram concentrations. Identification
• Fruit Extracts
on the FPD or NPD is made by retention time. Then a
• Food Safety second injection is made under identical GC conditions on
a mass spectrometer in Full Scan for confirmation. When
• Residual
the matrix ions coelute with a target compound, the
Pesticides
resulting spectrum is very complex, and the confirmation
is not always clearly seen. The signal to noise of the
primary ion used for quantitation is adversely affected by
these coeluting matrix ions, resulting in poor sensitivity.
Alternately, Single Ion Monitoring may be performed to
enhance the sensitivity, but this does not provide a
spectrum with sufficient ions for library matching. Instrument Parameters
The Thermo Scientific PolarisQ ion trap mass POLARISQ ION TRAP
spectrometer uses a scanning method where a precursor
Source Temperature: 250 °C
ion is selected from the analyte spectrum. This ion is
Ionization Mode: +EI, 70 eV
isolated in the ion trap and all other ions are expelled.
AGC: 50
Then sufficient energy is applied to the single ion for
Injection Waveform: 1 volt
fragmentation into a unique spectrum of its product ions,
MS/MS Parameters: See Table 1
generating response in the low picogram levels with a
signal to noise or sensitivity similar to that of the FPD or
NPD. This type of detection is called tandem MS/MS. The TRACE GC Ultra
adverse effects of the sample matrix are minimized while Column: Rtx™ 5 MS 0.25 mm x 30 meter, 0.25 µm
generating confirmational data for quantitation. The Oven: 40 °C, 1.0 min; 5 °C/min, 200 °C,
PolarisQ ion trap GC/MS was used to develop a 2.0 min;10 °C/min, 275 °C, 5.0 min
quantitative method for the analysis of organophosphorus PTV Inlet: Large Volume Mode
pesticides in apple extract. Liner: 2 mm straight Silcosteel
Carrier Flow: 1 mL/min helium, constant flow
Split Flow: 50 mL/min
Splitless Time: 0.75 min
Solvent Valve Temperature: 100 °C
Injection: 40 °C, 0.3 min at 10 psi
Evaporation Phase: 10 °C/sec, 56 °C, 0.2 min
Transfer: 10 °C/sec, 250 °C, 2.0 min at 15 psi
Clean: 14 °C/sec, 275 °C,38 min

AUTOSAMPLER

Injection volume: 5 uL
MS/MS
The ion trap mass spectrometer is very sensitive and may
be run in a tandem EI MS/MS mode. As the analyte enters
the ion trap (Figure 1a), a precursor ion is isolated (Figure 1b).
Then energy is applied to fragment the ion (Figure 1c) to
form a complete spectrum of product ions (Figure 1d).
The product spectrum is unique for each pesticide. The
sample matrix ions are excluded from the ion trap during
the isolation of the precursor ion and do not affect the
product spectrum or sensitivity of the analysis.

(a) (b) (c) (d) Figure 2: TIC of high standard - EI Full Scan

Figure 1: The process of Ion Trap MS/MS

Objective
A group of 20 organophosphorus pesticides listed in Table 1 Figure 3: Terbufos EI Full Scan spectrum
was examined by tandem MS/MS to determine the optimum
parameters in an apple extract matrix. A calibration curve
in the low picogram levels was run. Then replicate injections
were made to check the precision of the method. No internal
standard was used and all calculations were done by
external standardization.
First an injection was made in EI full scan to determine
the retention times of each analyte (Figure 2). Then the
spectrum of each analyte was reviewed for selection of a
precursor ion with an abundant intensity and high mass
Figure 4: Terburfos precursor ion 231 m/z
for greater product ion yield. For terbufos, the typical EI
full scan spectrum was reviewed in Figure 3. The second
run was set up with defined segments for isolation of the
precursor ion for each analyte. For terbufos, the precursor
ion was 231 m/z as shown in Figure 4.
Multiple scan events were set up for each MS/MS
segment for optimization of the appropriate collision
energy for the fragmentation. Figure 5 shows the product
ion spectrum for the optimum collision energy for
terbufos at 1 volt. You can still see a small amount of
231 m/z. Then the product ion scan range was reduced to
maximize the intensity of the product ions for quantitation.
Figure 5: Optimization of collision energy for generation of terbufos
For terbufos, this was set at a scan range from 165 to 213 m/z
product ions
(Figure 6). The MS/MS Total Ion Chromatogram (TIC)
for the method is shown in Figure 7.
The injections were made using a temperature
programmable injector with an injection volume of 5 µL
of apple matrix spiked with the target list in acetone. The
linearity was evaluated and replicates made to prove the
robustness of the method in matrix. Ion ratios of the top
two product ions were tested.

Figure 6: Defining scan range (165 to 213 m/z) for detection of terbufos
MS/MS product ions
Figure 7: TIC run of high standard - MS/MS

Pesticides in Apple Extract

Figure 8: Terburfos MS/MS spectrum, quan ions and linearity (30,60,150,300 pg)

Figure 9: Diazinon MS/MS spectrum, quan ions and linearity (60,120,300,600 pg)

Figure 10: Fenthion MS/MS spectrum, quan ions and linearity (50,100,250,500 pg)

Figure 11: Methyl chlorpyrifos MS/MS spectrum, quan ions and linearity (25,50,125,250 pg)
Results Conclusion In addition to these
The MS/MS parameters for the pesticides are listed in A tandem MS/MS method was developed and validated offices, Thermo Fisher
Table 1. The standard was diluted in apple matrix extract on the Thermo Scientific PolarisQ ion trap GC/MS for the Scientific maintains
for generation of a calibration curve in the low to high analysis of organophosphorus pesticides in apple matrix. a network of represen-
picogram ranges. The method provided good sensitivity at the low picogram tative organizations
Some typical curves are shown for terbufos, diazinon, range and excellent ion ratio confirmational data of the throughout the world.
fenthion, and methyl chlorpyrifos. The mass chromatogram product ion spectra.
of the ions used for quantitation for the low point and the A linear working range was established at levels normally
product spectrum for each pesticide are also shown in run on a GC specific detector like the NPD or FPD. A
Figures 8-11 on page 3. single injection generated a report for quantitation and
The precision for replicate injections and calibration confirmation of the presence of organophosphorus Australia
curve linearity are shown in Table 2. The ion ratio pesticides in apple matrix. +61 2 8844 9500
confirmation data for the product ions was within the Austria
+43 1 333 50340
needed criterion of ±20 %. Acknowledgements: Belgium
We would like to thank John Meola of the New York State Department of +32 2 482 30 30
Agriculture for providing the sample extracts. Canada
Authors: Jessie Butler, Carl Feigel +1 800 530 8447
China
COMPOUND RETENTION TIME PRECURSOR ION (M/Z) COLLISION ENERGY (V) PRODUCT ION SCAN RANGE QUANTITATIVE IONS (M/Z) +86 10 5850 3588
methamidophos 18.72 94 1 60-104 64,80 Denmark
acephate 22.98 136 0.5 35-100 42,94 +45 70 23 62 60
TEPP 26.11 263 1 200-245 207,235 France
omethoate 26.58 156 0.5 100-150 110,141 +33 1 60 92 48 00
dimethoate 29.43 125 0.5 55-90 62,79 Germany
terbufos 30.34 231 1 165-213 175,203 +49 6103 408 1014
diazinon 30.94 304 0.75 150-190 162,179 India
methyl chlorpyrifos 32.71 286 1 200-280 210,271 +91 22 6742 9434
malathion O analog 32.91 127 1 90-110 99,109 Italy
pirimiphos 34.02 305 1 170-300 180,290 +39 02 950 591
fenthion 34.8 278 1.5 125-255 135,245 Japan
+81 45 453 9100
parathion 34.92 291 0.5 132-270 142,263
Latin America
methidathion 37.26 145 0.5 50-95 58,85
+1 608 276 5659
tetrachlorfenvinphos 37.63 331 2.5 165-341 109,316
Netherlands
DEF 38.36 202 0.5 80-160 89,147 +31 76 587 98 88
ethion 39.7 231 1 170-213 175,203 South Africa
carbophenothion 40.22 342 1 190-300 199,276 +27 11 570 1840
fenamiphos sulfoxide 41.45 304 0.5 186-286 196,276 Spain
phosmet 41.65 160 1.5 95-145 105,133 +34 91 657 4930
azinphosmethyl 42.49 160 0.5 94-142 104,132 Sweden / Norway /
Finland
Table 1: MS/MS parameters for pesticides in apple matrix +46 8 556 468 00
Switzerland
LINEARITY PRECISION FOR 6 REPLICATE INJECTIONS OF HIGH SPIKE
+41 61 48784 00
COMPOUND AVG. RF STD. DEV. %RSD AVG. AREA CTS. STD. DEV. - CTS. % RSD UK
methamidophos 75 6.1 8 25634 1210 5 +44 1442 233555
acephate 71 5.7 8 31768 3910 12 USA
TEPP 75 11.5 15 35207 3461 10 +1 800 532 4752
omethoate 123 8.0 7 80956 7095 9
www.thermo.com
dimethoate 183 22.6 12 62358 5023 8
terbufos 2237 88.8 4 565996 45090 8
diazinon 259 18.5 7 144968 5099 4
methyl chlorpyrifos 546 33.8 6 116779 5200 4
malathion O analog 391 88.7 13 171177 22477 13
Thermo Fisher Scientific,
pirimiphos 824 123.2 15 146692 16456 11 Austin, TX USA is ISO Certified.
fenthion 726 77.7 11 308088 24343 8
parathion 87 12.5 14 25485 2656 10 ©2007. Thermo Fisher
Scientific Inc. All rights
methidathion 924 104.1 11 294206 31105 11 reserved. Rtx is a registered
tetrachlorfenvinphos 239 21.9 9 107317 6256 6 trademark of Restek
Corporation. All other
DEF 672 83.9 13 146192 14092 10 trademarks are the property
of Thermo Fisher Scientific
ethion 7315 603.4 8 1121116 166329 15 and its subsidiaries.
carbophenothion 51 8.1 16 18648 1518 8 Specifications, terms and
fenamiphos sulfoxide 569 90.0 16 347068 67890 20 pricing are subject to change.
Not all products are available
phosmet 844 181.0 19 921658 66839 7 in all countries. Please
consult your local sales
azinphosmethyl 57 32.9 22 90594 8132 9 representative for details.

Table 2: Linearity and precision of the MS/MS Method AN10080_E 10/07C

Part of Thermo Fisher Scientific

Thermo Scientific
TRACE 1300 Series
Gas Chromatograph

powerful breakthroughs

for increased productivity
and performance
 the productivity you need
the performance you want
Reducing cost of ownership while increasing productivity is one of today’s tough challenges faced by
laboratories using automated analytical equipment. For QA/QC and routine chemical laboratories, improvements
in this area are intimately connected with the simplification of analytical workflows, optimization of technical
resources and waste reduction.
The Thermo Scientific TRACE 1300 Series Gas Chromatograph is the latest technology breakthrough conceived
to substantially elevate performance in QA/QC and routine laboratories. Developed around key innovations driven
by customer needs, including user-exchangeable instant connect injectors and detectors, enhanced robustness
of components, scaling down of injector and detector solutions, customer-driven design of the user interface,
and optimization of all electronic elements, the TRACE™ 1300 Series GC results in an extremely fast, easy to
use, compact GC, delivering an incredibly high lab productivity at reduced cost of ownership.

Ultimate Productivity Invest for the Future

A breakthrough in lab productivity is provided by: A substantial reduction of ownership costs is ensured by:
• E asy adoption of standard GC methods – • Tailorable GC configurations – Proprietary user-exchangeable
Eliminate the difficulties of method development during start-up instant connect injectors and detector modules offer budget-
with the aid of enhanced injectors, integrated electronic gas conscious laboratories the flexibility to start with a single-channel
control (IEC) and an easy-touch, icon-driven instrument instrument investment with the opportunity to expand to
interface, no matter how complex the analytical method. multi-injector/detector configurations. Accommodate new
• Increased robustness of injector technology – applications or increased throughput requirements simply by
Reduce sample clean-up prior to analysis and exploit time- installing new modules within two minutes.
saving benefits of the injector backflush. • Reduced training requirements – Install the system with
• Shorter sample cycle time – Ensure fast cycle time with low minimal effort. Just easily plug in injector and detector modules
thermal mass injectors and detectors, combined with fast GC oven and connect gases. The icon-driven touch screen or intuitive
proprietary technology. Thermo Scientific Dionex Chromeleon software guides the
instrument and method set-up instantaneously.
• Unmatched detector sensitivity in trace analysis –
A completely new range of micro volume GC detectors, ideal for •S
 mart maintenance – Access injectors and detectors with
trace analysis, to limit sample re-concentration requirements minimal effort using unprecedented tool-free options and eliminate
or reduce injected sample amount. For positive detection and any downtime during maintenance with back-up injectors and
identification, the most advanced range of mass spectrometers detector modules.
complement conventional GC detectors.
•C
 onsumable adaptability – Systems are compatible with
•S
 eamless automation – Automate analyses with liquid,
existing standard consumables to reduce inventory costs in the
headspace and solid-phase micro extraction (SPME) injections
laboratory populated by multiple GC brands.
and sample preparation cycles with the most modern robotic
sample handling in the industry. • Energy-saving – Limit power usage during operations with
•S
 calable, simply intelligent chromatography data system – reduced instrument thermal mass allowing quick start-up.
Efficiently analyze data with full Thermo Scientific Dionex • Minimal sample and solvent consumption –
Chromeleon Chromatography Data System compatibility. Optimize sample and solvent usage with the powerful combina-
tion of large volume and back-flush capabilities of injectors,
extraordinary detector sensitivity and micro-volume sample
handling with robotic automation.

2
 “Instant Connect”
Injectors and Detectors
With the TRACE 1300 Series GC, changing the configuration requires only
two minutes, just the time needed to remove three screws and put the
new injector or detector module in place.
With these proprietary Thermo Scientific “Instant Connect” (IC) modules,
special training, dedicated tools or on-site service engineers are not
required to tailor the GC configuration to your workload or to run a specific
The TRACE 1310 local user method. This unique modularity design offers the following advantages to
interface can be easily translated the operator:
into any language. Chinese, and
Brazilian Portuguese versions • Exceptional throughput –
are shown. Upgrade a GC from single to multiple channels to satisfy rapid incremental
business needs and enhance laboratory productivity
• Postpone preventive maintenance for continuous operations –
Remove contaminated injector/detector bodies, replace them with clean
Complete Solution for your Needs ones and start running samples in a few minutes while programming
basic maintenance when the laboratory schedule allows.
The TRACE 1300 Series GC is available in two models designed to
meet the specific needs of all laboratories. The TRACE 1300 GC
system is the ideal budget-conscious investment for the basic routine
laboratory when lower operator expertise requires ease of use with
minimal instrument interaction. Its simplified user interface is also
ideal for 24/7 operations, as needed in petrochemical plants or
remote laboratories that require single-button start/stop/mainte-
nance local interactions while maintaining full programmability
through the networked control software.
Larger routine QA/QC laboratories will benefit from the
TRACE 1310 GC which features a complete icon-based touch
screen interface which is ideal for direct instrument control when
method development is required. While retaining all of the
capabilities and performance of the TRACE 1300 GC model, it also
provides local status update of the oven, injectors and detectors,
maintenance commands, run log, multiple language capabilities
and video tutorials to drive simple instrument interaction.

Thermo Scientific TRACE 1300 GC with a two-button Thermo Scientific TRACE 1310 GC with an icon-based
user interface offers simplicity for laboratories touch screen is ideal for direct instrument control and
where local instrument interaction is not necessary. for method development. 3
 reliability, robustness and up time
the advantages of Instant Connect injectors
The Highest Versatility through the Instant Connect (IC) Injector Modules
A full range of liquid injection modes are available on the TRACE 1300 Series GC to cope with the most demanding
sample analysis. Starting with the conventional Split/Splitless and extending through the Programmable Temperature
Vaporizing (PTV) injector for wider boiling point sample ranges, up to on-column capability if a more gentle injection
technique is needed, the flexibility of the instant connect injectors is maximum.
Further versatility is achieved by adding back-flush or large-volume capabilities for reduced sample clean-up
steps or increased system sensitivity. All of these injection techniques are available as user-exchangeable plug-in
modules, featuring compact, tubing-free injector manifold design for easier maintenance and fully integrated
electronic carrier gas control. Maximum flexibility is guaranteed with the ability to switch the injector module quickly
when a different injection technique is required.

Instant Connect-SSL Module

The Instant Connect-SSL injector features a further optimized
thermal profile developed to avoid sample discrimination in split and
splitless mode, thus allowing the broadest range of analytes to be
accurately injected. The unique injector head guarantees minimum
thermal stress to the septum, therefore reducing its bleeding and
extending septa lifetime. Injector design also guarantees complete
oxygen diffusion-free operation, a prerequisite for accurate injections
into high performance mass spectrometers.
The flexible injector configuration enables a quick and easy
implementation of existing and validated methods to become
Instant Connect-SSL free from discrimination: Hydrocarbon Florida immediately productive. Its large compatibility with standard
mix % recovery versus C20, average of 20 injections. consumables allows the use of generic liners, septa and ferrules
often available in the lab and used on different GC models and
brands, therefore cutting operational costs.
This new proprietary injector design also ensures easy and
immediate access to the septum and liner for simple and quick
maintenance. Moreover, when difficult sticky samples require
extra care, users can extract the injector body without specific
iC-SSL injector tools, thoroughly cleaning it and immediately restoring routine
temperature operations – a quick, trouble-free two-minute operation!
profile

Exceptional Retention Time Stability

Hydrocarbon Mean RT Min. Std. Dev. Min. Hydrocarbon Mean RT Min. Std. Dev. Min.

C12 C28 12.4725 0.0005

4.6200 0.0011
C14 C30 13.1348 0.0008
6.0192 0.0005
C16 C32 13.7557 0.0007
7.2268 0.0005
C18 C34 14.3395 0.0007
8.3051 0.0006
C20 C36 14.8908 0.0007
9.2825 0.0006
C22 10.1767 0.0006
C38 15.4118 0.0008
C24 10.9997 0.0005
C40 15.9063 0.0006
C26 11.7629 0.0005

Retention time stability on 20 consecutive runs of hydrocarbon mix.

Retention time standard deviation ≤1/1000 minute.
4
 IC-PTV Module
The Instant Connect-PTV injector combines the “discrimination-free” performances of
a cold injector with the robustness of the vaporizing injectors. Merging together fast
heating and cooling performance with the inertness of the injector chamber and large
sample capacity, this injector is the ideal choice for trace analysis in dirty matrices and
for thermally labile compounds. Its unique design and multi-mode operations enable the
preservation of sample integrity in all situations.
Unlike other PTV injectors using liquid coolant, fast cooling is achieved by a compact
and limited thermal-mass design, combined with an efficient forced air circulation system.
When initial sub-ambient temperatures are required by the application, a convenient iC-PTV: On-column chromatogram of Decabromodiphenylether
(MW= 959 m/z). Its full elution shows the lack of discrimination
cryogenic option is available. Easy liner removal from the top and complete access to even for very high molecular-weight components.
the injector chamber makes maintenance quick and trouble-free.

Backflush and Large Volume

Capabilities
The capabilities of Instant Connect-SSL and Instant
Connect-PTV modules are further enhanced by the
available backflush options. These solutions enable the

microVolts
user to reverse the flow inside the injector, eliminating
heavy or “undesired” compounds, protecting the column
and detector while cutting down non-productive times,
thus increasing throughput.
Additional enhancement on system performance is
reached by the large volume capabilities available on
both Instant Connect-SSL and Instant Connect-PTV
modules. Splitless injections of up to 50 µL are possible
Minutes
on a standard Instant Connect-SSL injector equipped
microVolts

with a liner packed with glass wool and a pre-column, Real gasoline sample
using the proprietary Concurrent Solvent Recondensation chromatogram with (left)
and without (right) backflush.
technique. Suitable for volatile samples, this technique
Without changing the analytical
guarantees strong robustness when analyzing con- method, the heavier matrix
taminated matrices. A PTV Large Volume solvent split components are eliminated.
injection of up to 250 µL is the alternative for Considerable time is
saved while maintaining
higher sensitivity improvements. the analytical column and
Minutes
iC-SSL backflush. The T-piece detector cleaner.
for column connections and
its carrier gas control are
integrated within the module.

Outstanding retention time stability is achieved compact, self-sufficient fully-featured devices, To further enhance analytical performances,
even in the most complex GC and GC/MS deliver strictly controlled pressure or flow to the IEC module also supports the automated Leak
applications through the use of innovative and columns and detectors. Check of the injector and column installed and
unique IEC (integrated electronic control) modules. Setting constant or ramped pressures and Column Evaluation procedures.
This guarantees 0.001 psi response through the flows is easy through the software or the local user Additional IEC channels can be implemented
entire working range. interface while the electronic control maintains at any time, providing further functionality for
These miniaturized gas controls, integrated the stability during every run for exceptional multidimensional applications.
within every injector or detector module for retention time accuracy and precision.

5
 exceptionally sensitive
and stable response in trace analysis
Instant Connect Detectors Designed for
Enhanced Productivity
A complete set of newly designed, miniaturized Instant Connect
detectors available with the TRACE 1300 Series GC guarantees
fast peak detection and maximum sensitivity. Small cell volumes
and rapid acquisition response, standard at 300 Hertz, make
them ideal for standard and fast chromatography applications,
ready to boost laboratory productivity at any time. The highest
sensitivity levels are easily achieved even in the most demanding
TCD determination of water in industrial solvent
trace analyses in dirty matrices, such as the determination of
halogenated or nitrogen/phosphorous-containing pesticides in
food or environmental samples. As for the other modules of the TRACE 1300 Series GC,
Ultra-modern digital electrometers enable wider dynamic swapping detectors or upgrading from single to multi-detector
ranges to be captured automatically by the software into a single takes only a few minutes to execute, and laboratory personnel
run without cumbersome manual method settings. In this way, can perform the replacement without any special training needed.
the quantification of trace contaminants and highly-concentrated The GC can be quickly configured to address evolving laboratory
analytes in a single run is possible without the need for needs and increased sample requirements by installing the
extensive method development, multiple standards injections necessary modules for a specific method; a time and expense-
or sample dilutions. saving benefit for routine and contract laboratories.

Mass Spectrometer Solutions

The TRACE 1300 GC and TRACE 1310 GC are fully compatible
Instant Connect Detectors with any Thermo Scientific mass spectrometer representing the
ideal front end for every GC/MS need. Transfer lines can be
Instant Connect-FID
connected on either side of the GC oven, thus providing the
The Instant Connect-FID (Flame Ionization Detector) offers high sensitivity maximum flexibility when coupling the TRACE 1300 Series GC
and a wide dynamic range with rapid acquisition speed, making it ideal for with one of these mass spectrometers.
extremely fast GC applications. Ranging from ion traps to single and triple quadrupoles
Instant Connect-TCD to high-resolution instrumentation, Thermo Scientific mass
The newly-designed micro-volume Instant Connect-TCD (Thermal Conductivity spectrometers deliver increasingly higher mass resolving capa-
Detector) is used in a wide variety of capillary and packed column applications. bilities, selectivity and sensitivity offering unsurpassed analytical
Due to its exceptional thermal stability and fast response, this non-destructive performance even for the most difficult matrix challenges.
detector provides exceptional sensitivity over the widest range of applications.

Instant Connect-ECD
The new Instant Connect-ECD (Electron Capture Detector) is excellently
developed for trace analysis in challenging samples. Its miniaturized cell
equipped with a purged, removable anode, has been designed to maximize
robustness towards the matrix effect while guaranteeing the utmost sensitivity.

Instant Connect-NPD
Built upon the proven sensitivity of the Thermo Scientific Nitrogen Phosphorous
Detectors (NPD), the new Instant Connect-NPD brings exceptional flexibility
to the determination of specific components with the adoption of multiple
dedicated sources.

6
green by design
Minimizing Environmental Impact
Thermo Scientific products responsibly contribute to the consci-
entious management of our environment and its resources. To
minimize the environmental impact of using GC instrumentation, Warm-up Times. From OFF Conditions to Readiness (minutes)

the TRACE 1300 Series GC has been designed to guarantee TRACE 1300 Series GC Standard GC
Oven at 50 °C
lower power consumption and the quickest start-up in gas Injector and Detectors at 250 °C 3.5 10.2
chromatography instrumentation. Due to the reduced thermal
mass design, heated zones reach their set-point from power-off
conditions in only a few minutes, thus further reducing electricity
consumption and limiting non-productive wait times.
Shorter sample cycle time and exceptional throughput are
achieved through the fast heating and cooling capabilities of the
new proprietary oven. Enabling an astonishing thermal stability,
this fast new oven is ideal for standard, as well as multi-columns
applications. Even with a full-sized oven, the TRACE 1300 Series
GC features one of the smallest footprints in the industry, thus
reducing laboratory bench space requirements.
Fewer parts are required to manufacture the TRACE 1300 GC,
and the reduced size and lower weight enable lighter transport.
Combined with ongoing recycling initiatives, all of these factors
The TRACE 1300 Series GC quickly reaches near-ambient temperatures.
contribute to a reduced carbon footprint that creates a positive
impact on our environment.
For laboratories adopting alternative carrier gases in order
to maintain lower operational costs and to preserve the limited
Helium natural sources, the TRACE 1300 Series GC is also
fully compatible with the use of Hydrogen and Nitrogen, and
is compliant in all its components with the latest Restriction of
Hazardous Substances (RoHS) requirements for electrical and
electronic equipment.

The significant width diffference between the

TRACE 1300 Series GC and the other dual-column
GCs saves precious bench-top space.

7
 expand throughput
with liquid, solid and gas sampling devices
Autosampling and Autoinjection Solutions
For maximum ease of use when executing liquid injections, the new Thermo Scientific AI 1310 Autoinjector
and the new Thermo Scientific AS 1310 Autosampler guarantee the desired flexibility, throughput and robustness.

The AI 1310 Autoinjector is an eight-position The AI/AS 1310 Autosampler uses a revolving
sampling module. It combines the high precision of turret that allows the syringe to swing from the
an automatic injection system with the ease of use sample tray to the injection port. This approach
of the Plug and Play concept and represents the keeps the injection syringe away from the inlet’s
ideal answer to those labs requiring highly reliable temperature influence, thus preserving low boiling
results for small batches of samples. compound sampling efficiency and leaving the inlet
A tool-free upgrade is available for the AI 1310 unobstructed. This option is valuable for manual
Autoinjector to extend sample capacity to the 105 sampling, for using an external device’s transfer
positions found on the AS 1310 Autosampler. Both line, or basic maintenance.
of these samplers feature removable trays and can Syringe installation is an easy task, and the
serve any type of GC injectors guaranteeing the utmost system automatically aligns without any need for
robustness while allowing true unattended operations. manual intervention. Just load the samples, and the
When dual column confirmation or double GC runs the analyses with no additional down time.
productivity is required, two AS 1310 Autosamplers Both of the AI/AS 1310 systems can be installed
are easily installed on the TRACE 1300 Series GC either on the front or on the rear GC injector just
system enabled by the AS 1310 Gemini Kit, allowing by swapping the sample container or sample tray’s
simultaneous injections on two ports, for higher side. The automatic recognition of the position does
analysis capacity of up to 210 samples. the rest. This completely independent, compact
sampling system can be placed on any Thermo
Scientific GC or GC/MS system in the lab.

8
Thermo Scientific
AI 1310 Autoinjector

Robotic Sample
Handling Solutions
For additional productivity requirements, including liquid, headspace or Solid
Phase Micro Extraction (SPME) injections or when unattended automated
sample and standard preparation is needed, the Thermo Scientific TriPlus
Robotic Sample Handling (RSH) autosampler offers the most innovative solution.
This modern sampling system is able to automatically switch between
injection modes during a single sequence to analyze, for example, liquid
samples, followed by headspace analyses, then SPME. The autosampler
enables the simultaneous automation of two adjacent Thermo Scientific
GC systems increasing laboratory productivity and executing standard or
sample dilutions, internal standard addition and derivatization.
These operations are possible using proprietary “custom cycles” and can
be run just before the injection, directly on the GC without wasting precious lab
bench space. Your results benefit from improved precision and reproducibility,
while your laboratory gains unique advantages from the TriPlus™ RSH
autosampler unattended operations and sample handling flexibility.

For volatile organic analysis, headspace, purge and trap, and thermal
desorbers can be connected through the injection port or into the oven, thus
allowing solid, liquid and gaseous sample quantitations with conventional or
mass spectrometry detectors.

Thermo Scientific
TriPlus RSH Autosampler

TriPlus RSH Autosampler installed onto a TRACE 1310 GC including the Automatic
Tool Changing station

9
 Chromeleon software
from samples to results quickly and easily,
boosting your productivity

The TRACE 1300 Series GC is fully controlled by the The Chromeleon 7.1 software package uses
Chromeleon™ 7.1 Chromatography Data System, the eWorkflows™ to accelerate chromatography analysis
Simply Intelligent™ chromatography package that minimizing operator tasks. An eWorkflow is a set of
streamlines your path from samples to results. rules that captures all of the unique aspects of a
Whether your needs are simple or complex – chromatography procedure and guides the operator
whether your scope is a single instrument, a global through a minimal number of choices needed to
enterprise, or anything in between – Chromeleon 7.1 execute the analysis. Using an eWorkflow, the operator
Operational Simplicity™ makes your job easy and simply selects an instrument, specifies the number
enjoyable. of samples and the starting vial position in the
The software’s intuitive, easy-to-navigate user autosampler, and begins the analysis. The software
interface guides you effectively towards your goals then runs the chromatography, processes the data,
with just a few clicks and allows for the quick training and produces final results and reports.
of new users. All detector signals are acquired digitally Chromeleon 7.1 software is the next-generation
without any analog/digital converter. Gas pressures, Chromatography Data System that adapts to your
injectors, oven temperatures, and additional instrument needs with its simplified user interface, innovative
status information are stored in the acquired data files. eWorkflows, powerful data mining and analysis tools,
and unrivaled reporting capabilities.

Additional
software platforms
The TRACE 1300 Series GC is also controlled by
other software platforms, including Thermo Scientific
Chrom-Card data system, Xcalibur data system, and
Chrom-Card and ChromQuest software. Chrom-Card™
software is a cost-effective software solution for rapid
instrument control and localized data acquisition and
handling. ChromQuest™ software is a multi-technique
chromatography software platform easily scalable from
a single system to an enterprise-wide network where
any instrument configured as part of the enterprise can
be monitored and controlled by any authenticated client.
The Xcalibur™ data system is the common platform
for all Thermo Scientific mass spectrometry systems.
It provides confident control of the TRACE 1300 Series
GC from method development to reporting and is used
to provide tools for generating and maintaining your
own spectral libraries.

10
Thermo Scientific
columns and consumables

The TRACE 1300 Series GC has been designed without

the need for customized consumables so it will be largely
compatible with most of the existing consumables that
injectors and detectors of other brands require. This allows
further operational cost savings without the need to buy
additional sets of dedicated consumables.
For today’s chromatographer, Thermo Scientific
TraceGOLD, TRACE and TracePLOT columns provide
excellent quality and performance, with guaranteed
reproducibility. Syringes, injection port liners, ferrules,
gas filters, o-rings, and septa are designed to complement
our innovative GC and GC/MS systems. This wide range
of consumables and accessories is designed to offer
application-focused solutions to customers in the petro-
chemical, food and beverage and environmental industries.
• Wide range of low-bleed, high temperature columns
• Consumables tested and certified on Thermo Scientific
instruments
•G
 as filters to improve column lifetimes and system stability
• Vials guaranteed for the Thermo Scientific
autosampler systems

11
 Thermo Scientific solutions
for your gas chromatography needs

Thermo Scientific ISQ Single Quadrupole GC-MS

The ISQ™ GC-MS system offers rugged and reliable performance and nonstop
productivity. The ISQ GC-MS features a new source design ideal for continuous
high-throughput operation. The vacuum interlock enables source removal
without venting the system, for unstoppable productivity.

Thermo Scientific ITQ Series GC-Ion Trap MS

The ITQ™ Series GC-Ion Trap MS offers outstanding full-scan electron ionization
sensitivity and upgradeability. From a small-footprint entry-level QA/QC instrument
to a fully-featured, research-grade system with advanced MSn functionality, the
ITQ Series GC-MS system offers a broad range of standard features along with an
impressive list of options.

Thermo Scientific TSQ Quantum XLS Series

Triple Quadrupole GC-MS/MS
The TSQ™ Quantum XLS Ultra is the new “Gold Standard” in GC-MS/MS.
Thermo Scientific HyperQuad technology delivers highly increased mass
resolving quadrupoles for ultra-selective SRM, with best-in-class sensitivity, and
allows unsurpassed analytical performance for the most difficult matrix challenges.

Thermo Scientific DFS High Resolution GC-MS

The DFS™ high resolution GC-MS system is the most advanced magnetic sector,
high-resolution mass spectrometer ever built for target compound analysis and for
solving general organic analytical questions. Its revolutionary ion optics and intuitive
user interface make operation of the DFS GC-MS easy and straightforward.

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Austria +43 1 333 50 34 0 +46 8 556 468 00 Middle East +43 1 333 50 34 0 UK +44 1442 233555
Belgium +32 53 73 42 41 France +33 1 60 92 48 00 Netherlands +31 76 579 55 55 USA +1 800 532 4752
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Application
Note: 52185
Maximizing Speed and Separations for Lowering
Drinking Water Detection Limits with GC/MS
Jessie Butler, Terry Jeffers, David Steiniger, Eric Phillips, Pat O’Brien, Thermo Fisher Scientific, Austin, TX, USA

Overview
Key Words Ever increasing drinking water regulations demonstrate
• ISQ Single the need for sensitivity and dynamic range of a GC-MS
Quadrupole system in order for the laboratory to provide usable data
GC-MS over the long term. The California Department of Public
Health published new notification levels for a list of
• Purge and Trap compounds including 1,2,3-trichloropropane (1,2,3-TCP)
• Single Ion at 0.005 µg/L.1 Uses for these compounds includes paint
Monitoring and varnish remover, cleaning and degreasing agent, a
(SIM) cleaning and maintenance solvent, and more currently
as a chemical intermediate. Its use as a pesticide was in
• US EPA formulations with dichloropropenes in the manufacture
Method 524.3 of D-D, a soil fumigant. EPA Method 524.3 allows for the
• Volatiles use of Single Ion Monitoring (SIM) to reach ppt levels.2
The determination of lower level MDLs in compliance
with the volatile notification levels listed below was run
on a Thermo Scientific ISQ GC-MS with an OI Analytical
Eclipse Purge-and-Trap Sample Concentrator. Purge and Trap Parameters
Sample Volume 5 mL
Experimental Sample Purge Temperature 40 °C
The Thermo Scientific Trace GC Ultra Multi-Channel Sample Mount 90 °C
Gas Chromatograph was configured for use with a Trap #10 (Tenax, silica gel, cms)
purge and trap by installing the purge and trap adapter. Purge Flow 40 mL/min
A Thermo Scientific TraceGOLD TG-VMS GC 20 meter × Purge Time 11 min
0.18 mm × 1.0 µm capillary column (PN 26080-4950) was Water Management Purge 110 °C, desorb 0 °C, transfer line 150 °C,
installed on the Split/Splitless inlet. The split vent was set Temperature valve oven 150 °C, bake 240 °C
to 30 mL/min and the column flow to a constant flow of Desorb Preheat 180 °C
1 mL/min. The instrument parameters are listed in Table 1 Desorb Temperature 190 °C
for the ISQ™ GC-MS and Table 2 for the Eclipse in the Desorb Time 0.5 min
analysis of the calibration curve and spiked samples. Bake Rinse Cycles Twice
Bake Cycle 210 °C for 12 min
Table 2: Parameters of the OI Analytical Eclipse for EPA Method 524.3
GC Parameters
Column TG-VMS GC 20 meter × 0.18 mm, 1.0 µm
Carrier 1 mL/min constant flow A 1 µL injection of 25 ng/µL BFB was analyzed based
Oven 40 °C, 4.5 min; 8 °C/min,100 °C, 0.0 min; on the 524.3 quality control criteria, before the calibration
25 °C/min, 230 °C, 2 min curve. BFB is no longer required every twelve hours as in
Split Inlet 200 °C previous versions of the method. BFB must pass the tune
Spit Flow 30 mL/min criteria before any new curve is established, or after any
ISQ MS Parameters
major maintenance is performed on the instrument.
Standards were prepared in organic free water. A 5 mL
Transferline 200 °C
aliquot was transferred from the 40 mL VOA vial into the
Ion Source 230 °C
fritted sparger automatically. The linearity was evaluated
Ionization Mode EI
from 0.020 to 20 µg/L and MDLs were determined by
Filament Delay 0.5 min
running seven replicates at 0.1 µg/L and 0.020 µg/L.
Chrom Filter Width 2.5 sec
Detector Gain 5 × 10 5
Table 1: GC and MS parameters for EPA Method 524.3
Results Conclusion
The ISQ GC-MS easily met the QC criteria for the The ISQ GC-MS easily met the quality control
method. Figure 1 demonstrates the results of the requirements and the lower detection limit requirements
BFB tune check criteria. for this drinking water method. The average MDL
The entire GC run time was 16 minutes as shown for drinking water was 0.035 µg/L. The following
by the TIC of the 2 µg/L standard in Figure 2. three compounds were run in SIM with MDLs:
The TIC of the scan segments are shown in dibromomethane – 2 ppt, 1,2,3-trichloropropane – 2 ppt,
Figure 3 along with the SIM ions that were utilized. and 1,2-dibromo-3-chloropropane – 4 ppt.
The instrumentation and the column provided
excellent chromatography which is demonstrated by References
the Gaussian-shaped peaks for the early eluting gases 1. Drinking Water Notification levels and Response Levels: An Overview,
seen in Figure 4. California Department of Public Health Drinking Water Program, Nov. 2010
http://www.cdph.ca.gov/certlic/drinkingwater/Pages/default.aspx
A comparison of the MDLs from EPA Method
2. EPA Method 524.3, Measurement of Purgeable Organic Compounds in Water
524.2 and those produced by EPA Method 524.3 are by Capillary Column by Gas Chromatography/Mass Spectrometry (GC-MS),
shown in Table 3. Revision 1.0 Jan. 2009

m/z Ion Abundance Criteria % Relative Abundance Pass/Fail

95 equals 100% of Base Peak 100.0 Pass
96 greater than or equal to 5% AND less than or equal to 9% of m/z 95 5.9 Pass
173 less than 2% of m/z 174 0.6 Pass
174 greater than 50% of m/z 95 52.9 Pass
175 greater than or equal to 5% AND less than or equal to 9% of m/z 174 7.0 Pass
176 greater than 95% AND less than 105% of m/z 174 95.5 Pass
177 greater than or equal to 5% AND less than or equal to 10% of m/z 176 5.4 Pass

Figure 1: BFB criteria report from Thermo Scientific EnviroLab Forms 3.1

Figure 2: TIC of a 2 µg/L standard in full scan

 524.2 524.3 524.2 524.3 524.2 524.3 524.2 524.3
Component Avg Conc (µg/L) Avg Conc (µg/L) Std Dev Std Dev % RSD % RSD MDL (µg/L) MDL (µg/L)
dichlorodifluoromethane 0.34 0.024 0.016 0.004 5 17 0.051 0.012
chloromethane 0.43 0.094 0.028 0.008 7 9 0.087 0.026
vinylchloride 0.40 0.020 0.020 0.004 5 20 0.062 0.012
bromomethane 0.45 0.091 0.047 0.001 11 16 0.148 0.045
chloroethane 0.45 0.067 0.077 0.012 17 17 0.242 0.036
trichlorofluoromethane 0.41 0.113 0.039 0.007 9 6 0.121 0.021
diethylether 0.45 0.429 0.031 0.021 7 5 0.097 0.068
1,1-dichloroethylene 0.42 0.116 0.049 0.013 12 11 0.154 0.041
carbon disulfide 0.43 0.400 0.032 0.063 7 16 0.099 0.198
allyl chloride 0.42 – 0.041 – 10 – 0.130 –
methylene chloride 0.46 – 0.032 – 7 – 0.101 –
acetone 4.789* – 0.399 – 8 – 1.255 –
trans-1,2-dichloroethylene 0.41 0.097 0.046 0.009 11 9 0.144 0.027
methyl tert-butyl ether 0.42 – 0.019 – 4 – 0.058 –
1,1-dichloroethane 0.42 0.021 0.027 0.002 7 8 0.085 0.005
acrylonitrile 0.41 0.118 0.036 0.023 9 20 0.115 0.073
cis-1,2-dichloroethylene 0.41 – 0.029 – 7 – 0.092 –
2,2-dichloropropane 0.38 – 0.026 – 7 – 0.08 –
bromochloromethane 0.39 0.090 0.042 0.007 11 8 0.131 0.021
chloroform 0.41 0.019 0.021 0.004 5 19 0.066 0.011
carbon tetrachloride 0.42 0.104 0.030 0.006 7 6 0.096 0.019
tetrahydrofuran 0.48 0.564 0.087 0.104 18 18 0.272 0.326
methyl acrylate 0.39 0.089 0.041 0.011 11 12 0.128 0.034
1,1,1-trichloroethane 0.43 0.024 0.035 0.004 8 18 0.112 0.014
1,1-dichloropropylene 0.39 0.091 0.030 0.006 8 6 0.093 0.019
2-butanone 0.52 – 0.099 – 19 – 0.310 –
1-chlorobutane 0.35 – 0.096 – 28 – 0.302 –
benzene 0.40 0.092 0.011 0.004 3 4 0.036 0.011
propionitrile 0.43 0.108 0.062 0.027 14 25 0.194 0.085
methacrylonitrile 0.42 0.095 0.053 0.026 13 27 0.166 0.082
1,2-dichloroethane 0.40 0.098 0.024 0.010 6 11 0.076 0.033
fluorobenzene (ISTD) – – – – 5 4 – –
trichloroethylene 0.41 0.095 0.025 0.004 6 5 0.077 0.014
dibromomethane 0.39 0.023 0.050 0.001 13 3 0.156 0.002
1,2-dichloropropane 0.40 0.086 0.026 0.007 7 8 0.082 0.021
bromodichloromethane 0.38 0.097 0.024 0.005 6 5 0.075 0.016
methyl methacrylate 0.38 0.100 0.032 0.008 8 9 0.099 0.025
cis-1,3-dichloropropene 0.38 0.094 0.024 0.008 6 9 0.077 0.026
toluene 0.38 0.017 0.008 0.003 2 17 0.025 0.010
chloroacetonitrile 0.31 0.025 0.086 0.005 28 20 0.270 0.015
1,1-dichloro-2-propanone 0.35 0.077 0.082 0.014 23 18 0.257 0.044
tetrachloroethylene 0.39 0.023 0.079 0.004 20 15 0.248 0.011
2-nitropropane 0.41 0.106 0.035 0.010 8 9 0.109 0.031
4-methyl-2-pentanone 0.41 0.098 0.031 0.009 8 9 0.098 0.029
trans-1,3-dichloropropene 0.38 0.092 0.021 0.008 5 9 0.065 0.025
1,1,2-trichloroethane 0.40 0.098 0.022 0.010 5 10 0.069 0.030
ethyl methacrylate 0.37 0.578 0.022 0.097 6 17 0.069 0.304
dibromochloromethane 0.37 0.019 0.027 0.003 7 16 0.084 0.010
1,3-dichloropropane 0.38 0.016 0.019 0.003 5 18 0.059 0.009
1,2-dibromoethane 0.38 0.095 0.019 0.009 5 10 0.061 0.030
2-hexanone 0.47 0.100 0.061 0.010 13 10 0.193 0.030
chlorobenzene 0.39 0.022 0.016 0.003 4 14 0.052 0.100
ethylbenzene 0.40 0.020 0.019 0.004 5 22 0.059 0.014
1,1,1,2-tetrachloroethane 0.42 0.092 0.038 0.005 9 6 0.119 0.016
m&p-xylene 0.79 0.021 0.041 0.002 5 10 0.130 0.006
o-xylene 0.40 0.021 0.009 0.002 2 12 0.030 0.008
bromoform 0.37 0.019 0.047 0.003 13 17 0.147 0.010
styrene 0.39 0.092 0.017 0.006 5 6 0.055 0.018
isopropylbenzene 0.39 0.017 0.018 0.001 5 9 0.057 0.005
4-bromofluorobenzene (surr) 5.27 – 0.234 – 4 – 0.735 –
bromobenzene 0.40 0.081 0.029 0.011 7 13 0.092 0.034
n-propylbenzene 0.39 0.022 0.020 0.003 5 13 0.064 0.009
1,1,2,2-tetrachloroethane 0.38 0.085 0.022 0.010 6 11 0.069 0.030
2-chlorotoluene 0.40 0.016 0.022 0.002 6 13 0.069 0.006
1,2,3-trichloropropane 0.41 0.020 0.017 0.001 4 4 0.053 0.002
1,3,5-trimethylbenzene 0.39 0.019 0.018 0.004 5 20 0.057 0.012
trans-1,4-dichloro-2-butene 0.40 0.094 0.025 0.011 6 12 0.078 0.036
4-chlorotoluene 0.36 0.017 0.014 0.002 4 12 0.045 0.007
p-isopropyltoluene 0.36 0.080 0.014 0.005 4 6 0.045 0.016
tert-butylbenzene 0.36 0.089 0.012 0.008 3 9 0.039 0.024
1,2,4-trimethylbenzene 0.40 0.023 0.017 0.002 4 8 0.053 0.006
pentachloroethane 0.40 0.337 0.051 0.013 13 4 0.160 0.041
sec-butylbenzene 0.39 0.019 0.017 0.002 4 8 0.053 0.005
1,3-dichlorobenzene 0.44 0.017 0.013 0.003 3 18 0.042 0.010
1,4-dichlorobenzene 0.52 0.075 0.019 0.005 4 7 0.061 0.017
n-butylbenzene 0.39 0.026 0.017 0.003 4 12 0.054 0.010
hexachloroethane 0.37 0.511 0.019 0.045 5 9 0.059 0.142
1,2-dichlorobenzene 0.42 0.023 0.025 0.003 6 14 0.078 0.010
1,2-dichlorobenzene-d4 (surr) 5.44 – 0.435 – 8 – 1.369 –
1,2-dibromo-3-chloropropane 0.38 0.026 0.046 0.001 12 5 0.144 0.004
nitrobenzene 0.02 – 0.046 – 19 – 0.144 –
hexachlorobutadiene 0.39 0.091 0.039 0.006 10 6 0.122 0.018
1,2,4-trichlorobenzene 0.42 0.018 0.023 0.002 5 12 0.071 0.006
naphthalene 0.38 0.020 0.012 0.003 3 13 0.038 0.008
1,2,3-trichlorobenzene 0.42 0.020 0.025 0.003 6 15 0.079 0.009
Table 3: Comparison of the MDL results from EPA Method 524.2 and EPA Method 524.3 Average 12 0.140 0.035
*lab air contamination
Figure 3: TICs showing In addition to these
the full scan and SIM offices, Thermo Fisher
segments used to analyze Scientific maintains
for the compounds in FS 47 to 300 a network of represen-
EPA Method 524.3
tative organizations
throughout the world.
FS 35 to 300

SIM 75,110 1,2,3–trichloropropane Africa-Other

+27 11 570 1840
Australia
+61 3 9757 4300
Austria
SIM 75,155,157 1,2-dibromo–3–chloropropane +43 1 333 50 34 0
Belgium
+32 53 73 42 41
Canada
+1 800 530 8447
SIM 93,95,174 dibromomethane China
+86 10 8419 3588
Denmark
+45 70 23 62 60
Europe-Other
+43 1 333 50 34 0
Finland / Norway /
Sweden
Figure 4: TIC of the +46 8 556 468 00
early eluting gases
France
from EPA Method 524.3 +33 1 60 92 48 00
dichlorodifluoromethane
Germany
+49 6103 408 1014
India
cloromethane +91 22 6742 9434
Italy
+39 02 950 591
vinychloride Japan
+81 45 453 9100
Latin America
+1 561 688 8700
bromomethane
Middle East
+43 1 333 50 34 0
Netherlands
cloroethane +31 76 579 55 55
New Zealand
+64 9 980 6700
trichlorofluoromethane Russia/CIS
+43 1 333 50 34 0
South Africa
+27 11 570 1840
Spain
+34 914 845 965
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+41 61 716 77 00
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+44 1442 233555
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+1 800 532 4752

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Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Austin, TX USA is ISO Certified.

AN52185_E 08/11M

Part of Thermo Fisher Scientific

Technical
Note: 10239
Multi-residue Pesticide Analysis in Rice by a
Modified QuEChERS Extraction and Ion Trap
GC/MSn Analysis
David Steiniger, Jessie Butler, Eric Phillips, Thermo Fisher Scientific, Austin, TX, USA

Introduction
Key Words Recently formulated pesticides are quite different in
• ITQ 700 their physical properties from their predecessors such as
4,4'-DDT. Most recently formulated pesticides are smaller
• Food Safety in molecular weight and designed to break down rapidly
• GC/MSn in the environment. Therefore, to successfully identify and
quantify these compounds in foods, careful consideration
• Pesticide Analysis must be placed on the sample preparation for extraction
• QuEChERS and the instrument parameters for analysis. This study
covers preparation of extracts and optimization of
analytical parameters for injection, separation, and detection.
The determination of pesticides in fruits and vegetables all of the various functional groups and different physical
has been simplified by a QuEChERS (Quick, Easy, Cheap, properties of most pesticides. A surge splitless injection
Effective, Rugged and Safe) extraction method, published was made into a Thermo Scientific TRACE TR-527
recently as AOAC Method 2007.01.1 The sample preparation 35% diphenyl/65% dimethyl polysiloxane column,
is simplified by using a single-step buffered acetonitrile (0.25 mm x 30 meter, and a film thickness of 0.25 µm
(MeCN) extraction and liquid-liquid partitioning from water with a 5 m guard column).
in the sample by salting out with sodium acetate and
magnesium sulfate (MgSO4).1 Analysis was performed by Item Descriptions
gas chromatography/tandem mass spectrometry (GC/MSn) TRACE TR-527 35% diphenyl/65% dimethyl polysiloxane column,
on the Thermo Scientific ITQ 700 GC-ion trap mass 0.25 mm x 30 meter, 0.25 µm w/5 m guard column
spectrometer. 5 mm ID x 105 mm L liner (pk of 5)
The study determined the linear ranges, quantitation 10 µL syringe
limits and detection limits for a list of pesticides that are Septa (pk of 50)
commonly used on rice crops. A splitless injection of Liner graphite seal (pk of 10)
33 pesticides was made with detection in electron ionization Ion volume - EI open
(EI) MS/MS. Since the extracts are prepared in MeCN, a
Ion volume holder
solvent exchange was made to hexane/acetone (9:1) prior
Graphite ferrule 0.1-0.25 (pk of 10)
to conventional splitless injection.2 Once the calibration
Ferrule 0.4 mm ID 1/16 G/V (pk of 10)
curve was constructed, multiple matrix spikes were analyzed
at a levels of 160, 320, or 480 ng/g (ppb) and low level Blank vespel ferrule for MS interface (pk of 10)
spikes at 16, 32, 40, 80, or 120 ng/g (ppb) to verify the 2 mL amber glass vial, silanized glass, with write-on patch (100/pk)
precision and accuracy of the analytical method. Blue cap with ivory PTFE/red rubber seal (100/pk)
Acetonitrile analytical grade (4L)
Experimental Conditions Hexane GC Resolv* (4L)
The sample preparation involves careful homogenization Acetone GC Resolv* (4L)
of the sample. Extraction solvents must be buffered and Organic bottle top dispenser
the powdered reagents measured at appropriate amounts HPLC grade glacial acetic acid
for the size of sample prepared. Careful addition of the 50 mL Nalgene FEP centrifuge tubes (pk of 2)
reagents must be taken since some reagents cause an Clean up tube: 15 mL tubes ENVIRO 900 mg MgSO4, 300 mg PSA 150 mg C18
exothermic reaction when mixed with water, which can (pk of 50)
adversely affect the recoveries of target compounds. The 50 mL PP Tubes 6 g MgSO4, 1.5 g CH3CHOONa (anhydrous) (pk of 250)
recommended consumables required for sample preparation Clean up tube: 2 mL tubes 150 mg MgSO4, 50 mg PSA (pk of 100)
and analysis were rigorously tested (Table 1). A list of the
Table 1: Consumables for QuEChERS Sample Prep and Analysis
pesticides to be studied was created that would address
Sample Extraction and Clean Up solutions were spiked into this MeCN layer for the
The QuEChERS sample prep procedure consists of the steps method validation (MVD) and method detection limit
shown in Figure 1. There are three parts: the extraction, (MDL) samples.
the clean-up, and solvent exchange. The solvent exchange The tube was capped and vortexed for 30 seconds.
provides a final solvent that is more amenable to splitless The cap was removed and the powder reagents were poured
injection. Care must be taken to adequately and thoroughly slowly into the MeCN layer. The cap was tightened securely
homogenize the sample. When analyzing grains such as on the 50 mL extraction tube, and was vortexed for 30
rice, water must be added during the homogenization step seconds until all of the powder reagents were mixed with the
and taken into consideration in the final calculations of liquid layers. The tube was placed on a mechanical shaker
spikes and standards. To perform liquid-liquid extraction for 5 minutes and then centrifuged for 5 minutes at 3000
requires water. Also, the water helps mix the rice during rpm. Next, 11 mL of the top MeCN layer was removed and
the homogenization step. transferred to a 15 mL clean-up tube. This tube was capped
A thoroughly homogenized 15 g sample of rice was and vortexed for 30 seconds and centrifuged for 5 minutes
weighed into this extraction tube. Then 15 mL of 1% glacial at 3000 rpm. A 5 mL aliquot of the top layer was transferred
acetic acid MeCN extraction solvent was poured into the into a clean test tube for solvent exchange.
tube on top of the sample. The surrogate and the pesticide

Figure 1: Flow diagram of QuEChERS sample preparation procedure

Page 2 of 8
Solvent Exchange
The 5 mL aliquot of cleaned up extract was blown down to
dryness with a gentle stream of nitrogen at 40 °C in about
one hour. Care was taken to remove the tube immediately
when dried. A 900 µL aliquot of hexane/acetone (9:1) was
added and 100 µL of the internal standard, d10-parathion,
was spiked into the organic solution. The tube was capped
and vortexed for 15 seconds. The 1 mL of extract was
transferred to a 2 mL clean-up tube, capped tightly, and
vortexed for 30 seconds. After centrifuging for 5 minutes
at 3000 rpm, 200 µL of the clear extract was transferred
to an autosampler vial with a small glass insert for analysis
on the ITQ 700 system. The individual calibration levels
were spiked into each extract for the calibration curve in
matrix before the final cleanup step (Figure 1).

Injection
The ITQ 700 is paired with the Thermo Scientific
FOCUS GC gas chromatograph, which is a single-channel
GC with a standard split/splitless (SSL) injection port.
The SSL inlet temperature was set to 250 °C. A 5 mm i.d.
splitless liner with a volume of 1.6 mL was selected for the
surged pressure injection. For the surge splitless injection,
the inlet pressure was held at an elevated pressure of
250 kPa for the 0.5 minute injection (splitless) time. This
technique reduces the vapor cloud of a 2 µL injection
from 0.37 mL to 0.19 mL. At an elevated injection flow
rate of 4.6 mL/ min., the liner was swept several times
during injection. The target compounds moved through
the inlet rapidly thus reducing the time to interact with the
inside walls of the liner. This minimized the amount of
breakdown of the more fragile pesticides.
A Performance Solution consisting of endrin and Figure 2: Endrin and DDT breakdown analysis, showing < 5% breakdown
4,4'-DDT was analyzed as a daily check to determine system
activity. The analysis of endrin, DDT, and their breakdown
Separation
products as part of daily quality control can alert the analyst
that the system has developed active sites and maintenance Chromatographic separation was achieved by using a
is needed. Without performing a breakdown analysis the 35% diphenyl/65% dimethyl polysiloxane column
laboratory may need to continually maintain the equipment (0.25 mm x 30 meter, and a film thickness of 0.25 µm with
and replace consumables, even when it may not be needed. a 5m guard column). This column was chosen to provide
Monitoring breakdown can decrease the cost of running sufficient resolution of the more polar compounds. The
the analysis and save significant amounts of time. Endrin oven was programmed as follows:
breakdown is determined by adding up the response for Initial Temp: 40 °C, 1.5 min., 25 °C/min. to 150 °C,
the two breakdown products: endrin aldehyde and endrin 0.0 min, 5 °C/min. to 200 °C, 7.5 min., 25 °C/min. to
ketone and dividing by the total response for the breakdown 290 °C with a final hold time of 12 min. and a constant
products and endrin in percent. The breakdown products column flow rate of 1 mL/min.
of DDT are DDE and DDD and are calculated similarly. The entire set of instrument parameters is listed in Table 2.
The breakdown check results showed < 5 % breakdown
for both compounds on a daily basis (Figure 2). For
routine use the liner would be changed when the
breakdown of either compound reaches > 20%. The
injection port liner tested showed very good results over a
long period of time without the need for maintenance.

Page 3 of 8
AS 3000 II Autosampler
Sample Volume 2 µL
Plunger Strokes 5
Viscous Sample No
Sampling Depth in Vial Bottom
Injection Depth Standard
Pre-inj Dwell Time 0
Post-inject Dwell Time 0
Pre-inject Solvent Wash Vial Position A+B
Pre-inject Solvent Wash Cycles 3
Sample Rinses 3
Post-inject Solvent A
Post-inject Solvent Cycles 3

FOCUS GC
Column TRACE TR-527 w/ guard column
0.25 mm x 30 meter, 0.25 µm
Column Constant Flow 1 mL/min.
Oven Program 40°, 1.5 min., 25°/min.; 150°,
0.0 min., 5°/min., 200°, 7.5 min.,
25°/min., 290°, 12 min.
S/SL Temperature 250 °C
S/SL Mode Splitless with Surge Pressure
Surge Pressure 250 kPa
Inject Time 0.5 min.
Split Flow 50 mL/min.
Figure 3: MS/MS scan of 160 ng/g in rice matrix
Transferline Temperature 290 °C

ITQ Mass Spectrometer

Damping Gas Flow 2
Source Temperature 250 °C
Ion Volume EI
Emission Current 250 µA
Detector Gain 3 (1367 V)
Lens 1 -25V
Lens 3 -25V
Gate Lens On -100
Gate Lens Off 100
Electron Lens On 15V
Electron Lens Off 85
Electron Energy -70eV
Trap Offset -10
Waveforms Off

Table 2: Selected instrument parameters for the ITQ 700 system

Figure 4: Full scan chromatogram of 160 ng/g of pesticides in rice

Page 4 of 8
Detection energy to further fragment and are then scanned. This
The detection of the pesticides was performed using the process provides the product ion spectrum. This is done
ITQ 700 ion trap mass spectrometer with optional MSn by setting up a stable field for the pesticide precursor ion.
mode. This scanning mode offers enhanced selectivity Once the precursor ion is isolated from the matrix ions,
over either full scan or selected ion monitoring (SIM). In Collision Induced Dissociation (CID) energy is applied to
SIM at the elution time of each pesticide, the ratio of the fragment it into its respective product ions. Finally these
intensity of matrix ions increases exponentially versus that unique product ions are scanned out to generate the
of the pesticide ions as the concentration of the pesticide product ion spectrum. Because of the elimination of
approaches the detection limit, decreasing the accuracy at matrix interferences, this process produces more accurate
lower levels. The ITQ 700 operated in the MSn mode results at the lower levels. The MSn parameters for the ITQ
performs tandem MS functions by injecting ions into the 700 are listed in Table 3. Figures 3 and 4 show a
ion trap and destabilizing matrix ions, isolating only the comparison between a Full Scan and MSn TIC.
pesticide ion. These pesticide ions are given sufficient

RT Precursor Width Collision Max. Excitation Range Product Ion Qualifiers

Compound (Minutes) (m/z) (amu) Energy (Volts) Energy (q) (m/z) (m/z) (m/z)
Dichlorvos 8.49 185 1 3 0.225 53-195 93 131, 109, 170, 63
Molinate 13.04 126 2 3 0.3 45-136 98 83, 55, 82, 81
Trifluralin 13.33 264 2 3 0.225 150-274 206 188, 160, 171, 177
Tebuthiuron 13.35 156 1 3 0.225 52-166 89 74, 62, 87, 125
Propachlor 14.47 120 2 4 0.3 67-130 92 91, 103, 77, 93
Phorate 15.73 231 2 3 0.225 165-241 203 175, 185
Propyzamide (Pronamide) 16.79 173 2 3 0.225 135-183 145 146
Diazanon 17.51 179 1 4 0.225 86-189 137 164, 138, 161, 96
Gamma BHC (Lindane) 17.89 219 4 3 0.225 171-229 181 183, 182, 184
b-BHC 18.34 181 2 3 0.225 135-191 145 146
Heptachlor 19.97 272 2 3 0.225 225-282 237 235, 268
Chlorothalanil 20.09 266 1 5 0.225 193-276 231 203, 205, 233, 229
d-BHC 20.35 181 2 3 0.225 135-191 145 146
Aldrin 22.12 263 1 5 0.225 217-273 229 228, 227, 230, 249
Metalaxyl 22.79 160 1 3 0.225 120-170 145 130, 132
Terbutryn 23.29 185 3 3 0.225 142-195 170 157, 152
Metolachlor 23.57 162 2 4 0.225 110-172 133 134, 120, 144, 147
Metribuzin 23.68 198 2 4 0.225 93-208 151 103, 110, 153, 128
Thiobencarb 24.09 100 3 5 0.45 52-110 72 71, 73, 99, 62
Sevin (Carbaryl) 24.12 144 1 3 0.3 105-154 116 115
Dursban (Chlorpyrifos) 24.14 314 5 3 0.225 248-324 286 258, 287, 288, 285
Malathion 24.31 173 3 4 0.225 125-183 136 145, 137, 138, 135
Parathion-d10 24.34 301 2 3 0.225 105-311 269 147, 115, 148, 271
Methiocarb 24.4 168 1 3 0.225 99-178 153 109
Terbufos (Sulfone) 26.01 199 7 3 0.225 133-209 171 172, 153, 143, 173
cis-Chlordane 26.06 373 5 4 0.225 254-383 301 337, 299, 264, 335
Dieldrin 26.67 277 3 4 0.225 197-287 241 242, 239, 207, 217
Endrin 27.25 263 1 5 0.225 183-273 228 230, 226, 229, 193
Endosulfan B 27.56 195 2 4 0.225 148-205 159 160, 158
p,p-DDT 27.97 235 4 4 0.225 156-245 166 200,199,201, 202
Bifenthrin 28.3 181 7 4 0.225 143-191 166 165, 167, 178, 153
Methoxychlor 29.44 227 7 5 0.225 175-237 212 195, 196, 185, 197
trans-Permethrin 31.2 183 3 4 0.225 143-193 168 165, 155, 153, 181
Fluridone 37.32 328 2 6 0.225 249-338 259 288, 313, 308, 268
Table 3: MS/MS parameters for pesticide analysis

Page 5 of 8
Compound (R2) Compound (R2) Results and Discussion
Dichlorvos 0.9999 Metribuzin 1.0000
Molinate 0.9997 Thiobencarb 1.0000 Linearity
Trifluralin 0.9983 Sevin (Carbaryl) 0.9982 The calibration curve was spiked into the rice matrix. Levels
Tebuthiuron 0.9995 Dursban (Chlorpyrifos) 0.9979 ranged from 1 ng/g to 1200 ng/g, depending on the compound
Propachlor 0.9995 Malathion 0.9996 and its MRL in rice. The linearity for all compounds was
Phorate 0.9996 Methiocarb 0.9999 R2 > 0.995. The results of the linearity are shown in Table 4.
Propyzamide (Pronamide) 0.9983 Terbufos Sulfone 0.9997 Figures 5 and 6 are two examples of calibration curves.
Diazanon 1.0000 cis-Chlordane 0.9995
Gamma BHC 0.9999 Dieldrin 0.9971 Limits of Detection and Quantitation
b-BHC 0.9989 Endrin 0.9989 The actual limit of detection (LOD) and limit of quantitation
Heptachlor 0.9997 Endosulfan B 0.9998 (LOQ) were determined by preparing matrix spikes at a
Chlorothalinil 0.9997 p,p-DDT 0.9983 level near or below the MRL. Concentrations of 16, 32, 40,
d-BHC 0.9988 Bifenthrin 1.0000 80, and 120 ng/g were analyzed in seven matrix samples,
Aldrin 0.9988 Methoxychlor 0.9986 and the LOD and LOQ were calculated from these results
Metalaxyl 0.9992 trans-Permethrin 0.9998 by multiplying the standard deviation of the calculated
Terbutryn 0.9993 amounts by 3 and 10 respectively. The results are shown
Metolachlor 0.9996 Average 0.9993 in Table 5.
Table 4: Calibration curve results

ng/g (ppb) ng/g (ppb) Lowest MRL ng/g (ppb) ng/g (ppb) Lowest MRL
Specified Amount Calculated Amount %D Japan Specified Amount Calculated Amount %D Japan
16 19 -20.0 8 9 -12.5
32 31 3.1 16 17 -3.8 20
80 79 1.0 40 38 6.3
160 154 3.6 200 80 81 -0.9
320 325 -1.6 160 160 -0.1
1280 1280 0.0
Figure 6: MS/MS calibration curve for tebuthiuron, from 8 to 160 ppb
Figure 5: MS/MS Calibration curve for dichlorvos, from 16 to 1280 ppb
 Japan1 US-EPA2 EU3 EU3 WHO4
Ave. Conc. LOD LOQ MRL MRL MRL MRL
Component (ng/g) Std. Dev. % RSD (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) LOD3 (ng/g)
Dichlorvos 60 3.6 6.1 11 36 200 2000
Molinate 40 1.1 2.9 4 11 100
Trifluralin 41 2.6 6.3 8 26 50
Tebuthiuron 33 2.8 8.8 9 28 20
Propachlor 34 2.6 7.5 8 26 50
Phorate 45 1.5 3.3 5 15 50 50 50
Propyzamide (Pronamide) 31 2.6 8.2 8 26 20 20 20
Diazanon 86 2.4 2.8 8 24 100 20 20
Gamma BHC 85 1.6 1.8 5 16 300 10 10
b-BHC 48 1.1 2.4 4 11 200 10 10
Heptachlor 14 2.0 14.4 6 20 20 10
Chlorothalinil 27 1.4 5.4 4 14 100 10
d-BHC 43 1.1 2.6 3 11 200 10 10
Aldrin 44 1.1 2.5 3 11 ND
Metalaxyl 30 4.7 15.4 15 47 100 50 50
Terbutryn 51 1.9 3.7 6 19 100
Metolachlor 38 1.4 3.7 4 14 100 100
Metribuzin 46 3.7 8.1 12 37 50
Thiobencarb 44 2.2 5.1 7 22 200 200
Sevin (Carbaryl) 32 3.7 11.6 12 37 1000 5000
Dursban (Chlorpyrifos) 113 2.9 2.6 9 29 100 50 50 500
Malathion 31 4.4 14.3 14 44 100 8000
Methiocarb 26 2.4 9.2 8 24 50
Terbufos Sulfone 36 1.4 3.8 4 14 5
cis-Chlordane 18 1.6 9.1 5 16 20
Dieldrin 45 4.4 9.8 14 44 ND
Endrin 41 6.4 15.7 20 64 ND
Endosulfan B 39 5.3 13.7 17 53 100
p,p-DDT 44 2.3 5.2 7 23 200
Bifenthrin 34 2.0 5.7 6 20 1000 50 50
Methoxychlor 44 3.4 7.7 11 34 2000 2000
trans-Permethrin 33 4.6 14.2 14 46 2000 50 50
Fluridone 30 8.0 26.3 25 80 100
Average 7.9 9 29

1. CODEX alimentarius (www.codexalimentarius.net/mrls/pesticides/jsp/pest-q-e.jsp)

2. Japanese Food Chemical Research Foundation (www.m5.ws001.squarestart.ne.jp/foundation/search.html)
3. Informal coordination of MRLs established in Directives 76/895/EEC, 86/362/EEC, 86/363/EEC, and 90/642/EEC (5058/VI/98
4. 40CFR180 (www.access.gpo.gov/nara/cfr/waisidx_02/40cfr180_02.html)

Table 5: Comparison of LODs and LOQs vs MRLs

Method Validation Results user to identify, confirm, and quantify in one analytical
The method validation (MVD) calculations were performed run. The injector demonstrated low endrin and DDT
using five matrix samples spiked at concentrations of 160, breakdown (< 5%) on a daily basis, proving that the
320, or 480 ng/g per pesticide. Samples had an average of system can analyze active compounds without the need for
98% recovery with an average % RSD of 5.9%. MVD continual, expensive, and time-consuming maintenance.
results are shown in Table 6. Calibration curves for the pesticides studied met a linear
least squares calibration with a correlation coefficient of
Conclusions R2 > 0.995 for all compounds. The Method Validation
Study generated an average % RSD of 5.9% for five replicate
The Thermo Scientific ITQ 700 GC-ion trap MS was
analyses at 160, 320, or 480 ng/g and a calculated average
thoroughly evaluated and showed excellent accuracy at
LOD of 9 ng/g in rice based on 7 replicate analyses of
low concentrations of 33 pesticide residues analyzed in
16, 32, 40, 80, and 160 ng/g.
rice. Using the instrument’s MSn functionality allows the

Page 7 of 8
Component Average Conc Theoretical Conc % Difference % RSD % Recovery In addition to these
Dichlorvos 272 320 -15.1 2.9 84.9 offices, Thermo Fisher
Molinate 158 160 -1.0 2.6 99.0 Scientific maintains
Trifluralin 207 160 29.3 7.5 129.3 a network of represen-
Tebuthiuron 149 160 -7.1 10.5 92.9 tative organizations
Propachlor 170 160 6.4 4.0 106.4 throughout the world.
Phorate 175 160 9.1 4.1 109.1
Propyzamide (Pronamide) 374 320 16.9 4.6 116.9
Diazanon 352 320 10.1 5.3 110.1
Gamma BHC 330 320 3.0 4.2 103.0
b-BHC 173 160 8.4 4.8 108.4
Heptachlor 178 160 11.1 6.6 111.1 Africa-Other
+27 11 570 1840
Chlorothalinil 280 320 -12.6 5.7 87.4
Australia
d-BHC 152 160 -5.0 4.6 95.0 +61 2 8844 9500
Aldrin 154 160 -3.5 4.8 96.5 Austria
Metalaxyl 177 160 10.4 6.9 110.4 +43 1 333 50 34 0
Terbutryn 168 160 5.1 4.4 105.1 Belgium
+32 2 482 30 30
Metalochlor 167 160 4.7 4.2 104.7
Canada
Metribuzin 172 160 7.5 5.7 107.5 +1 800 530 8447
Thiobencarb 154 160 -4.0 4.2 96.0 China
Sevin (Carbaryl) 149 160 -6.7 6.4 93.3 +86 10 8419 3588
Dursban (Chlorpyrifos) 487 480 1.5 4.0 101.5 Denmark
+45 70 23 62 60
Malathion 155 160 -2.9 7.0 97.1
Europe-Other
Methiocarb 119 160 -25.5 4.6 74.6 +43 1 333 50 34 0
Terbufos Sulfone 153 160 -4.5 4.5 95.5 Finland / Norway /
cis-Chlordane 153 160 -4.3 6.3 95.7 Sweden
+46 8 556 468 00
Dieldrin 172 160 7.7 5.7 107.7
France
Endrin 161 160 0.5 5.8 100.5 +33 1 60 92 48 00
Endosulfan B 157 160 -2.1 11.2 97.9 Germany
p,p-DDT 133 160 -16.9 8.2 83.1 +49 6103 408 1014
Bifenthrin 150 160 -6.5 8.2 93.5 India
+91 22 6742 9434
TPP (Surrogate) 161 200 -19.7 5.5 80.3
Italy
Methoxychlor 139 160 -13.4 6.8 86.6 +39 02 950 591
trans-Permethrin 271 320 -15.3 8.9 84.7 Japan
Fluridone 115 160 -28.4 10.3 71.6 +81 45 453 9100
Latin America
Average 5.9 98.1 +1 608 276 5659
Table 6: Results of method validation study Middle East
+43 1 333 50 34 0
Netherlands
+31 76 579 55 55
References South Africa
1. AOAC Official Method 2007.01 Pesticide Residues in Foods by Acetonitrile +27 11 570 1840
Extraction and Partitioning with Magnesium Sulfate, S. Lehotay, Journal Spain
of AOAC International Vol. 90, No. 2, (2007) 485–520 +34 914 845 965
2. Rapid Method for the Determination of 180 Pesticide Residues in Foods by Switzerland
Gas Chromatography/Mass Spectrometry and Flame Photometric Detection, +41 61 716 77 00
M. Okihashi, Journal Pesticide Science, 304 (4), (2005) 368–377 UK
+44 1442 233555
USA
+1 800 532 4752

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 Thermo Scientific TSQ 8000
Triple Quadrupole GC-MS/MS

uncompromised
MS/MS simplicity
Unstoppable productivity
 your next
competitive advantage...
The role of the analytical laboratory has changed in recent times as analytical data has
become more commoditized. At the same time, competitive pressures have increased the
demand for analytical cost savings while maintaining compliance with regulatory guidelines
by producing high quality data with confidence on time, every time.

The Thermo Scientific TSQ 8000 triple quadrupole GC-MS/MS system is designed
to provide the modern laboratory with unstoppable productivity and uncompromised MS/MS
simplicity without sacrificing the high level performance you come to expect from Thermo
Scientific mass spectrometers.

The TSQ™ 8000 GC-MS/MS is a system built for laboratories seeking a competitive edge
to accelerate the turnaround time to get from raw sample to final report while reducing
overhead costs. This GC-MS/MS system delivers high performance selected reaction
monitoring (SRM) through the use of software that alleviates the usual concerns associated
with the adoption of triple quadrupole GC-MS/MS technology.

Easy Operation, Brilliant Results 

• Uncompromised MS/MS simplicity
• Unstoppable productivity
• Automated SRM development
• Maximum uptime with vent-free source removal

2 3
 unstoppable productivity More Robustness, More Conforming Results
The TSQ 8000 GC-MS/MS system combines high

powerful MS/MS results, routinely sensitivity with system robustness to ensure that your
results stay within regulatory limits. Less maintenance
intervention is needed, and the instrument can remain
The Thermo Scientific ISQ single quadrupole GC-MS has a strong reputation in allowing laboratories in production to report results for longer periods.
to tackle the demands of producing results on time, every time. Building upon the success of this
proven technology, the TSQ 8000 triple quadrupole GC-MS enables laboratories to take the next
step in productivity through the power of selected reaction monitoring (SRM).
The compound selectivity advantage that SRM brings to GC-MS technology allows:

• Simple sample preparation • Consolidation of methods into a single analysis

• Lower detection limits • Faster data processing
• Faster analytical run times • High confidence in compound identification and quantitation

Precision at 10 pg/µL before and after 1,400 vegetable matrix injections

More Compounds, Less Time
The speed, sensitivity and selectivity delivered by the TSQ 8000
GC-MS/MS system allows more compounds to be run in shorter
analysis times.

The structurally-selective nature of SRM reduces the likelihood

of interference from matrix and other target compounds,
allowing for the rapid analysis of more compounds in a single
run. SRM has the ability to deliver clean peak detection along Less Method Maintenance, More Uptime
with automatic peak integration through Thermo Scientific Retention-Timed SRM (t-SRM)
TraceFinder software, and this solution enables the user to spend
As more compounds are added to a single run, more complexity arises with the management of acquisition
less time adjusting peak integrations while realizing faster
windows. The TSQ 8000 GC-MS/MS system dramatically reduces this complexity by automatically
result-reporting.
Multiple single quadrupole pesticides methods consolidated into a single optimizing the targeting of a particular compound. Simply enter the retention time, and the time required
40 minute 350 pesticides (700 transitions) run to capture the peak and t-SRM takes care of the rest. This ensures that compound detection is optimized
for maximum sensitivity, and more compounds can be added to the method without compromising the
excellent sensitivity of each individual analyte. The mass spectrometer is not consuming valuable resources
Less Sample Prep, Faster Results scanning for compounds at the times when those compounds are not eluting.
The selectivity of the TSQ 8000 GC-MS/MS
system provided through SRM enables the ability
to cut through heavy matrix background for clean Timed SRM automatically optimizes
peak detection and quantitation with confidence, methods for maximum sensitivity
in contrast to traditional single quadrupole GC-MS
analysis. Therefore, costly sample preparation
procedures can be optimized to deliver an
efficient laboratory workflow.

Selectivity of SRM (bottom) vs

SIM (Top) for malathion at 5 ppb
in soil extract

4 5
 MS/MS simplicity
The advantages of selected reaction monitoring (SRM) and multiple reaction
monitoring (MRM) analysis often come with challenges, especially with respect to
adoption of new technology in the laboratory. These challenges are due to the
complexity of mass spectrometer set-up and optimization and management of AutoSRM Workflow
complex methods. Laboratories that strive to realize the benefits of MS/MS must
Step 1: Selects your precursor ions from full scan
overcome some barriers in the tuning, set-up and optimization to reduce the time
required to achieve a smooth operating routine.
Your Starting Point
The TSQ 8000 GC-MS/MS was built to guarantee an easy start-up. Whether you
are managing retention times, starting a completely new analysis, transferring a
method from a single quadrupole, or porting a known MRM method from another Unknown SRM transitions
instrument, the TSQ 8000 system ensures the fastest route to routine high • Start from the very beginning
performance results through its integrated software tools.
• Start with existing SIM method
• AutoSRM: A purpose-built software for automated SRM method creation and AutoSRM
optimization from full scan to the complete analytical method setup.

SRM Optimization
• TSQ 8000 GC-MS/MS Instrument Method: Offers true timed-SRM operation,
allowing for high sensitivity and ease of use for the most complex SRM methods

• TraceFinder™ Software: State of the art, multi-platform user-friendly Step 2: Selects your product ions from product ion scans
chromatography analysis software

From the Start

For new compounds, choose to begin the AutoSRM function, ask for a precursor ion study
Known SRM transitions
(Step 1), and place a vial in the autosampler. The AutoSRM function will report back to you a • From an existing method
full scan chromatogram of the injection, and you simply identify your peaks and choose the most TSQ 8000 • From SRM transition list
appropriate precursors that AutoSRM presents. Instrument
From an Existing SIM Method Method
Method Sync

If you are moving from a single quadrupole GC-MS method using SIM analysis, or you already know
which ions you want to select for your precursor ions, you can start with Step 2. The AutoSRM Step 3: Optimizes the collision energy for selected
function will perform product ion scans for each selected precursor and present the results in a transitions
single window. Simply select (or ask AutoSRM to select) the best product ion for that compound.

From a Previous MS/MS Method

If you have already performed previous AutoSRM steps or are simply moving from another MS/MS
system, then Step 3 can take you to the final fully optimized SRM transitions for your compounds.
Fully automated collision energy optimization is performed to ensure that transitions are as sensitive Optimized SRM transitions
as they can be. These optimized SRM transitions are then exported into a finished MRM method
• From Compound Data Store
ready for analyzing real samples in routine.
• From existing TSQ 8000
TraceFinder GC-MS/MS method
Software

6 7
 % RSD Ion
Compound % RSD Ratio LOD 95 (ppb)

high performance in
Dichlooraniline 8.5 2.5 1.56 Aldrin: r 2 0.9952 Azinphos-Ethyl: r 2 0.9976

Azinphosethyl 4.6 1.0 0.84

routine applications
Bifenazate 5.2 5.7 0.95
Bromopropylate 4.7 2.7 0.86
Carbaryl 5.0 1.2 0.92
Cyfluthrin 4.4 2.0 0.81
For Contract Analysis, Food Take Environmental Analysis to the Next Level of Productivity
Deltamethrin 6.0 2.4 1.10
Safety, Environmental Monitoring, Triple quadrupole GC-MS/MS instruments are quickly becoming a competitive
and Bioanalysis Endosulfan a 4.6 8.2 0.84 Endosulfan: r 2 0.9994 Disulfoton: r 2 0.9951
requirement for environmental laboratories. Method consolidation into large Etrimfos 3.8 4.5 0.70
The analytical advantages of high multi-analyte tests with generic sample Phosmet 4.9 1.4 0.90
performance SRM are not limited to a preparation assist laboratories in the Tecnazene 5.1 6.1 0.93
single application area. Laboratories maintenance of low sample overhead. Tetraconazole 2.8 4.1 0.51
working in food safety, environmental The TSQ 8000 system is the fastest Vinchlozolin 3.3 7.4 0.60
monitoring and bioanalysis can all benefit route to productive triple quadrupole
from the unique advantages that the analysis available. It has been designed Quantitative performance at 10 ppb vegetable matrix for a selection of target pesticides and matrix calibration curves
TSQ 8000 system brings. This is to ease the transition from single
especially true for laboratories working quadrupole methodologies and enable
in a production environment, such as easy method and results management.
performing contract analysis where
delivering on-time, high quality, low cost 1,3-Dichlorobenzene, High-throughput in Pesticide Analysis
analysis is high priority. 1,4-Dichlorobenzene
The TSQ 8000 system is well prepared for routine analysis of pesticides. It provides
and 1,2-Dichlorobenzene
high robustness of the chromatographic system and MS ion source, thus reducing
(peaks left to right) in
the need for frequent maintenance and avoiding system downtime for high sample
soil extract at 5 ppb
throughput and productivity. The high matrix tolerance, sensitivity, and selectivity
for target compounds allows for multi-residue confirmatory analysis to be
performed. Clean detection and automatic integration of peaks results in faster data
processing and result production.

Confidence in Routine Forensic Toxicology Applications

Confident drug detection, whatever the matrix, is paramount even with high sample
volumes. The high selectivity and robustness of the TSQ 8000 system means you
can expect defendable and quality results every time you inject.

Peaks at 4 ppb from Tolylfluanid (left, 238.1 > 137.0, CE 15 V) and Pyridaben
(right, 309.1 > 147.1, CE 15 V) in lemon peel extract after 500 matrix injections

Multi-drug class screen in oral fluid. Cocaine and BZE at 0.8 ng/mL. THC and THCA at 0.2 ng/mL.
Quantitative SRM transitions on top row, confirmatory SRM transitions on bottom row.

8 9
 powerful MS/MS; powerful MS Simultaneous Acquisition Modes
Offered in MS mode, as well as MS/MS, is
the possibility to combine acquisition modes,
such as SIM/full scan (and SRM/full scan) to
Add capability with GC-MS/MS, don’t compromise... understand the detail in each injected sample
and identify unknowns by library search.
Single quadrupole (SQ) instruments have for years delivered
Whether you monitor clean-up efficiency or
excellent results in targeted and scanning determinations alike.
Regulatory Tuning catch an untargeted contaminant, a targeted-
Some regulations and methodologies still require the reporting
The Target Tune function on a TSQ 8000 system ensures spectra created in a full non-targeted approach can provide an edge
of data based on the capabilities of single quadrupole analysis.
scan analysis match “classic” single quadrupole GC-MS spectra. This facilitates without compromising the ability to measure
Adding triple quadrupole technology enhances the capability of
comparison of spectra to standard libraries and makes it easy to meet regulations low concentrations.
the laboratory. This is why the TSQ 8000 system was built to
be a high performance instrument regardless of whether you that specify excepted ion ratio ranges for certain compounds.
wanted to perform MS or MS/MS experiments.

Pentachlorophenol full
scan spectrum TSQ 8000
Hexachlorocyclopentadiene 100 ppb in
selected ion monitoring (SIM) mode
Classic spectra for DFTPP (Decafluorotriphenylphosphine) obtained using
Target Tune on a TSQ 8000 system

NIST ’08 Library search Lindane isomers 40 ppb in cucumber in simultaneous

Pentachlorophenol full scan/SIM
>900 match
Retention Timed SIM (t-SIM)
Retention Timed SIM is available
for the first time on the TSQ 8000
system. t-SIM makes complex SIM
methods more efficient, more
sensitive and easier to manage.
Simply enter the mass and the
retention time of your compound,
and the TSQ 8000 GC-MS/MS
takes care of the rest.

10 11
 Industry-Leading Detector Linearity

brilliant design for brilliant results The Thermo Scientific DynaMax detection system standard on the TSQ 8000 system
offers industry best linearity. Combined with the low detection limits attainable by
SRM, this detector makes this mass spectrometer
the ultimate quantitative instrument.
Remove the Source of your Downtime
Allows full source removal without venting,
the MS including ion guide surface. No wire No Helium Required
connections are necessary, and venting is Never Clean or Replace Quads Choose between nitrogen or
never necessary to clean your instrument.
Heated ion guide protects the main argon for collision gas. No
quadrupole sets so they never expensive helium is required.
require cleaning or replacement.

Thermo Scientific
ExtractaBrite wireless ion
source cartridge

Dual Heated Zones on Source

Maximize Ruggedness Switch Between Full Scan, SIM and MRM
in Milliseconds
Dual independently-heated zones on the
Effortless switching is made possible with the fast
TSQ 8000 system allow for thousands
scanning capabilities of the TSQ 8000 GC-MS/MS.
of injections of the dirtiest matrices
Pictured on the left is a simultaneous full scan/MRM
before source cleaning is required.
acquisition of the dichlorobenzene isomers. On the right,
Fast Scanning Allows Fast Chromatography a comparison of the 1,2-dichlorobenzene spectra
obtained from the full scan/MRM acquisition (top)
Dynamic ion expulsion in the collision cell allows
against the NIST full scan library spectra (bottom)
hundreds of MRM transitions per second. Combined with
is shown.
t-SRM for efficiently managed transition scheduling,
the TSQ 8000 system can analyze hundreds of
compounds with multiple transitions each over short
chromatographic runs.

No Noise is Good Noise

On-Axis (in-line) Optics
Off-axis optics provided by Off-Axis Optics
the curved ion guide effectively
eliminate neutral noise, resulting
in less background and lower
Dual Filaments for limits of detection. noise reduced
Prolonged Uptime to ~ 2 ions/sec
Intelligent dual filament
design keeps you
running longer between
maintenance intervals.
Noise reduction due to off-axis ion optics
12 13
 Intuitive software platforms streamlined, results-oriented workflow
for confident control Software designed for the high-productivity laboratory

Solutions designed to meet the specific needs of your laboratory TraceFinder software is designed to provide workflow guidance for those
areas which benefit most from automation, while also providing sophisticated
manual tools for those areas where the analyst’s expertise is key to completing
Maximum Versatility Optimized Productivity
that task at hand. From the very start, TraceFinder software is focused on
Thermo Scientific Xcalibur Data System TraceFinder Software
efficient method development, rapid batch set-up, fluid data review, and
The Xcalibur data system is the core ‘operating system’ for
™
The TraceFinder software application provides a streamlined
flexible reporting.
each of the broad range of Thermo Scientific mass spectrometry workflow for the needs of a wide variety of high-throughput
systems. Offering a common set of easy-to-use, yet powerful, quantitative applications. Customized versions target the critical
tools for GC/MS and LC/MS alike, Xcalibur software provides needs for key application areas, such as environmental, food Getting Started
a unified experience for every user of every system. It offers safety, clinical research and forensic toxicology. Configuring the software to meet the unique needs of your
instrument control, sample sequencing, and a set of programs laboratory is straightforward. Once set up, developing an optimized Application Configuration
Beginning at the dashboard and working through each of the
for both qualitative and quantitative applications. Xcalibur method is quick and easy through the use of Method Forge, an
specialized modules, TraceFinder software guides the user
software is also compatible with commercially-available mass automated tool to take you from a standard data file to a
through a highly productive workflow that is focused on the
spectral libraries, such as NIST, Wiley and Maurer-Pfleger-Weber, complete method.
fastest route from sample to report.
and others. It also includes
tools for generating and
Method View
maintaining your own spectral
libraries. Supporting each
type of autosampler and GC Getting Productive
available, Xcalibur software
Once the method is in place, the Batch Wizard accelerates
allows each mass spectrometer
the process of loading samples onto the system, targeting
to be utilized to its fullest.
specific compounds, and selecting automated report options.

Easy Interpretation of Mass Spectral Data

Thermo Scientific Mass Frontier software provides a suite of
options that allows the MS operator to interpret mass spectral Batch View
data. This optional software package assists the operator in Automated processing and QA/QC review, coupled to a reporting
structural identification, isotope pattern determination, and system capable of both standard and customized hardcopy and
spectral classifications. electronic deliverables, this software provides a comprehensive Getting Results
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Following acquisition, automated
processing kicks in, generating results
sets for intuitive data review using a
variety of customized views. Easy to see Report View
flags are available in the datasets,
making it quick to determine which
Data Review
samples and compounds require attention Active View
based on concentrations, recoveries,
ion ratios and a wide variety of user-
defined metrics. On-the-fly modifications are immediately
carried into the results, and no manual reprocessing is
necessary. Once done, automated reporting can be
started to generate print-outs and electronic files.

14 15
 excellence in GC-MS begins with the GC
Match GC and autosampler options to your lab’s needs

Ground-Breaking Plug-In Modularity Less is More

Why stop at a removable source design? The oven, injectors, and detectors of the
With the TRACE 1300 TRACE™ 1300 Series GC have a very low
Series GC paired with Tailor the Thermo Scientific TRACE 1300
ther­mal mass. This enables faster heat-up
your TSQ 8000 GC-MS, Series GC to your needs with its proprie-
and cool-down times, reducing your inject- TriPlus RSH
tomorrow’s opportunity tary user-exchangeable Instant Connect
to-inject time and increasing your sample Liquid
is in your hands today. injector and detector modules. Swapping Autosampler
throughput.
modules is easily done by the removal and
replacement of three screws, accessible Increase sensitivity with a completely new
from the top of the GC. The entire process range of micro volume GC detectors. The
takes less than five minutes. This enables unmatched sensitivity of these new instant
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expand their capabilities to accommodate required initial sample amounts. Precise sample introductions are required
new applica­tion and throughput demands. to achieve the best reproducibility and
Easy Operation, Easy Maintenance accuracy. The Thermo Scientific AI/AS
The TRACE 1310 GC offers an intuitive, 1310 autosampler and TriPlus™ RSH
icon-based touch screen interface which autosampler offer ideal solutions for
allows for local control of the GC. In automated injections.
addition, tool-free access to septa and liners TriPlus RSH Autosampler – Offers
allows for quick routine maintenance. When optimized liquid injection modes to support
more aggressive injector cleaning is needed, a wide range of sample types, inlets, and
easy removal of the entire injector body techniques for syringe-filling and injection.
allows for sonication in solvent. Finally, two AI/AS 1310 Autosampler – Offers a basic
easily accessible carbon traps ensure the configuration for up to 8 vials at a time, while
injector and flow lines stay clean. Combined, the AS 1310 holds 105 standard liquid
these new features on the TRACE 1300 autosampler vials. Excellent precision and
Example of plug-in module installation by user
Series GC make it the most serviceable tool-free alignment make this sampler a
GC in the world. perfect choice for routine liquid sample
injections.

Thermo Scientific chromatography

columns and consumables
The perfect partner for optimal analytical performance

Thermo Scientific chromatography columns and consumables are

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systems. Get the most out of the TSQ 8000 GC-MS
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applications-focused solutions in the environmental,
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China +86 10 8419 3588 India +91 22 6742 9434 Russia/CIS +43 1 333 50 34 0
Denmark +45 70 23 62 60 Italy +39 02 950 591 South Africa +27 11 570 1840 BR52289_E 05/12M
Application
Note: 52098
Confirmation and Quantitation of Six Opiates in
Urine Using the ISQ Single Quadrupole GC-MS
Dwain Cardona, Matthew Lambing, Thermo Fisher Scientific, Austin, TX, USA

Overview
Key Words Opioids compose a chemical class of both naturally
• ISQ Single occurring compounds produced from the opium poppy plant
Quadrupole GC-MS (Papaver somniferum) and similar synthetic analogues.
Substances within this class are commonly recognized as
• ToxLab Forms potential drugs of abuse.1 Biological matrices containing
• Forensic heroin (diacetyl morphine), codeine, morphine, hydrocodone,
Toxicology hydromorphone, oxycodone and oxymorphone and thebaine
are regularly monitored by drugs of abuse analysis
• Opiate laboratories. Structural similarities of opiate metabolites
• Selected Ion introduce challenges to the accurate separation, confirmation
Monitoring and quantitation of these substances.2
A forensic toxicology method for the confirmation
and quantitation of codeine, morphine, hydrocodone,
hydromorphone, oxycodone and oxymorphone in human
urine was developed using the Thermo Scientific ISQ single
quadrupole GC-MS system. Multiple analytical procedures
can be replaced by this method, which combines a dual
derivatization technique with the selection of the correct
analytical column.

Methods
All samples were prepared as batches using a 2 mL sample ion monitoring mode (SIM), collecting three ions for each
size. Standard materials were obtained for calibration, target compound, and two ions for each deuterated internal
and separate sources of the opiates were used as controls. standard (Table 1 and Figure 2). Thermo Scientific ToxLab
Deuterated internal standards were employed. Batches Forms software provided automated acquisition and
included a matrix-matched single point calibrator processing of all data, including quantitation and ion ratio
(300 ng/mL), quality control samples set to contain each confirmation calculations. The method was assessed for
target compound at 40% and 125% of the calibrator specificity, linearity, precision, recovery and interference.
(120 ng/mL and 375 ng/mL respectively), and a negative
control, which was blank urine with internal standard only.
A hydrolysis step was performed to release the bound
drug from the glucuronide. Samples underwent a first
derivitization with hydroxylamine to convert the keto
moieties to their oxime derivatives. Thermo Scientific
HyperSep Verify-CX solid-phase extraction columns
(200 mg, 10 mL) were used for sample extraction. Sample
extracts were derivatized a second time with Thermo
Scientific BSTFA and 1% TMCS.
A Thermo Scientific AS 3000 II autosampler and a
Thermo Scientific TRACE GC Ultra gas chromatograph,
equipped with a split/splitless injection port, provided
sample introduction into the ISQ mass spectrometer. 10
10
A 15 m × 0.25 mm I.D. × 0.25 µm Thermo Scientific
0
TraceGOLD TG-1MS analytical column was used to 0

enhance separation of the target opioid class compounds

from each other and from matrix components (Figure 1). Figure 1: Total ion chromatogram of the six opiate compounds from an
The ISQ™ mass spectrometer system was operated in selected extracted urine sample at the cutoff (300 ng/mL)
 Retention Quan Ion Qual Ion(s) Batches were reviewed for conformance to quality
Analyte Time (min) (m/z) (m/z) control criteria regarding both quantitative and qualitative
Codeine-D6 2.67 377 349 performance, based on accrediting agency guidelines. All
quality controls within a batch demonstrated quantitative
Codeine 2.69 371 372, 313
results within ±20% of their expected (theoretical) con-
Morphine-D6 2.90 435 435 centration. Additionally, ion ratio ranges for qualifier ions
for target compounds were established using ±20% of the
Morphine 2.91 429 414, 401
ratios calculated for the 300 ng/mL calibration standard.
Hydrocodone-D6 2.98 392 377 These ranges were used to assess ion ratio performance.
Hydrocodone 3.00 386 371, 297 ToxLab™ Forms performed ion ratio confirmations,
retention time checking, and quality control conformance
Hydromorphone-D6 3.10 450 435 automatically as a part of batch acquisition and processing.
Hydromorphone 3.12 444 429, 355 For precision analyses, a coefficient of variation (CV) of
<10% of the average calculated quality control amounts
Oxycodone-D6 3.34 480 465
were required for each analyte.
Oxycodone 3.36 474 459, 475
Oxymorphone-D3 3.46 535 520
Oxymorphone 3.47 532 533, 517
Table 1: Retention times and ions monitored for the opioid analytes and their
deuterated internal standards

Figure 2: Extracted ion overlays of the six opiate compounds at the cutoff (300 ng/mL). Note that no interference is seen from coeluting matrix ions.
Results Codeine Concentration CV for Batch 1 CV for Batch 2 Inter-batch CV

• The dual derivatization technique 120 ng/mL 1.5% 1.8% 1.7%

led to the successful separation of
375 ng/mL 2.7% 3.1% 2.9%
all six opiate compounds (Figure 1).
• Optimized instrument parameters Morphine Concentration CV for Batch 1 CV for Batch 2 Inter-batch CV
and the selection of the 1 phase 120 ng/mL 4.3% 2.4% 3.9%
column versus a 5 phase analytical
column eliminates compound 375 ng/mL 2.5% 2.5% 3.9%
coelution and interference (Figure 3).
Hydrocodone Concentration CV for Batch 1 CV for Batch 2 Inter-batch CV
• Linearity ranged between
30–5000 ng/mL for codeine, mor- 120 ng/mL 1.5% 2.4% 2.2%
phine, hydrocodone, hydromorphone, 375 ng/mL 2.1% 3.3% 3.0%
oxycodone and 30–1200 ng/mL
for oxymorphone (Figure 4). This Hydromorphone Concentration CV for Batch 1 CV for Batch 2 Inter-batch CV
supports both directed assay and
retest samples while also reducing 120 ng/mL 1.4% 3.1% 3.5%
the need to re-extract concentrated 375 ng/mL 2.5% 2.8% 3.8%
samples.
Oxycodone Concentration CV for Batch 1 CV for Batch 2 Inter-batch CV
• Correlation coefficients (R2) better
than 0.9960 for all analytes based 120 ng/mL 1.7% 2.8% 3.4%
on a one point calibration further
demonstrates sample concentration 375 ng/mL 2.6% 3.3% 4.3%
range capability. Oxymorphone Concentration CV for Batch 1 CV for Batch 2 Inter-batch CV
• Intra and inter-batch precision of
120 ng/mL 1.5% 2.1% 2.4%
<5% CV (Coefficients of Variation)
at 120 ng/mL and 375 ng/mL 375 ng/mL 1.8% 2.2% 2.5%
ensure confidence in the measured
result and achieve reliable results Table 2: Results of the precision study for 120 ng/mL and 375 ng/mL quality control samples.
Note: Individual quantitative values from each batch combined for calculation of inter-batch CV.
day in and day out.
• Limit of quantitation at 30 ng/mL
using a 2 mL sample size allows the
analyst to achieve required detection
limits with limited sample volume.

Figure 3: Comparison of chromatography using a TG-1MS (top) to a TG-5MS (bottom) analytical column
 In addition to these
offices, Thermo Fisher
Scientific maintains
a network of represen-
tative organizations
throughout the world.

Africa-Other
+27 11 570 1840
Australia
+61 3 9757 4300
Austria
+43 1 333 50 34 0
Belgium
+32 53 73 42 41
Canada
+1 800 530 8447
Figure 4: Linearity study results for six opiates comparing calculated concentrations to the expected amounts at each level.
China
The regression analysis for this study gave a correlation coefficient of 0.9960 or higher for each analyte. +86 10 8419 3588
Denmark
+45 70 23 62 60
Conclusion References Europe-Other
A method was developed to demonstrate the performance 1. Baselt, R., Disposition of Toxic Drugs and Chemicals in Man, Eighth +43 1 333 50 34 0
Edition, Biomedical Publications: Foster City, 2008. Finland / Norway /
of the ISQ GC-MS system for the confirmation and Sweden
2. Ropero-Miller, J.D.; Lambing, M.K.; Winecker, R.E. Simultaneous
quantitation of six opiate compounds in a urine matrix. Quantification of Opiates in Blood by GC-EI-MS Analysis Following
+46 8 556 468 00
The chromatographic performance of the TraceGOLD™ Deproteination, Detautomerization of Keto Analytes, Solid Phase France
Extraction and Trimethylsilyl Derivatization. J. Anal. Toxicol. 2002, 26, +33 1 60 92 48 00
TG-1MS analytical column with the optimized parameters
524–528. Germany
of the ISQ GC/MS provided fast run times without coelution +49 6103 408 1014
interference, ensuring accurate confirmation and quantitation. India
+91 22 6742 9434
The assay described offers broad linearity to cover a wide
Italy
range of analyte concentrations, with R2 values of 0.9960 +39 02 950 591
or higher for all analytes. The coefficient of variation for Japan
+81 45 453 9100
intra- and inter-batch precision was shown to be less than
Latin America
5% for 120 ng/mL and 375 ng/mL concentrations, +1 561 688 8700
demonstrating excellent precision. Limits of detection and Middle East
quantitation at 30 ng/mL ensure sensitive performance for +43 1 333 50 34 0
Netherlands
retest and directed assay samples. With the final analyte, +31 76 579 55 55
oxymorphone, eluting at a retention time of less than five New Zealand
minutes, the methodology offers a productive means for +64 9 980 6700
confirming the six-opiate panel. Laboratories save time Russia/CIS
+43 1 333 50 34 0
and money by using a single extraction, single injection South Africa
and a fast analysis time, while achieving results that are +27 11 570 1840
quantitatively accurate and precise across a broad Spain
+34 914 845 965
concentration range. Switzerland
+41 61 716 77 00
UK
+44 1442 233555
USA
+1 800 532 4752

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Legal Notices: ©2011 Thermo Fisher Scientific Inc. All rights reserved. ISO is the registered trademark of the International Standards Organization.
All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities
of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual Thermo Fisher Scientific,
property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Austin, TX USA is ISO Certified.
Please consult your local sales representative for details.
AN52098_E 11/11M

Part of Thermo Fisher Scientific

 IC Theory and Principles

Name :- Thaker Chanakya M.

Title :- Senior Applications Specialist, Dionex Products
Date :- 25/02/2013
 Definitions

Chromatography is the general term for a

physical--chemical separation method, which is
physical
based on analyte distribution between a mobile
and a stationary phase.

2 Proprietary & Confidential

 Terminology

• What are Ions?

An ion is an atom or molecule in which the total number of
electrons in an atom is not equal to the total number of protons, giving
the atom a net positive or negative electrical charge.
If a neutral atom loses one or more electrons, it has a net positive
charge and is known as an cation.
If an atom gains electrons, it has a net negative charge and is
known as an anion.
An ion consisting of a single atom is an atomic or monatomic ion;
if it consists of two or more atoms, it is a molecular or polyatomic ion.

3 Proprietary & Confidential

 Terminology

• What is Ion Exchange?

Ion exchange is an exchange of ions between two electrolytes or
between an electrolyte solution and a complex.
In most cases the term is used to denote the processes of
purification, separation, and decontamination of aqueous and other ion-
containing solutions with solid polymeric or mineralic 'ion exchangers'.
Typical ion exchangers are ion exchange resins (functionalized
porous or gel polymer), zeolites, montmorillonite, clay, and soil humus.
Ion exchangers are either cation exchangers that exchange
positively charged ions (cations) or anion exchangers that exchange
negatively charged ions (anions).

4 Proprietary & Confidential

 Types of HPLC Techniques

Liquid Chromatography (HPLC)

Reversed
Reversed--phase Chromatography

Normal
Normal--phase Chromatography

Ion-
Ion-Exchange Chromatography

Ion-
Ion-Exclusion Chromatography

Ion-
Ion-Pair Chromatography

5 Proprietary & Confidential

 Ion Exchange Chromatography

• In ion-exchange chromatography (IC), retention is based on the

attraction between solute ions and charged sites bound to the
stationary phase.
• Solute ions of the same charge as the charged sites on the column
are excluded from binding, while solute ions of the opposite charge of
the charged sites of the column are retained on the column.
• Solute ions that are retained on the column can be eluted from the
column by changing the solvent conditions (e.g. increasing the ion
effect of the solvent system by increasing the salt concentration of
the solution, increasing the column temperature, changing the pH of
the solvent, etc...).

6 Proprietary & Confidential

 Ion Exclusion Chromatography

• Ion exclusion chromatography (IEC) is mainly used for the separation of

weak acids or bases.
• The greatest importance of IEC is for the analysis of weak acids such
as carboxylic acids, alcohols, phenols etc.
• Retention mechanism are donnan exclusion, steric exclusion and
adsorption partition.
• Those having high molecular weight elute first while those with low
molecular weight elute at the end.

7 Proprietary & Confidential

 Types of Ion Chromatography

1) Ion Exchange (IC)-

Ion Exchange chromatography is based on the ion exchange
process occurring between the mobile phase and ion-exchange groups bonded
to the support material. This is the primary mode of separation in IC for
inorganic anions and cations.
3) Ion exclusion chromatography (IEC)-
Those having high molecular weight elute first while those with
low molecular weight elute at the end. This is governed by Donnan exclusion
and used for selective determinations of weak anions (i.e. organic acids)

8 Proprietary & Confidential

 Distinguish between HPLC and IC

HPLC IC

1 The ‘hardware’ of HPLC is made up of SS The ‘hardware’ of IC is made up of PEEK

material.(Poly Ether Ether Ketone)

2 Generally non ionic mobile phase is use. Ionic mobile phase is required

3 Stationary phase used in HPLC is mostly Polystyrene base ion exchange resin
bonded Silica copolymer with styrene and dvb is
generally used as stationary phase in
IC.
4 Only molecule is determined by HPLC Charged species as well as some
molecule can be determined.

5 Absorbance is measured in HPLC Primarily Conductance is measured in

IC

6 Can not used as IC. Can be used as RP- HPLC.

9 Proprietary & Confidential

 What is an IC?
Eluents
(NaOH, H2SO4)

Chromatogram

Column

Injector
Suppressor
Pump Conductivity Cell

Waste

Regenerant
(H2SO4, TBAOH)
14209
10 Proprietary & Confidential
 Species Detected by Ion Chromatography

Conductivity DC Amperometry Integrated UV-Vis

Amperometry
Inorganic anions Catecholamines Carbohydrates Silicate

Inorganic Cations Phenols Aliphatic amines 10, 20,

Bromate
30

Carboxylic acids Aromatic amines Amino acids Chromate

Sulfonic acids Thiols Alcohols Transition metals

Phosphonic acids Cyanide Aldehydes Lanthanides

Amines, 10, 20, 30 Sulfide S species

(except S[VI])

Iodide Sulfite
Sulfite Iodide
11

11 Proprietary & Confidential

 Advantages of Ion Chromatography

Speed
- Complete anion and cation profiles in about 10 minutes

Sensitivity
- Analyses in the lowest µg/L-
µg/L-range without pre
pre--concentration
- Analyses in the lowest ng/L-
ng/L-range after pre-
pre-concentration
- Limiting factor: contaminations by ubiquitous ions such as
chloride and sodium

Selectivity
- Huge variety of stationary phases
- Specific detection (suppression, UV, fluorescence, MS, ICP)

12 Proprietary & Confidential

 Advantages of Ion Chromatography / 2

Simultaneity
- Simultaneous analysis of many sample components
(In contrast to AAS, photometry, titration, etc.)
- Limiting factor: extreme concentration differences between the sample
components.

Costs
- Cost effective as Mobile phase like coustic or Acids in diluted form is
required for analysis.

Robustness
- pH and solvent compatible separators allow a variety of applications
- Analysis of complex matrices such as wastewater, foods, body fluids, etc.

13 Proprietary & Confidential

Modes of Ion Chromatography System

• 1) Delivery Mode

• 2) Separation Mode

• 3) Detection Mode

• 4) Data Mode

14

14 Proprietary & Confidential

 1) Delivery Mode – In this mode delivery of mobile phase as
well as sample is done .It consist of

I) Eluent Reservoir – It is nothing but mobile phase container. This can be made up of good
quality of glass or polypropylene depending upon the nature of mobile phase.

II) Pump – For optimal system performance it is essential that the pump provide smooth,
accurate, and precise eluent delivery .The pump material as well as fluid path in the
system is made up of Peek (Poly ether ether ketone) material. This peek material is
compatible with 0 to 14 PH and with most of organic solvents.
Flow Range – 0.001 to 10 mL / min.
Pressure Range – 50 to 5000 psi.

III)Sample Injector – This can be manual or auto to load the fixed amount of standard as
well as sample in to the column.

15

15 Proprietary & Confidential

2) Separation Mode – The separation of ions take place in this
mode. It consist of

• I) Guard Column – generally guard column is made up of same material as

analytical column. Guard column prevents direct shock to main analytical
column.

• II) Analytical Column – The column is the heart of ion chromatography.

Physically, it consists of a chemically inert tube packed with a polymeric
resin. IC columns are available in different sizes and packed with different
resins depending upon the application and desired mode of separation.

16

16 Proprietary & Confidential

The Mechanism of separation -

• Ion exchanger normally consists of solid phase on whose surface ionic groups
are fixed. (e.g. stationary phase of analytical column is made up of polystyrene
base ion exchange resin copolymer with styrene and divinylbenzene with ionic
group such as quaternary ammonium group, Sulphonated group etc)

In anion chromatography the separation is based upon the exchange of

ions between mobile phase and cationic sites of stationary phase. The
anions from the solutions compete for the cationic sites of stationary
phase. At any point during the ion exchange process, the neutrality of
the fixed ionic charge (resin) is preserved by the exchangeable eluting
ions; same time the sample ion exchanges with the eluent ion. When it
is paired with the fixed charge the ion does not move down the column
because ions have different affinities for fixed exchange site.

17

17 Proprietary & Confidential

 Ion Exchange Separation

Anion A
Anion B
Anion C
Injection

Initial

18 Proprietary & Confidential

 Pellicular Anion Exchange Bead (Latex)

Ion Exchange
0.1 µm Diameter Core Particle Surface Outer Surface

R3 N+ N +R 3
SO-3
R3 N+ N +R 3
N +R 3
SO-3
R3 N+ N +R 3
SO-3
R3 N + N +R 3
SO-3 R3 N+ N +R 3
SO-3 R3 N+ N +R 3

R3 N+ N +R 3
SO-3
R3 N+ N +R 3
SO-3

3183--03
3183
19 Proprietary & Confidential
 Mechanisium of Separation

Typical Eluents
Anion Exchange

Anion Exchange –
S- NaOH, Na2CO3 etc.

Stationary
NR+3 S-
Phase Cation Exchange –
H2SO4, MSA etc.
S-

Mobile
Phase

20

20 Proprietary & Confidential

 Ion Exchange Separation – Na2CO3 and NaHCO3 Eluent
In case of NaOH or KOH Eluent “OH” will be the available ions for Interaction
CO32- SO42- CO32-
HCO3- ＋ ＋ HCO3- ＋ HCO3-
Equilibrium ＋ CO32- ＋
＋ CO
＋
SO324-2- ＋ SO 2-
＋
4

＋ HCO3- CO32- ＋ HCO3- CO32- ＋ HCO3- CO32-

＋ 2- ＋ 2- ＋ 2-
CO3 CO3 CO3
＋ HCO3- ＋ Cl- ＋ HCO3-
＋ HCO3- ＋HCO3- ＋ HCO3-
＋ HCO - ＋ HCO
Cl- 3- ＋ Cl-
3
＋ HCO - ＋ HCO - ＋ HCO -
3 3 3
＋ ＋ ＋ HCO3-
＋ CO 2-
HCO3- ＋ CO 2-
HCO3- ＋ CO 2-
3 3 3

HCO3- CO32- HCO3- CO32-

＋ ＋
＋ SO 2- ＋ SO 2-
4 32-
CO
4
＋ ＋
＋ HCO3- CO32- ＋ HCO3- SO42-
＋ ＋ 2-
2-
CO3 CO3
＋ ＋
HCO3- HCO3-
＋
HCO3- ＋HCO3-
＋ Cl
CO- 2- ＋ HCO3-
3
＋ HCO - ＋ HCO -
3 3
＋ Cl- ＋
＋ CO 2- ＋ CO32- HCO3-
3

21 Proprietary & Confidential

 Progress of ions in IC System

Eluents
Eluent： Na2CO3 / NaHCO3

NaF,NaCl,NaNO3
Na2HPO4,Na2SO4

Counter ions
G. Pump
NaF NaCl NaNO3 Na2SO4
Na2HPO4
Columns
Eluent form： Na2CO3 / NaHCO3

Waste
HF HCl H2SO4

Suppressor HNO3
H3PO4

Eluent form： Na2CO3 / NaHCO3

22 Proprietary & Confidential

 The Role of Suppression

Eluent Without Suppression

(Na2CO3) Counter Ions
Sample F-, CI-, SO42 -

F- CI - SO42 -

Analytical µS
Column

NaF, NaCl, Time

Na2SO4 in Na2CO3 With Suppression
Waste Waste CI- SO42 -
Na+
Anion F-
H2 O H+
Suppressor OH- H2 O

Anode Cathode µS

HF, HCI, H2SO4

23 in H2CO3 Time
12943
23 Proprietary & Confidential
 Chemistry and Ion Movement in an Anion SRS®

Anode Cathode
Waste/Vent Waste/Vent
Na+, X-
in NaOH Eluant

H2O, O2 NaOH, H2
-
OH
H+ Na+
+ -
H + O2 H+ + OH H2 O H2 + OH-
+ -
H ,X
H2 O in H2O H2 O

H2 O To Detector H2 O

H2 O
+
2H + ½ O2 + 2e
- Cation-
Cation- 2H2O + 2e
- -
2OH + H2
Exchange
Membranes
24

24 Proprietary & Confidential

Factors affecting on Retention time

• In this example the counter ion is OH- in the case of the anion exchange process and H+
in the cation exchange. The equilibrium established is represented by equation-

Resin Solute Eluent

[ R4N+ X- ] [ OH- ]
• K= -----------------------------------
[ R4N+ OH-] [ X- ]
Resin Eluent Solute

• Higher the value of the distribution coefficient (K), more strongly the ionic solute
interacts with the ion exchanger, and longer its retention time. This distribution
coefficient is a function of ionic charge, ionic size, and ionic strength of the eluent, pH
and resin type.

25

25 Proprietary & Confidential

 Retention Determining Parameters:
Properties of Ions

• Valency
• Size
• Polarizability

26 Proprietary & Confidential

 Retention Determining Parameters (cont.)

• Valency

Rule:
The higher the valency of an analyte ion, the larger the
retention time.
Example:
tms (Nitrate) < tms (Sulfate)
Exception:
Retention of multivalent ions such as orthophosphate
is depending on eluent pH.

27 Proprietary & Confidential

 Retention Determining Parameters (cont.)

• Size

Rule:
The larger the radius of the analyte ion, the larger the
retention time.
Example:
tms : F– < Cl– < Br– << I–

28 Proprietary & Confidential

 Retention Determining Parameters (cont.)

• Polarizability

Rule:
The higher the polarizibility of the analyte ion, the larger the
retention time.
Example:
tms (Sulfate) < tms (Thiocyanate)
Reason:
Adsorption processes contribute to the ion-exchange process.

29 Proprietary & Confidential

Conductivity Cell –

• Conductivity detection is based upon the electrical conductivity of ionic

solution when placed between two oppositely charged electrodes. This
electrode mounted in a cell body. The distance between the two
electrodes is around 0.8µl to 1.0µl. the presence of ions in the solution
allows electrical current to flow between the electrodes completing the
circuit. Detector monitors the conductance of the sample ions as they
emerge from the suppressor.

30

30 Proprietary & Confidential

 4) Data Mode –

• Signal generated by detector is transmitted to Recorder or directly

displayed as a chromatogram on computer. The concentration of ionic
analytes are automatically determined and tabulated.

31

31 Proprietary & Confidential

Anion Standard on IonPac AS12A

Column: IonPac AS12A

10 Eluent: 0.3 mM Sodium bicarbonate
2.7 mM Sodium carbonate
Flow Rate: 1.5 mL/min.
8 1
Inj. Volume: 25 µL
2 Detection: Suppressed Conductivity
6 5 Peaks: 1. Fluoride 3
10
µS 4 mg/L
4 2. Chlorite 10
3 3. Bromate 10
6 8
7 4. Chloride 4
2
9 5. Nitrite 10
6. Bromide 10
0 7. Chlorate 10
8. Nitrate 10
-2 9. Orthophosphate 10
10. Sulfate 20
0 2 4 6 8 10 12 14
32
Minutes
10495
32 Proprietary & Confidential
 Separation of Alkali and Alkaline-
Alkaline-Earth Metals and Ammonium

Separator: IonPac CS12A

20 4 Eluant: 18 mmol/L MSA
Flow rate: 1 mL/min
1 Inj. volume: 25 µL
2 Detection: Suppressed conductivity,
Recycle Mode
7 8 Peaks: 1. Lithium 1 mg/L
2. Sodium 4
µS 3. Ammonium 5
5 4. Potassium 10 5.
Rubidium 10 6. Cesium
3 6 10 7.
10 Magnesium 5 8. Calcium
9
10 9.
Strontium 10 10. Barium
0
10
0 5 10 15 20 25
Minutes
33

33 Proprietary & Confidential

 Amperometry

• Not all analytes separated by ion chromatography are amenable to

conductivity detection.
• Amperometric detection takes advantage of some analytes’ capacity to
undergo chemical reactions when subjected to an applied potential.
• An amperometric cell is composed of a small-volume channel flowing
between a pair of electrodes.
• A potential is applied across these electrodes and causes either the
oxidation or reduction of the analyte, thereby rendering it capable of
conducting an electrical current.
• This current is referenced to a separate electrode and the result is
compared to a standardized value to generate a viable measurement.

34 Proprietary & Confidential

 Direct-Current (DC) Amperometry

How it works: • Constant Potential is applied to an electrode

• Redox of analyte generates current which is
measured.

Catecholamines
Applications:

Some inorganic anions

35

35 Proprietary & Confidential

 Integrated Amperometry

• Rapidly repeating pattern of potentials

How it works: (“waveform”) applied to electrode
• Current integrated at analytical potential
• Electrode continuously reconditioned

Applications:

Carbohydrates

Amino acids

Alcohols

Sulfur--containing compounds
Sulfur

Many electroactive inorganic ions

36

36 Proprietary & Confidential

 Integrated Amperometry: Waveform and Integration Period

1 1

Potential (V)
Potential (V)

0 0
-1 -1
-2 -2
-3 -3
0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0
Time (s) Time (s)

Carbohydrate Waveform Amino Acid Waveform

integration period
37

37 Proprietary & Confidential

 Example :- HPAE-IPAD detection of Sugars(Carbohydrates)

• Many carbohydrates are weak acids with pKa values in the range 12–
14, and, consequently, at high pH values their hydroxyl groups are
partially or totally transformed into oxyanions.
• This enables sugars to be selectively eluted as anions by high-
performance anion exchange chromatography in a single run.
• Under alkaline conditions, carbohydrates are readily separated by
quaternary-ammonium-bonded pellicular anion exchange columns,
where the order of increasing retention is correlated with decreasing
pKa value.
• Monosaccharides possess several potentially ionisable hydroxyl groups
having, taking glucose as reference, the following hierarchy of acidity:
1-OH > 2-OH≥6-OH > 3-OH > 4-O.

38 Proprietary & Confidential

 Chromatogram : Example

Detection of food sugars and sugar alcohols

39 Proprietary & Confidential

 Resolving Power of HPAE-IPAD

Comparison of chromatographic
techniques used for the analysis of
a hydrolyzed glucose syrup
illustrates the remarkable resolving
power of the HPAE-PAD technique.
Elution order is reversed with the
CarboPac® PA1 column as
compared with conventional metal
loaded cation exchange columns.

40 Proprietary & Confidential

Absorbance Detection

• Absorbance detection relies on the ability of molecular bonds within an

analyte to absorb electromagnetic radiation at specific wavelengths.
• This radiation, usually in the visible or ultraviolet spectrum, causes the
promotion of outer valence electrons in certain bonds within the sample
analyte to a higher energy state.
• This results in a change in energy, or intensity, of the applied radiation,
which can then be detected with a photometer.
• Beer’s Law states that, in a fixed cell path, the absorbance of a solution
will be proportional to its concentration.
• Although deviations from this law do occur, for most analytes measured
by this method, it is possible to determine an effective range of linear
response.

41 Proprietary & Confidential

Transition Metals Analysis

• Metal ions can exist in several different forms. The factors which
determine the form of the metal ion are the extent of complexation and
the oxidation state.
• Hydrated and weakly complexed transition metals can be separated as
cations on a cation exchange column.
• By adding a carboxylic acid chelating agent to the eluent, the net
charge on the metal is reduced, since the carboxylic acids are anionic
in solutions above their pKa s.
• The selectivity of the separation is actually due to the different degrees
of association between the metals and the chelating agents producing
different net charges on the metal complexes.
• If strong enough chelating agents are used in high enough
concentration, the net charge of the metal complexes can be negative.
• These anionic metal complexes are separated by anion exchange.

42 Proprietary & Confidential

Transition Metals Analysis ….. Continued

• The IonPac CS5A column has both cation and anion exchange
capacity, allowing metals to be separated as cations or anions on a
single column.
• This is called a mixed mode separation.
• Most hydrated and weakly complexed metals will precipitate in a
suppressor and, therefore, cannot be detected by conductivity.
• Also, with a few exceptions, transition metals cannot be detected by
direct UV absorbance.
• Therefore, the metal complexing agent 4-(2-pyridylazo)resorcinol (PAR)
is added postcolumn to form a light-absorbing complex.
• Speciation of many transition metals are also possible with this
technique.

43 Proprietary & Confidential

 Example :- Transition metals analysis

44 Proprietary & Confidential

 Chromium Analysis :- Flow Chart for EPA Method 218.6

45 Proprietary & Confidential

 Example : Chromium Speciation

46 Proprietary & Confidential

 Thermo Scientific Dionex IC modules

1) ICS 900 :- Basic Module

2) ICS 1100
3) ICS 1600
4) ICS 2100 :- RFIC enabled with LCD screen
5) ICS 5000 :- RFIC enabled; Higher end module capable
of IPAD detection

RFIC :- Reagent Free Ion Chromatography; Just add

water technique  no need to prepare eluent

All systems are controlled by Chromeleon CDS

software

47 Proprietary & Confidential

 Demo Video :- ICS 2100 with RFIC

48 Proprietary & Confidential

 Thank You!

IC, RFIC, Cap IC

49

49 Proprietary & Confidential

 HPLC – Theory and Principles

Name :- Thaker Chanakya M.

Title :- Senior Applications Specialist, Dionex Products
Date :- 25/02/2013

Proprietary & Confidential

 Which Separation technique?

2
History of Liquid Chromatography

• Chromatography was first discovered by Russian

botanist Mikhail Tswett who separated plant pigments
on chalk columns in 1903
• British chemists A.T. P Martin and R. L. M. Synge
developed partition chromatography in 1942; Martin
and A. T. James developed gas chromatography (GC)
in 1952
• Since 1940’s, chemists used gravity-fed silica columns
to purify organic materials
• In late 60’s, LC turned high performance with small-
particle columns that require high-pressure pumps
• Development of on-line detectors allows HPLC to
become a sensitive and quantitative technique for
diverse applications

Classical LC (Gravity flow)

3
 Overview

• HPLC is :-
• A physical separation technique in which a sample dissolved in liquid is
injected into a column packed with small particles and is separated into
its constituent components
• The most important and widely used analytical technique for the
quantitative analysis of organics and biomolecules
• Applicable to many sample types
• Most useful for pharmaceuticals, biomolecules, and labile organics
(also some ionic compounds)

4
Introduction – The Chromatographic Process

Continous stream of mobile phase

Mobile
Column Phase
Stationary
Phase
● The stationary phase retains analytes due to various interactions.
● When different chemical components pass through the column at different rates they
become separated in single zones.

5
 Advantages of HPLC

• Amenable to diverse samples including labile organics, biomolecules, and

ions - Can handle > 70% of all existing compounds vs. 15% for GC
• High-resolution
• Resolves hundreds of components in complex samples
• High sensitivity detection
• pg - ng detection limits
• Rapid and precise analysis
• 1 - 60 min analysis, Precision < 1% RSD
• Automated analysis
• Using autosampler and data system for unattended analysis and report
generation
• Quantitative sample recovery
• Preparative technique from mg to kg quantities

6
 The Chromatographic Process

Mobile Phase

• In LC, analytes to be separated is distributed Analyte

• between two phases

• Mobile phase (a flowing liquid) Stationary Phase

• Stationary phase (a column packed with porous particles)

• Components is separated by differential interactions (repetitive
sorption/desorption) with the porous support
• An on-line detector monitors the concentration of each eluting
component and generates a trace called the chromatogram

7
 Introduction – Characteristics of a Chromatogram

1 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 UV_VIS_1

2 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 Pump_Pressure
600 140,0
mAU bar
Retention time

2 - 6,573
1 - 5,363
500 Detector
signal 130,0

3 - 8,863
400
Peak Height
Pressure
120,0

4 - 12,930
signal and
300 ripple

200 110,0

Peak area
100
%D: 0,0 % 100,0
2 %C: 0,0 % Baseline
Methanol: 70,0 %
1
Flow: 0,50 ml/min
min
-50 90,0
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0

8
 HPLC: Basic Terminology

• Retention time (tR)

• Peak width (Wb)
• Capacity Factor (k’)
• Column Efficiency
• Plate number (n)
• Height Equivalent of a Theoretical Plate (HETP)
• Selectivity (α)
• Resolution (Rs)

9
 Retention Time (tR)

• tR= Retention time t0 = Retention time of an unretained solute

• Wb = Base width of peak (4 σ width)

Peak tends to be gaussian and broadens with time - Wb becomes larger with longer tR.

10
 Capacity Factor (k’)

• k’ is a measure of peak retention or how many times the peak is retained vs an

unretained peak (to )
• k’ = (tR - to ) / to = tR ‘ / to

11
 Separation Factor or Selectivity (α)

• a (separation factor) = k2’ / k1’ = 2.1” / 1.5” = 1.4

• a is dependent on the column and mobile phase
• a is measure of differential retention of two analytes by the column and
a must be > 1.0 for peak separation

12
 Column Efficiency (n)

• Plate number (n) is a measure of column efficiency

• n = 16 (tR / Wb ) 2 = 16 (135 / 10)2 = 2916
• n is determined by particle size (dp ), column length (L) and flow rate (F)

Wb is base width by the tangent method. Alternately, use n = 5.54 (tR / W0.5 ) 2

13
 Resolution (Rs)

• Rs (resolution) = 2(tR1 - tR2 ) / Wb = ∆ t / Wb = 23 / 14 =1.5

• Rs is a measure of the degree of separation of two peaks.
• Rs = 0 (no separation); Rs = 1 (partial separation);
Rs = 1.5 (baseline separation)

Rs = 1.5

14
 The Resolution Equation

• Rs = (k’/1+k’) (α −1/ α) (n)0.5 / 4

retention selectivity efficiency

• The goal of most LC separations is to achieve baseline resolution for all

key analytes (Rs > 1.5)
• k’ should be kept between 1 to 10
α is maximized with by selecting the column and mobile phase that
resolve all critical analytes
• n is increased using high-efficiency columns of a reasonable length

15
 Glossary of HPLC Terms
A Review

• Column - a tube that contains packed sorbents

• Mobile phase - a liquid that carries the sample through the
column
• Retention: the tendency of a solute to be retained by the
stationary phase in the column
• Resolution (Rs) - the degree of separation of 2 peaks
• Efficiency - a measure of the narrowness of peak widths
produced by a column
• Selectivity (α) - the ratio of retention of 2 adjacent peaks
• Sample capacity - the maximum sample load for the column

16
 Introduction – HPLC-System: General Design

● Pump with Degasser

● Autosampler
● Column (installed in a Column Compartment)
● Detector
● Computer with control software

Column
Autosampler

17
 Detectors for HPLC

• An HPLC detector is equipped with a small flow cell

connected to the column outlet and monitors the concentration
(or mass) of the analyte component
• Common detectors are:
• UV/Vis absorbance (UV/Vis)
• Fixed or variable wavelength
• Rapid scanning diode array detector
• Fluorescence (Fl)
• Refractive index (RI)
• Mass spectrometry (MS)
• Other detectors
• Electrochemical (ECD), conductivity, infrared, mass
(evaporative), radioactivity and post-column reaction systems

18
 UV/Vis Absorbance Detector
Schematic

19
 Characteristics of a UV/Vis Detector

• UV/Vis absorbance detector typically consists of :

• A deuterium source (190 - 600 nm)
• A monochromator involving a moveable grating controlled by stepper motor
to select a certain wavelength through the exit slit to a small flow cell
• Two photodiodes to measure the light intensity of the sample and reference
beam
• Principle for absorbance detector is the Beer’s Law
• Absorbance = molar absorptivity x pathlength x concentration
• A = e b c = - Log I / I0 where I0 = Initial light intensity
• Is the most common detector for HPLC, capable of ng detection
• Noise/drift characteristics important for sensitivity

20
Photodiode Array Detector
Schematic

• Principles and components similar to that of UV/Vis detector

• A flow cell is placed before a stationary grating and passes the
entire light spectrum of the light source
• A diode array with many elements measures the spectral intensity
of each wavelength
• Records spectra to assist the determination of peak identity and
purity

21
 Diode Array Detection
UV Spectrum of the First Peak (Uracil)

22
 Fluorescence Detector (FL)
Schematic

• A fluorescence compound absorbs a

photon (e.g, p - p * electronic
transition) and emits another photon
at a longer wavelength
• Types of fluorescence detectors are
• Filter/filter
• Filter/monochromator
• Double monochromator
• Programmable
• Fluorescence is 100-1000 times
more sensitive than absorbance
detection (pg detection)

Fluorescence emission is measured at

right angle to the incident light

23
 HPLC/Mass Spectrometry
(LC/MS)
• LC/MS is the ultimate analytical technique which combines the
versatility of HPLC with the sensitivity and identification power of
mass spectrometry
• Interfacing a liquid stream at atm. pressure into the high vacuum of
MS is a challenging task and is made possible with innovations in
interface technology (electrospray or IonSpray)
• Applications of LC/MS is universal though is limited by instrument
cost and operational complexity
• LC/MS is currently prevalent in bio-research, drug discovery,
pharmacokinetic assays

24
 5. HPLC Applications

• Applications Categories

• Pharmaceutical
• Environmental
• Life Sciences and Biotechnology
• Food applications
• GPC/Plastics/Chemicals

25
 Introduction – Size of Stationary Phase Particles

● The smaller the particles, the better the separation performance

● But: Smaller particles generate higher back pressure

Typical Typical Typical

Particle Size Back Pressure Column Diameter
Preparative HPLC 100 – 10 µm 10 - 100 bar 21 mm
Conventional HPLC 5 – 3 µm 100 – 300 bar 4.6 mm
UHPLC ≤ 3 µm > 500 bar 2.1 mm

● HPLC: High Performance Liquid Chromatography

● UHPLC: Ultra High Performance Liquid Chromatography

26
 Column Chemistry – The Column

Continous stream of mobile phase

Mobile
Column Phase
Stationary
Phase

Particles are mostly porous materials and not just spheres with an outer
surface

27
 1. LC Modes and Mechanism

• Normal Phase or Liquid-solid (NP, LSC)

• Reversed Phase (RPC)
• Ion Exchange (IEC)
• Size Exclusion (SEC - GPC or GFC)

28
 Normal-phase or Liquid-solid
Chromatography (LSC)
• Uses silica, alumina, amino-, cyano-, or
phenyl bonded phases
• Uses nonpolar mobile phase such FLOW
as hexane, CH2Cl2 modified with
isopropanol
• Polar analytes has longer retention
times
• Excellent for non-polar analytes,
isomers, group separation, and for
sample clean-up

29
 Reversed-phase Chromatography
(RPC)
• Separation based on partitioning of the
analyte into a hydrophobic stationary FLOW
phase boned on silica support (e.g.,
C18, C8)
• Uses polar mobile phase such as
mixture of methanol/acetonitrile and
water; Polar analytes elute first
while non-polar analytes are
retained more
• Most common HPLC mode used in
> 60% all LC separations
• For polar (water-soluble), medium
polarity, and some non-polar
analytes
• Ionic analytes can be separated
using ion-pairing reagents

30
 Ion-exchange Chromatography (IEC)

• Used to separate ionic or ionizable

+
analytes SO3 Na
Polymeric
• Stationary phase is bonded cationic
or silica p+
(sulfonate) or anionic (Quaternary
support
ammonium) groups on polymeric
materials or silica SO- 3 Na
• Uses mobile phase consisting of
buffered solutions of different pH
and ionic strength
- +
• Common applications are analysis of
ions, amino acids, proteins/peptides,
polynucleotides, etc.
Cationic exchanger

31
 Size-exclusion (SEC or GPC)

• Separation based on sieving action of

cross-linked polystyrene with controlled
pore sizes FLOW
• Common mobile phase is toluene,
THF, CH2Cl2
• Large molecule elutes first while
small molecules penetrate into the
pores and elute later
• Useful for molecular weight
determination of polymers and
biomolecules

32
 Guidelines in LC Mode Selection
Mode is dictated by the structures of analytes

• Macromolecules (MW > 2000)

• Polymers GPC
• Biomolecules (proteins, DNA) SEC, RP, IEC

• Organics (MW < 2000)

• Polar RP, NP
• Medium polarity RP
• Non-polar RP, NP
• Ions, ionizable cpds IEC, RP-ion pair
• Group separation LSC, NP

• Note: For sample preparations -- LSC, GPC (RP) are used

33
 Theory of Chromatography
– Band Broadening (Van Deemter Plot)

B
H =A + + C ⋅ u
u
A
Plate height (HETP)

u
Resistance to mass
transfer C u
B
Eddy diffusion A
Longitudinal diffusion B/u
C
Mobile phase velocity (u)

34
Theory of Chromatography
– The Influence of the Particle Diameter on Resolution

100
Separation on 5 µm material

Response
10 µm particles
5 µm particles
3 µm particles
2 µm particles
H [µm]

Separation on 2 µm material

uopt, 5 µm uopt, 2 µm

Response
Hmin, 5 µm
Hmin, 2 µm
0
0 5 10 0
Time
Linear Velocity u [mm/s]

6 d p2 ⋅ u
H = 2⋅dp + +
u 20

35
Theory of Chromatography
– The Influence of the Particle Diameter on Speed

250 x 4.6 mm; 5 µm

100
L/dp = 50
10 µm particles 1.5 mL/min
5 µm particles
3 µm particles 150 x 4.6 mm; 3 µm
Speed 2.8x
H [µm]

2 µm particles
L/dp = 50
No loss in
uopt, 3 µm 2.5 mL/min resolution
uopt, 5 µm uopt, 2 µm
Hmin, 5 µm
Hmin, 3 µm 100 x 4.6 mm; 2 µm
Hmin, 2 µm Speed 6.2x
0 L/dp = 50
0 5 10 No loss in
Linear Velocity u [mm/s] 3.75 mL/min resolution min
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

36
Theory of Chromatography
– Influence of the Particle Diameter on Pressure

Efficiency
100 1200
Pressure
1000
2 µm particles

Column pressure [bar]

10 µm particles
5 µm particles 3 µm particles
3 µm particles 800 5 µm particles
2 µm particles 10 µm particles
H [µm]

600

400

200
0
0 5 10
0
Linear Velocity u [mm/s]
0 2 4 6 8 10
u[mm/s]

For separation speed – We Need High Pressure and Fast Flows

37
 Theory of Chromatography – Summary

• Plate theory and column Efficiency

• The higher the plate number N the better the efficiency of the column
• Band broadening (van Deemter plot)
• Natural processes result in band broadening
• Resolution is a function of column efficiency, selectivity and retention
characteristics
• Influence of smaller particle diameter
• Higher resolution
• More speed possible
• Higher back pressure generated

38
 Thermo Scientific Dionex U3000 : Standard System

39
 Thermo Scientific Dionex Chromeleon 7 : CDS

40
41
carbohydrates
Carbohydrate Analysis
by IC and HPLC
High Performance Liquid Chro- mixtures existing in nature. Selection High-performance anion-
matography (HPLC) is an important of the optimal HPLC approach depends exchange chromatography with pulsed
tool to identify and quantify carbohy- on the sample matrix, carbohydrate amperometric detection (HPAE-PAD)
drates in food and beverage samples, concentration, selectivity, and sensitiv- and specialized CarboPac® columns
providing key metrics of product quality ity required. solve these chromatographic and
and related properties, contamination, HPLC on aminopropyl-bonded selectivity issues, while also
or adulteration. HPLC plays important silica or polymer-based metal-loaded allowing the determination of alcohols,
roles in quality control, nutritional cation-exchange resins, in conjunction glycols, and aldehydes. HPAE-PAD
labeling, authenticity testing, and with refractive index (RI) or low- can separate sugars, sugar alcohols,
production processes monitoring, for wavelength UV detection, provide oligo-, and polysaccharides with very
example, tracking the fermentation of simple isocratic methods. In most cases, high resolution, without derivatization
alcoholic beverages. HPLC on metal-loaded cation-exchange or pre-concentration. This approach
Separation and detection in resins with RI detection (HPLC-RI) is provides quantification to picomolar
high-concentration carbohydrate used to determine simple mono- levels.2
mixtures, as found in the food and and disaccharides in the g/L range. Dionex offers HPLC-RI and
beverage industry, are made challeng- However, some sample matrices require HPLC-PAD solutions optimized for a
ing by the wide variety of carbohydrate better resolution of sugars from sugar wide variety of research and monitoring
molecules and intricacy of carbohydrate alcohols, organic acids, and sodium applications.
chloride.1

Passion. Power. Productivity.

HPLC-RI for Mono- and Disaccharides
RI is the next most widely-used Column: Aminex HPX87C
1 Eluent: Water
detection method for carbohydrates, as 25
Temperature: 80 °C
other alternatives such as fluorescence Flow Rate: 0.5 mL/min
Inj. Volume: 20 μL
and UV-Vis detectors require Detection: RI
pre-column derivatization of sugars. �RIU 2 3 Peaks: 1. Sucrose
RI allows direct determination and 2. Glucose
3. Fructose
quantification of sugars present in the
percent range of most foods. -1
Metal-loaded cation-exchange 0 2 4 6 8 10 12 15
Minutes
columns provide a simple, non-destruc- 24416

tive method to separate carbohydrates

using a deionized water mobile phase, Figure 1. One-hundred consecutive chromatograms of a high-concentration
which is compatible with RI detection. carbohydrate standard.
These columns separate compounds
using a combination of size exclusion
and ligand exchange mechanisms. 50 Column: Aminex HPX87C
Apple Juice Eluent: Water
For oligosaccharide separations
Temperature: 80 °C
3
using metal loaded columns, size Flow Rate: 0.5 mL/min
Inj. Volume: 20 μL
exclusion is the primary separation Detection: RI
1
mechanism. For monosaccharides, Peaks: 1. Sucrose
ligand exchange dominates. This 2. Glucose
3. Fructose
mechanism involves the binding of
hydroxyl groups in the sugars with the μRIU

fixed counter-ion of the resin. Ligand

exchange is affected by the nature of the 2

counterion (Pb2+, Ca2+, etc.) and by the

spatial orientation of the carbohydrate’s
hydroxyl groups.
Figure 1 shows the good area
-4
repeatability of HPLC-RI, with RSD
of less than 0.8% for 100 consecutive 50
injections of a sugar standard (c = Orange Juice 1

10 g/L each, injection volume: 20 μL).

Peaks: 1. Sucrose
Carbohydrates often occur as major 2. Glucose
components. Levels of individual sugars 3. Fructose

up to 5% may be quantified
(Figures 2 and 3).
μRIU
2 3
HPAE-PAD Techniques
The different selectivity of
ion-exchange CarboPac columns with
respect to metal-loaded columns can
help to better separate carbohydrates
and other species in complex matrices.
-4
Carbohydrates are separated by anion 0 2 4 6 8 10 12 15
exchange chromatography at high pH, Minutes
24417
and detected by pulsed electrochemical
detection. Figure 2. Analysis of glucose, fructose, and sucrose in fruit juices.

2
 At high pH values, carbohydrates
are deprotonated. The resulting anionic 70 Column: Shodex Sugar SC1011
Red Wine Eluent: Water
species can be separated by anion-
Temperature: 80 °C
exchange mechanisms, typically using Flow Rate: 1.0 mL/min
aqueous mobile phases such as NaOH or Inj. Volume: 20 μL
Detection: RI
KOH. For oligosaccharide separations,
μRIU Peaks: 1. Glucose
the mobile phase also contains sodium
acetate. Concentration gradients of
sodium acetate facilitate the elution
of oligosaccharides. 1

High-pH eluents require the use -5

of polymeric columns. Dionex CarboPac
70 3
columns provide the basis for optimized White Wine
carbohydrate separations using
these conditions.

2
Innovative Resin Technology μRIU Peaks: 1. Sucrose
The CarboPac PA20 column uses 2. Glucose
3. Fructose
Dionex pellicular resin technology for
improved chromatographic resolution, 1
peak shape, and efficiency for the six
common monosaccharides. CarboPac -5
0 5 10 15 20
PA20 columns are packed with a Minutes
hydrophobic, polymeric, pellicular 24418

anion exchange resin that is stable Figure 3. Analysis of glucose, fructose, and sucrose in wine.
over pH 0–14. This unique pH-stability
allows the use of eluent compositions
that are conducive to oxidation of
12 Column: CarboPac PA20
carbohydrates at gold electrodes. Eluent: 2 mM NaOH, isocratic
The MicroBead™ latex particle Flow Rate:0.5 mL/min
Detection:Pulsed electrochemical
was optimized to further improve detection, Au electrode
column performance by imparting Waveform: Waveform A

a unique chromatographic selectivity. 1 Peaks: 1. Mannitol

This selectivity results in a significantly nC 4 2. Arabinose
3. Galactose
improved resolution between the 4. Glucose
2 5. Xylose
previously-problematic analytes 3 5
7 6. Mannose
galactose and glucosamine. 6 7. Fructose

Mono- and Disaccharide Separations -4

Using HPAE-PAD 0 2 4 6 8 10 12 14
Minutes
Mono- and disaccharides important 24418

in food analysis are typically separated

at eluent concentrations lower than Figure 4. Separation of coffee sugars using the CarboPac PA20 column.
100 mmol/L NaOH. Coffee sugars,
such as mannitol, arabinose, galactose,
glucose, xylose, mannose, and fructose,
can be separated with 2 mmol/L sodium
hydroxide (Figure 4) using waveform A,
which is described in Dionex Techni-
cal Note 21.3 For outstanding inter-run
consistency, this analysis can be run
using automatically-generated potassium
hydroxide eluent on a Reagent-Free™ IC
(RFIC™) system with Eluent Generation
(RFIC-EG™ system).

3
 The analysis of well-resolved
sugars can be made faster by increasing 50 Column: CarboPac PA20 (3 × 150 mm)
Gradient: 52 mM Sodium hydroxide
the hydroxide concentration. Mono- and Flow Rate: 0.5 mL/min
5
disaccharides important in dietary fiber Detection: Pulsed electrochemical,
disposable gold electrode
analysis require higher concentrations of Waveform: Waveform A
sodium hydroxide for timely elution and
Peaks: 1. Glycerol
are readily eluted in less than 12 min nC 2. Xylitol
2 3 8
with 52 mmol/L sodium hydroxide 3. Sorbitol
1 4
4. Mannitol
7
(Figure 5). This technique provides good 5. Glucose
6. Fructose
resolution between the sugar alcohols 6 7. Sucrose
and sugars in a single isocratic run. 8. Lactose

0
Predictable, High-Resolution 0 5 10 15
Separation of Oligosaccharides Minutes
There is a significant and 18874

increasing demand for reproducible, Figure 5. Separation of sugar alcohols, mono- and disaccharides.
fast, and simple methods to profile
oligosaccharides and homologous sugar
series such as inulins, amylopectins,
180 Columns: CarboPac PA200 (3 × 250 mm)
and maltooligosaccharides in the food Gradient: 120–320 mM NaOAc in 100 mM
industry. Most HPLC approaches NaOH over 40 min
Flow Rate: PA200: 0.5 mL/min
proposed for these applications are Detection: Pulsed amperometry, Waveform A,
limited by insufficient specificity and gold electrode
Samples: Inulin from chicory (Sigma)
high limits of detection.
The CarboPac PA200 is a
nonporous, high-efficiency, polymeric
nC
anion-exchange column that provides
the highest resolution available for
oligosaccharide mapping and analysis
through PAD. The resin consists of 5.5 μm
nonporous beads covered with a fine
layer of functionalized MicroBead latex PA200
particles. This pellicular resin structure
permits excellent mass transfer, result- –20
10 20 30 40 50 60
ing in high-resolution chromatography Minutes
and rapid re-equilibration after gradient 20243

elution. The 3 × 250 mm column format

provides fast separations. The recom- Figure 6. Inulin profile using the CarboPac PA200 column.
mended flow rate of 0.5 mL/min results
in significant savings in eluent
consumption. determined using HPAE-PAD with has developed a more direct HPAE-PAD
gradient elution (Figure 6). method that allows commercially
Linear Polysaccharide Profiling Commercial food ingredient available FOS and inulin products to be
Inulin and fructo-oligosaccharides products derived from the lower-molec- identified and quantified directly in a
(FOS) are increasingly used as functional ular-weight fractions of inulin (DP3-20) variety of foods: Application Note 150,
food ingredients. Chain-length distribution can be determined by AOAC Method Determination of Plant-Derived Neutral
profiles of commercial products such 997.08, an enzymatic preparation fol- Oligo- and Polysaccharides Using the
as those derived from inulin can be lowed by HPLC. However, Dionex CarboPac PA200.4

4
Amylopectins
HPAE-PAD with gradient 330 Column: CarboPac PA200 and guard
elution has been used for structural Eluent: Sodium acetate gradient in
100 mM Sodium hydroxide
studies on starch-derived materials such 70 to 300 mM in 30 min
as amylopectins, since the chain length Flow Rate: 0.5 mL/min
Inj. volume: 5 μL from 10 μL loop
distribution is an important parameter Temperature: 30 °C
Detection: Pulsed amperometry, gold electrode
for characterizing the molecular struc- Sample: Red Hook Amber Ale 1:50 dilution
S
ture. These distributions can be used as Waveform A: Quadruple potential
fingerprints for the amylopectin source
(Figure 7).

Systems for Carbohydrate Analysis 30

0 5 10 15 20
Dionex offers configurable sys- Minutes
tems to support carbohydrate analysis, 24420

from robust basic systems to dual-pump

Figure 7. Amylopectins separated on the CarboPac PA200 column.
models that support parallel, tandem,
and other high-productivity LC tech-
niques.
Optimized configurations for ICS-3000 Standard System for Carbohydrate Analysis by
HPAE-PAD
HPAE-PAD methods include the ICS-
Part Number Description
3000 basic and dual systems described
in the tables to the right. The ICS-3000 061706 SP Gradient Pump with degasser
dual configuration with autosampler 062629 EO Eluent Organizer (includes four, 2-L eluent bottles)
sharing supports one pump performing 063493 EO Regulator Accessory and holder
carbohydrate analysis, while the other 061790 DC module with one temperature zone and one injection valve, micro bore
with an optional ED or CD detector
061718 ED Amperometric Detector (without cell and working electrode)
is available for other ion-exchange
061756 ED Cell with reference electrode and spacer block
determinations for food and beverage
applications (e.g., amino acids, organic 061749 ED Au working electrode, with gasket and polishing kit
acids, inorganic anions and cations, 061360 Chromeleon® CHM-1 (including one timebase)
biogenic amines). PC OptiPlex 745 MT, standard model with 17” TFT, Windows XP Professional
For RI detection, Dionex features
the UltiMate® 3000 basic and x2 Dual
systems, detailed in the tables below. ICS-3000 Dual IC System for Food & Beverage Applications
The x2 configuration with autosampler Part Number Description
sharing supports one pump performing
061710 DP Dual Pump - gradient/isocratic with degasser
carbohydrate analysis with RI detec-
062629 EO Eluent Organizer (includes four, 2-L eluent bottles)
tion, while the other is available for
other gradient HPLC applications with 063493 EO Regulator Accessory and holder
UV detection (e.g., vitamins, organic 061793 DC module with two temperature zones and two injection valves, microbore
acids, PAHs, pesticides). 061718 ED Amperometric Detector (without cell and working electrode)
061756 ED cell with reference electrode and spacer block
061749 ED Au working electrode, with gasket and polishing kit
061714 EG Eluent Generator module
058900 EluGen® II KOH cartridge
060477 CR-ATC Continuously Regenerated Anion Trap Column
063353 EG/DP vacuum degas conversion kit
063104 AS simultaneous injection with no injection valves
061364 Chromeleon CHM-1 (includes 2 timebases)
060728 Chromeleon Server option: PDA licence (3D data acquisition)
PC OptiPlex 745 MT, standard model with 17” TFT, Windows XP Prof.

5
 References UltiMate 3000 Standard System for Carbohydrate Analysis
1. De Vries, J. W.; Nelson, A. L., Food by HPLC-RI
Technology 1994, July, pp. 76–77. Part Number Description
2. Dionex Corporation. Technical Note 5035.9250 SRD-3200 Solvent Rack with two degasser channels
20: Analysis of Carbohydrates by 5035.0010 ISO-3100A isocratic analytical pump
High-Performance Anion-Exchange
5035.0600 UltiMate 3000 Manual Injection Valve analytical/micro, with mounting kit
Chromatography with Pulsed Ampero- and 20 μL sample loop
metric Detection (HPAE-PAD). 2004. 5722.0000 TCC-3000 Thermostatted Column Compartment
3. Dionex Corporation. Technical Note 5060.0030 RI 101 Refractive Index Detector
21: Optimal settings for pulsed 5960.0067 Chromeleon CHM-1 (includes one timebase)
amperometric detection of carbo-
PC OptiPlex 745 MT, standard model with 17” TFT, Windows XP Professional
hydrates using the Dionex ED40
Electrochemical Detector. 1998.
4. Dionex Corporation. Application UltiMate 3000 x 2 Dual-Gradient HPLC System for
Update 150: Determination of Plant- Food & Beverage Applications
Derived Neutral Oligo- and Poly- Part Number Description
saccharides Using the CarboPac™ 5035.9230 Solvent Rack SRD-3600 with six degasser channels
PA200. 2005. 5035.0014 x2 Dual-Gradient Analytical Pump DGP-3600A
5822.0020 Analytical in-line split loop thermostatted autosampler WPS-3000TSL
5722.0010 Thermostatted Column Compartment TCC-3100 1x2P-6P with 2-position
6-port switching valve
6037.0004 Parallel Operation Capillary Kit, Dual-Gradient Analytical
5080.0020 Photodiode Array Detector PDA-3000, without flow cell
6080.0210 Absorbance Cell for PDA-3000, 13 μL, SST, 10 mm path
5060.0030 RI 101 Refractive Index- Detector
5960.0068 Chromeleon CHM-2 for two UltiMate 3000 LC systems
5960.0020 Chromeleon Server option: 3-D Data Acquisition
PC OptiPlex 745 MT, Standard Model with 17” TFT, Windows XP Professional

MicroBead, Reagent-Free, RFIC and RFIC-EG are trademarks and Chromeleon, UltiMate, EluGen, and
CarboPac are registered trademarks of Dionex Corporation in the U.S. and other countries.
Aminex is trademark of BioRad Corporation. Shodex is trademark of Showa, Ltd.

Passion. Power. Productivity.

Dionex Corporation
North America Europe Asia Pacific
1228 Titan Way U.S. (847) 295-7500 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768; (32) 3 353 4294 Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735
P.O. Box 3603 Canada (905) 844-9650 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190
Sunnyvale, CA Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Switzerland (41) 62 205 9966 Taiwan (886) 2 8751 6655
South America
94088-3603 United Kingdom (44) 1276 691722 LPN 1971 8M 09/07
Brazil (55) 11 3731 5140 www.dionex.com ©2007 Dionex Corporation
(408) 737-0700
6
Environmental Analysis Using the ICS-3000 Ion Chromatography System

Ion chromatography (IC) is The ICS-3000 delivers a new level When configured in the dual
well established as a routine method of performance for environmental IC IC mode, the ICS-3000 can easily
for the analysis of ionic analytes in analysis. Equipped with either single or deliver simultaneous anion and cation
environmental samples. IC has been dual eluent pumps, ultrastable detectors analyses, simultaneous multi method
incorporated into environmental (conductivity, amperometry, UV-Vis, analyses, as well as two-dimensional
regulatory methods, worldwide, for MS) and a versatile valving system (2-D) methods that significantly
quantifying contaminants in drink- allows the ICS-3000 to execute even the enhance analysis sensitivity and selec-
ing water, wastewater, surface and most complex IC methods with ease, tivity. Furthermore, the ICS-3000 is the
ground water, rain water, soil extracts, precision and accuracy. ideal RFIC system to couple with mass
and other environmental sample matrixes. The ICS-3000 can easily perform spectrometry (MS) detection, to deliver
Official methods from organizations all the traditional IC methodologies the ultimate in sensitivity and selectiv-
provide detailed instructions on the using, for example, carbonate eluents. ity for ion analysis.
application of IC methods to determine In addition, it can also perform the more
the concentration of regulated ionic advanced methodologies using
contaminants. Examples of such organi- Reagent-Free™ IC (RFIC™) systems.
zations include the U.S. Environmental RFIC systems bring a new level of pre-
Protection Agency (EPA), American cision and accuracy, as well as outstand-
Society for Testing and Materials ing ease of operation, to IC analysis.
(ASTM), International Standardization
Organization (ISO), Deutsches Institut
für Normung (DIN), Association Fran-
çaise de Normalisation (AFNOR), and
Japanese Institute for Standards (JIS).

Passion. Power. Productivity.

IC Analysis Examples
The following figures illustrate 2 4 Column: IonPac AG18, AS18, 4 mm
9 Eluent: 23 mM KOH for 7 minutes
the performance of the ICS-3000 for a 40 mM KOH for 12 minutes
Temperature: 30 °C
broad range of environmental analytical Flow Rate: 1.0 mL/min
applications. Inj. Volume: 50 µL
Detection: Suppressed conductivity,
Figure 1 shows the use of the ASRS® ULTRA II, 4 mm
ICS-3000 RFIC system with hydroxide recycled water mode
µS
eluent generation and IonPac® columns Peaks: 1. Fluoride 0.3 mg/L
2. Chloride 75
for the accurate and precise determina- 3 5 3. Carbonate –
tion of common anions in environmen- 4. Sulfate 40
5. Nitrate 4.0
tal water samples. 1 6. Phosphate 0.5
6
• This combination has approval 0
for drinking water and interim 0 2 4 6 8 10 12 14 16 18 20
Minutes
approval for wastewater compliance 21140
monitoring, using U.S. EPA Method
300.0(A) and 300.1(A). Figure 1. Determination of inorganic anions in drinking water.
Figure 2 shows the use of the
ICS-3000 RFIC system mode with the
hydroxide-selective IonPac AG19 and
AS19 ion-exchange columns, for the 1.5 Column: IonPac AG19, AS19, 4 mm
accurate and precise determination of Eluent: 10–45 mM KOH in 15 minutes
Temperature: 30 °C
common anions and oxyhalides, such Flow Rate: 1.0 mL/min
Inj. Volume: 200 µL
as bromate, chlorite, and chlorate, in Detection: Suppressed conductivity,
4
environmental water samples. ASRS ULTRA II, 4 mm
recycled water mode
• This combination is approved for µS 5
Peaks: 1. Chlorite 4.54 µg/L
compliance monitoring using U.S 2. Bromate 1.98
3. Nitrite –
EPA Method 300.0 (A and B) and 4. Chlorate 55.5
300.1 (A and B). 3
5. Bromide 33.5

• Using this combination, bromate can 1

2
be determined in municipal drinking
0.5
water and bottled drinking water, 0 5 10 15 20 25 30
with a detection limit (DL) of Minutes
21138
0.3 µg/L (ppb).
• The high sensitivity performance of Figure 2. Determination of oxyhalides and bromide at trace-level concentrations in drinking
water.
this method is more than adequate
to do compliance monitoring of
bromate in ozonated drinking water,
where the maximum contaminant 1.0 Column: IonPac AG19, AS19, 4 mm
3 4 Eluent: 10–45 mM KOH in 15 minutes
limit (MCL) is 10 µg/L. Temperature: 30 °C
Figure 3 shows the use of the Flow Rate: 1.0 mL/min
Inj. Volume: 500 µL
ICS-3000 with a large loop injection Detection: Suppressed conductivity,
(500 µL) for the determination of sub- ASRS ULTRA II, 4 mm
recycled water mode
ppb level bromate in drinking water. µS Peaks: 1. Chlorite 6.52 µg/L
1
• The high capacity of the AS19 2. Bromate
3. Chlorate
0.68
57.0
column, combined with the good 2 4. Bromide 44.5
separation of bromate from chlo-
ride, and the superior stability of the
ICS-3000 pump and conductivity 0.5
detector, ensures excellent low- 0 5 10 15 20
Minutes
level bromate analysis, even when
21139
analyzing high TDS drinking water
samples. Figure 3. Determination of bromate at concentrations below 1 ppb in drinking water.


 Figures 4a and 5 show the use of
the ICS-3000 dual IC system to deliver Pump
Cell 2
greater sensitivity and selectivity for EG Autosampler
selected ion analysis (e.g., bromate) Injection Valve 1
Large Loop
using 2-D IC. Figure 4a is a schematic Suppressor
diagram showing how the ICS-3000 is
configured in a 2-D mode for analysis Injection Valve 2
of low-level bromate in environmental Concentrator
Column
water samples. Two distinct separation 4 mm
Column
channels or columns are set up in the Cell 1
2 mm Column
DC Detector Chromatography module
of the ICS-3000 and used to automate
Suppressor Pump EG
this 2-D IC application. In this case,
the first IC channel is used to separate
bromate from other potentially interfer- Load Transfer to 2-D
Inject Load Concentrator
ing ions (e.g., chloride), using a high
capacity 4-mm AS19 column, and to
transfer it to a concentrator column. The Figure 4a. Configuration of the ICS-3000 for 2-D IC determination of trace-
level bromate and perchlorate in environmental water samples (see Figures 5c,
concentrator column contents are then
5d, and 9).
transferred to a lower capacity 2-mm
AS19 column for independent separa-
tion and detection in the second IC
channel. This mode of operation is best
accomplished using RFIC hydroxide
selective, high-capacity
column chemistry to:
• Enhance sensitivity, since the large,
500 µL sample is ultimately chroma-
tographed using a 2-mm column.
• Enhance selectivity, since the se-
lected ion (e.g., bromate) is chroma-
tographed a second time with fewer Figure 4b. The AM Automation Manager mounts inside the Detector
Chromatography compartment (DC) and integrates up to two high-pressure
matrix ions, which significantly valves, two solenoid valves, and a postcolumn heater. The entire AM mounts
improves peak shape and minimizes on a moveable slide for easy configuration for any application. All components
potential interferences. are completely controlled through Chromeleon.
The suppressed hydroxide elu-
ent is water, which provides an ideal
medium for trapping, focusing, and in the AM while injection is controlled ming and eliminating the use of
concentrating the ions of interest eluted by in Injection Valve 2 within the DC. TTL contact closures. Each valve is
from the first dimension (1-D) separa- This allows the entire process—sample individually named so that it is easy
tion onto the concentrator column in the preparation, injection, separation, and to synchronize fluidic pathways and
second dimension. analysis—to occur in the temperature- to simplify troubleshooting.
controlled DC compartment. The result
For complex applications (Figure • Use the AM as a separate injection
4a), the ICS-3000 delivers a solution: is greater reproducibility for complex
system. With a dual-heated zone,
the AM Automation Manager, shown applications.
you can run applications at different
in Figure 4b. Typically, adding extra • By placing the AM within the DC, temperatures. For example, channel
valves, plumbing and determining void samples are preequilibrated for the one can have the injection valve and
volumes is cumbersome and tedious. precise temperature of the separa- column in the lower chamber at one
With the AM, complex plumbing con- tion. This provides greater consis- temperature, while channel two can
figurations, such as that used in the 2-D tency from run to run. have the injection valve and column
preconcentration for bromate (Figure • Control all valves with Chromeleon® in the upper chamber at a different
4a) are easier to implement. software, simplifying program- temperature. Applications with typi-
Preconcentration is controlled cal loop injection or preconcentra-
independently with Injection Valve 1

 tion and matrix elimination can be
accomplished on any channel. (A) 5 ppb BrO3– in Low Salt Water Column: AG19, AS19, 4 mm
0.4 Eluent Source: EGC KOH
2 3
Flow Rate: 1.0 mL/min
Figure 5a shows the result of a Suppressor: ASRS ULTRA II, 4 mm
1-D separation on a 4-mm column of a Current: 113 mA
Loop: 500 mL
500-µL injected sample containing µS
Oven: 30 °C
5 µg/L bromate in a low salt matrix. 1
Gradient: Time mM
Here the bromate peak is small but 0 10
–0.1 10 10
quantifiable. 25 45
Figure 5b shows the result of a 30 45
(B) 5 ppb BrO3– Sample Spiked with 30.1 10
1-D separation from a 500-µL injected 250 ppm each Cl– and SO4– 35 10
0.4
sample also containing 5 µg/L bromate 2 3 Peaks A B
1. Bromate 0.005 mg/L –
in a high salt matrix. Here the bromate 2. Chloride 0.030 250– mg/L
peak is not distinguishable from base- µS
3. Sulfate 0.150 250

line variation.
Figure 5c shows the result of a
2-D separation of bromate from the low –0.1
salt matrix that was trapped on a con-
(C) 5 ppb BrO3– in Low Salt Matrix
centrator column and transferred to 2-D 0.4
Primary Secondary
Column: AG19, AS19, 4 mm AG19, AS19, 2 mm
column (2-mm) for further separation Eluent Source: EGC KOH EGC KOH
and detection. The bromate peak from Flow Rate: 1.0 mL/min 0.25 mL/min
Suppressor: ASRS ULTRA II, 4 mm ASRS ULTRA II, 2 mm
the secondary column is larger than µS Current: 113 mA 29 mA
from the 1-D column. Loop: 500 mL
1 Concentrator: TAC-ULP1
Figure 5d shows the result of a Oven: 30 °C 30 °C
–0.1
2-D separation of bromate from the high Gradient:
Time mM mM
salt matrix sample that was trapped on (D) 5 ppb BrO3 Sample Spiked with
– 0 10 10
a concentrator column and transferred 0.4 250 ppm each Cl– and SO4– 10 10
19.5 10
to a second column (2-mm) for further 25 45
separation and detection. The bromate 30 45
µS 30.1 10
peak from the second column is now 34.5 45
clearly distinguishable as compared to 35 10 45
1
the 1-D column results. Furthermore, Peaks: C D
–0.1 1. Bromate 0.005 mg/L 0.005 mg/L
this bromate peak is the same size as 0 5 10 15 20 25 30 35 2. Chloride 0.030 250
3. Sulfate 0.150 250
that obtained from the low salt sample. Minutes
21113-01
Thus, this mode of operation yields
greater sensitivity and minimizes inter-
Figure 5. The ICS-3000 dual IC system delivers greater sensitivity and selectivity for selected
ference from the high salt matrix. ion analysis (e.g., bromate).
This mode of operation is
sometimes called heart-cutting. This
technique is used to isolate unresolved
solutes from one separation and place
the mixture on another column that
resolves the solutes. In this example,
the technique is used as a form of
enrichment process where the solute
of interest, e.g., bromate, is non-
detectable due to the presence
of large amounts of chloride.


 Figure 6a shows the use of the
ICS-3000 in the RFIC mode with the 1.5
2 Column: IonPac CG16, CS16, 5 mm
A Eluent: 26 mM MSA
CG16 and CS16 cation-exchange Eluent Source: EGC KOH
columns, for accurate and precise Temperature: 30 °C
Flow Rate: 1.5 mL/min
determination of common cations 6 Inj. Volume: 10 µL
5 Detection: Suppressed conductivity,
and ammonium using ASTM Method
CSRS®, VLTRA II 4 mm
D6919-03. recycled water mode
µS
• This method has been proposed Peaks: 1. Lithium
2. Sodium 149
0.04 mg/L

by the U.S. EPA for compliance 4 3. Ammonium 0.04

4. Potassium 1.20
monitoring of drinking water and 1 5. Magnesium 2.19
wastewater. 3 6. Calcium 4.76

Figure 6b shows how the CS16 0

column gives sufficient resolution 1 2 4 5 6

0.22
of sodium and ammonium to allow B
quantification of ammonium at the µg/L
level, even in the presence of high mg/L
levels of sodium.
Figure 7 shows the use of the
ICS-3000 dual RFIC IC system to µS
3
deliver simultaneous anion and cation
analyses of the same sample, using
only one autosampler. In this case, the
autosampler loads the injection loop of
each IC channel with the same sample, 0.12
0 5 10 15 20 25 30
so that anions and cations can be ana- Minutes
22693
lyzed simultaneously, without cross-
contamination of eluents. By providing Figure 6. Determination of common alkali and alkaline earth cations
simultaneous analysis of anions and and ammonium using an RFIC system.
cations, the dual ICS-3000 increases IC
sample throughput without taking more
benchspace than a single system. Series Analysis ANIONS CATIONS 36 Injections—21 Hours
The Chromeleon data system
manages detection and quantitation and ANIONS
ICS-3000 18 Injections—12 Hours
delivers a report which coordinates the Simultaneous Analysis CATIONS

anion and cation results for each sample

in a convenient, easy to read format. 14 Chloride
Note that this simultaneous mode of Bromate
Sulfate
Phosphate
dual channel IC operation can also be µS Fluoride Nitrite Bromide
Chlorite
Chlorate Nitrate 7 minute gradient
used for simultaneous small loop and Carbonate equilibration
large loop anion or cation analysis. 0

• This mode of operation is especially 0 5 10 15 20 25 30 37

Lithium Sodium
beneficial when analyzing samples 4 Ammonium Potassium Magnesium Calcium
of unknown ionic concentration µS
levels, where some are suspected to
0
be of low concentration and others 0 5 10 15 20 26
of high concentration. For example, Minutes

one IC channel with a 100 µL loop

can be calibrated from 10 ppb to
1000 ppb (1 ppm), while the other Figure 7. The AS Autosampler with simultaneous analyses provides nearly twice the throughput
compared to traditional IC systems.
IC channel with a 10 µL loop
can be calibrated from 1 ppm
to 100 ppm.


• Using simultaneous dual channel IC 1
Column: IonPac AG20, AS20 2 mm
operation samples can be accu- Eluent: 0.5 mM NaOH 0–12 min
100.00
rately analyzed over four orders of 65 mM 12.1–28 mm, 100 mM
28.1–35 min
magnitude, i.e., 10 ppb to 100 ppm, Eluent Source: EGC NaOH with CR-ATC
without any advanced estimate of Temperature: 35 °C
65.00 Flow Rate: 0.25 mL/min
analyte concentrations. This virtu- Inj. Volume: 2 mL
ally eliminates sample dilution and µS Rinse Volume: 1 mL (10 mM NaOH)
B: Without CRD Concentration: IonPac Cryptand C1 (4 × 35 mm)
time consuming sample reruns. Detection: ASRS ULTRA II, 2 mm
2
1 AutoSuppression external water mode
Figure 8 shows the use of the Peaks: 1. Perchlorate 0.5 µg/L (ppb)
2. Unknown —
ICS-3000 system for the determina- 1 2
tion of sub-ppb levels of perchlorate in Concentration: 0.50 mM
A: With CRD
drinking water by EPA Method 314.1. 0
Typical levels of TDS in the drinking 0 5 10 15 20 25 30 35
Minutes
water range from 30 to 70 ppm each for 22685

chloride and sulfate, as well as residual

Figure 8. Chromatogram showing 0.5 ppb (µg/L) perchlorate in Sunnyvale drinking water.
ppm levels of carbonate. These anions
can interfere with the accurate deter-
mination of ppb levels of perchlorate.
Figure 8 chromatograms are shown with opportunity to confirm that the
and without the use of the new Dionex
• Whether you choose simultaneous
peak quantified is actually or sequential injection, one autosam-
Carbonate Removal Device (CRD). The perchlorate. pler is shared between two indepen-
CRD removes residual amounts of car-
bonate from the sample and facilitates
• The ICS 3000 dual RFIC IC system dent systems.
allows the simultaneous determina- • Combine detectors for conductivity,
enhanced detection and quantitation of
tion of perchlorate, using both the electrochemical, or UV detection
sub-ppb perchlorate in drinking water
primary and confirmatory columns, on the same sample or different
samples. Benefits of using the ICS-3000
simultaneously from only one injec- samples. Virtually any analysis com-
for perchorate analysis by Method 314.1
tion and one autosampler. bination is possible—anions/cations,
include the following:
• Using this instrument/column com- • Sequential analysis increases flex- anions/trace metals, etc.
ibility by using a diverter valve
bination, and sodium hydroxide elu-
with the AS autosampler to deliver
ent generation, 0.05 µg/L detection
any sample to any channel, one at
limits for perchlorate are achieved,
a time on a first come, first served
even with a common anion concen-
basis. With this capability, you can
tration of up to 1000 mg/L, each, of
introduce the same sample or two
chloride, bicarbonate, and sulfate.
different samples to two independent
• EPA Method 314.1 documents eluent streams.
this method and details the use of
a primary column set (AG16 and
AS16) and a confirmatory column
set (AG20 and AS20) to provide the


 As shown previously in Figure
A: 0.5 ppb ClO4– spiked with matrix ions (1000 ppm Cl–, HCO3–, SO42–)
4, the ICS-3000 can be configured to 2.0
perform 2-D IC for enhanced detection
sensitivity of low-level environmen-
tal contaminants such as perchlorate. µS 1
Primary Secondary
IonPac Column: AG20, AS20 4 mm AG20, AS20 2 mm
Figure 9 demonstrates the effectiveness Flow Rate: 1.0 mL/min 0.25 mL/min
of the ICS-3000 setup in the 2-D mode Concentration: 35 mM KOH 60 mM KOH
Suppressor: ASRS ULTRA II 4 mm ASRS ULTRA II 2 mm
with an IonPac AS20 4-mm column Current: 87 mA 38 mA
0.5
in the first dimension and a 2-mm Loop: 4000 mL
25 30 35 40 45
Concentrator: TAC-ULP1
AS20 column in the second dimension. B: 0.5 ppb ClO4– in Reagent Water Temperature: 30 °C
2.0
Chromatogram 9A shows the analysis Matrix A B
of 0.5 µg/L perchlorate in a simulated Chloride 1000 ppm 0
Bicarbonate 1000 0
high TDS matrix of 1000 ppm each of Sulfate 1000 0
µS
chloride, carbonate, and sulfate. Figure
1 Peaks: 1. Perchlorate: 0.5 µg/L (ppb)
9B shows the same analysis in reagent
water. The perchlorate peak areas in
these two analyses are virtually identi- 0.5
25 30 35 40 45
cal. Further, interference and tailing Minutes 22694
from the other high-concentration ma-
trix anions is significantly reduced. This Figure 9. Two-dimensional perchlorate analysis with the IonPac AS20 column.
demonstrates the power of 2-D IC on
the ICS-3000 for detecting and quanti-
fying sub-ppb levels of perchlorate in
challenging matrices.

What the ICS-3000 Means to You

The ICS-3000 brings a new level
of precision, accuracy, versatility, and
ease of operation to environmental
ion analysis. Dual pump and dual IC
configurations add new capability to
IC methodology. Heart-cutting meth-
ods, simultaneous method analysis and
postcolumn reagent addition methods
are easily and reliably performed using
the new ICS-3000.
The robust ICS-3000 delivers a
whole new level of productivity, power,
and sensitivity to the environmental
analysis laboratory.


 Reagent-Free and RFIC are trademarks and ASRS, Chromeleon, CSRS, and IonPac are registered trademarks of Dionex Corporation.

Passion. Power. Productivity.

Dionex Corporation North America Europe Asia Pacific

1228 Titan Way Sunnyvale, CA (408) 737-8522 Westmont, IL (630) 789-3660 Austria (01) 616 51 25 Belgium (03) 3-353 42 94 Australia (61) 2 9420 5233 China (852) 2428 3282
P.O. Box 3603 Houston, TX (281) 847-5652 Atlanta, GA (770) 432-8100 Denmark 36 36 90 90 France 01 39 30 01 10 India 91-22-28475235 Japan (06) 6885-1213
Sunnyvale, CA Marlton, NJ (856) 596-0600 Canada (905) 844-9650 Germany 06126-991-0 Italy (06) 66 51 50 52 Korea (2) 2653 2580
LPN 1783 8M 02/06
94088-3603 The Netherlands (0161) 43 43 03 Switzerland (062) 205 99 66 ©2006 Dionex Corporation
(408) 737-0700 www.dionex.com United Kingdom (01276) 691722
Related Dionex Applications Literature
Dionex has an extensive library of methods and techniques for determining a wide variety of analytes im-
portant to chemical industries. Below is a selected list of applications covered in this brochure. For more in-
formation, please visit www.dionex.com, click on the Markets tab, and select Chemicals. All of the literature
below and more can be found by clicking the Documents tab and selecting Application Notes and Updates,
or by contacting your local Dionex representative. Corporate Headquarters
Chemical Analysis Literature Dionex Corporation
1228 Titan Way
Contaminant Matrix Resource
P.O. Box 3603
Alcohols, carbohy- AN 122: The Determination of Carbohydrates, Alcohols, and Glycols in Sunnyvale, CA 94088-3603
Fermentation broths
drates, glycols Fermentation Broths
Tel: (408) 737-0700
AN 188: Determination of Glycols and Alcohols in Fermentation Broths Using Fax: (408) 730-9403
Alcohols, glycols Fermentation broths
Ion-Exclusion Chromatography and Pulsed Amperometric Detection
Worldwide Sales and Service
AU 155: Determination of Cations and Amines in Hydrogen Peroxide by
Amines, cations Hydrogen peroxide North America
Ion Chromatography Using a RFIC (Reagent-Free) System
US/Canada (847) 295-7500
AN 141: Determination of Inorganic Cations and Ammonium in Environmental Waters
Amines, cations Wastewater
by Ion Chromatography Using the IonPac CS16 Column South America
Brazil (55) 11 3731 5140
Anions Concentrated acid AN 78: Determination of Trace Anions in Concentrated Hydrofluoric Acid
AN 93: Determination of Trace Anions in Concentrated Bases Using Europe
Anions Concentrated base
AutoNeutralization Pretreatment/Ion Chromatography Austria (43) 1 616 51 25
AU 163: Determination of Trace Anions in Organic Solvents Using Benelux (31) 20 683 9768
Anions Organic solvents (32) 3 353 42 94
Matrix Elimination and Preconcentration
Denmark (45) 36 36 90 90
Anions Toothpaste AN 156: Determination of Anions in Toothpaste by Ion Chromatography
France (33) 1 39 30 01 10
Anions Wastewater AN 135: Determination of Inorganic Anions in Wastewater by Ion Chromatography Germany (49) 6126 991 0
AN 123: The Determination of Inorganic Anions and Organic Acids in Ireland (353) 1 644 0064
Anions, organic acids Fermentation broths
Fermentation Broths Italy (39) 02 51 62 1267
Anions, organic acids Personal care AN 104: Analysis of Personal Care Products by Ion Chromatography Sweden (46) 8 473 3380
Switzerland (41) 62 205 9966
Anions, oxalate Bayer liquor AN 206: Determination of Oxalate and Other Anions in Bayer Liquor Using IC
United Kingdom (44) 1276 691722
Anions, perchlorate Fertilizer AN 144: Determination of Perchlorate in High Ionic Strength Fertilizer Extracts by IC
Asia Pacific
Aromatic antioxidants Polymers AN 331: Accelerated Solvent Extraction (ASE) of Additives from Polymer Materials
Australia (61) 2 9420 5233
Carbohydrates Alternative fuels AN 363: Using Accelerated Solvent Extraction in Alternative Fuel Research China (852) 2428 3282
AN 225: Rapid Method for the Estimation of Total Free Monosaccharide Content of India (91) 22 2764 2735
Carbohydrates Biomass Japan (81) 6 6885 1213
Corn Stover Hydrolysate Using HPAE-PAD
Korea (82) 2 2653 2580
AN 203: Determination of Cations in Biodiesel Using a Reagent-Free Ion
Cations Biodiesel Singapore (65) 6289 1190
Chromatography System with Suppressed Conductivity Detection
Taiwan (886) 2 8751 6655
AN 94: Determination of Trace Cations in Concentrated Acids Using
Cations Concentrated acids
AutoNeutralization Pretreatment/Ion Chromatography
Chloride, sulfate Ethanol AU 161: Determination of Sulfate and Chloride in Ethanol Using Ion Chromatography
Chloride, sulfate Methanol AN 201: Determination of Chloride and Sulfate in Methanol Using Ion Chromatography www.dionex.com
AN 80: Determination of Dissolved Hexavalent Chromium in Drinking Water, Ground-
Chromium (VI) Wastewater
water, and Industrial Wastewater Effluents by Ion Chromatography
Corrosion inhibitors Engine coolants AN 204: Improved Method for Determination of Corrosion Inhibitors in Engine Coolants
Dionex products are designed,
Cyanide Alkaline solutions AU 107: Determination of Cyanide in Strongly Alkaline Solutions developed, and manufactured
under an ISO 9001 Quality System.
Ethanolamines Industrial waters AU 138: Determination of Ethanolamines in Industrial Waters by Cation-Exchange
Halides, Sulfur All Brochure: Combustion IC System
Phenols Wastewater AU 119: Determination of Phenols
AN 336: Accelerated Solvent Extraction (ASE) of Plasticizers from MSQ and MSQ Plus are trademarks of Thermo Fisher Scientific.
Plasticizers Polyvinyl chloride
Poly(vinyl chloride) Polymer Venture is a trademark of W. R. Grace and Co.
Link
DCMS , Reagent-Free, RFIC, and ultra are trademarks and Acclaim, ASE,
Metal cyanides Wastewater AU 147: Direct Determination of Metal Cyanides by IC with UV Absorbance Detection
ASRS, AutoSuppression, AutoTrace, CarboPac, Charged Aerosol Detection,
Sulfate Brine AN 53: Method for the Determination of Trace Sulfate in Brine Chromeleon, Corona, CAD, CSRS, IonPac, and UltiMate are registered

Thiosulfate, trademarks of Dionex Corporation.

Wastewater AN 138: Determination of Thiosulfate in Refinery and Other Wastewaters
thiocyanate
© 2010 Dionex Corporation
Transition metals Complex matrices TN 25: Determination of Transition Metals in Complex Matrices by Chelation IC LPN 2388 10M 01/10 Printed in U.S.A.

Passion. Power. Productivity.

 Quality, Safety, and
the Environment

Chemical manufacturers are under increasing pressure to expand their markets and launch new products in the face of tough
environmental and economic conditions. This puts renewed focus on process improvement, quality assurance, and the challenge to
develop new analytical methods that ensure the safety and efficacy of chemicals and the products that are made from them.

Industrial manufacturers such as petroleum important to ensure product quality and Dionex has developed products and
refineries and chemical producers struggle safety. For example, phthalates used as solutions that address the challenging task
to obtain reliable, low-cost sources of plasticizers are potential carcinogens. Cation of isolating and identifying contaminants
raw materials for their process needs. contamination of acids, such as phosphoric in chemical and petrochemical products to
Trace contaminants present in solvents or acid used in the production of soft drinks, protect manufacturing equipment, increase
starting reagents such as bases, acids, or has been linked to increased urinary calcium productivity, and ensure only safe, reliable
feed gasses used during manufacturing can which is associated with hypertension. products come to market. Dionex innovation
introduce contaminants, which can not only Ions such as bromate or chromium VI are coupled with years of experience working
cause corrosion damage to costly chemical carcinogenic at very low concentrations. with industry and regulatory experts has
infrastructure and reduce product yields, they produced a broad range of liquid sample
The pollution emitted by internal combustion
can be potentially dangerous. prep, extraction, IC, and HPLC solutions for
engines greatly impacts the quality of our air.
the analysis of trace-level contaminants in
Ensuring the quality of monomers used in With more stringent regulatory standards by
raw materials, chemical intermediates, and
food-grade plastics, as well as the chemical the US EPA limiting the sulfur content of fuels
finished products.
formulations used in the production of such as diesel, gasoline, and ethanol, the
consumer products like adhesives, paints, analysis of total sulfur and individual sulfur-
inks, cosmetics, detergents, and many containing components is becoming more
pharmaceutical products is extremely challenging and important.

page 2
System Solutions

Solvent Extraction Ion Chromatography Liquid Chromatography

Extraction of plasticizers from PVC polymers, Ion chromatography (IC) is well established The Dionex UltiMate® 3000 series of liquid
water-soluble compounds in plant biomass, as a routine analytical technique, and is chromatography systems are ideally suited
azo dyes in textiles, and active ingredients specified in many required ASTM regulatory for the determination of monomer quality for
in consumer product formulations, including methods for the production of fuels and polymer production, PAHs in fuels, chemical
chemicals. Dionex is the leading provider of
adhesives, paints, inks, cosmetics, detergents formulations in consumer products such as
IC instrumentation, with innovative solutions
and many pharmaceutical products, is herbicides, disinfectants, and cosmetics, and
that fit your workflow and your budget.
required to ensure the safety and efficacy monitoring workplace contaminants to ensure
of industrial products. Dionex Accelerated • Reagent-Free™ IC (RFIC™) systems reduce compliance with regulatory methods. Dionex
Solvent Extraction (ASE®) systems use eluent and regenerant preparation while offers a wide variety of modules, detectors, and
providing greater reproducibility and
elevated temperatures and pressures to configurations to suit your application needs.
confidence in your results
extract analytes quickly and efficiently.
• Dual systems allow double throughput and • Excellent retention time precision, detector
Alternative techniques, including Soxhlet
simultaneous analysis sensitivity, linearity, and drift specifications
extraction, microwave-assisted extraction,
• Configurable for on-line process monitoring • x2 dual systems for doubled throughput or
and supercritical fluid extraction, are not only
using Integral™ process analytical systems multidimensional separations
much slower than ASE systems, they also
require the handling and disposal of large • Suppressed conductivity, pulsed • Rapid Separation LC (RSLC) systems for
volumes of hazardous organic solvents, and amperometry, UV-vis, fluorescence, and fast, high flow-rate, ultrahigh-pressure
MS detectors applications
additional cleaning steps after extraction.
• Ion-exchange, ion-exclusion, carbohydrate, • Configurable for on-line process monitoring
• Extraction of sample sizes from 1 to 100 g using Integral process analytical systems
and organic acid columns
in minutes, not hours
• Integrated control and data processing • Reversed-phase, ion-exchange,
• Automation reduces operator time mixed-mode (HILIC, reversed-phase,
with the Chromeleon® Chromatography
and labor and ion-exchange combinations), and
Data System
• Dramatic solvent reduction compared to monolith columns
traditional extraction methods • Diode array, multi-wavelength,
• Compatible with a wide variety of matrices, fluorescence, electrochemical, and
including acidic and alkaline MS detectors

ASE Series Solvent Extractors ICS Series UltiMate Series

IC/RFIC Analytical Systems HPLC/RSLC Analytical Systems

page 3
 Fine and Specialty Chemicals

Fine and specialty chemical manufacturers produce a broad range of products used in many different industrial applications. From
reagents and solvents to monomers for acrylates and other polymeric materials, from intermediate chemicals such as alcohols, amides,
ethers, and acids to pigments, inks, paints, adhesives, and coatings. Dionex provides instrumentation to separate, isolate, and identify
components and contaminants for a variety of chemicals.

Analysis of Ionic Liquids Trace Anions in Organic Solvents EMIM

10
+
N N

Ionic liquids (IL) are a new generation of Anion contamination introduced during
HMIM
solvents with a substantial potential for manufacturing processes can damage BMIM
+
N
+
N
N N

replacing conventional organic solvents in equipment and ruin final products. This is 7
OEIM
numerous technical applications. They are especially important in the manufacturing of
+
N N

organic salts with a melting point below semiconductors and computer components, 5
30 °C

100 °C. They have very low vapor pressure where trace contaminants can cause short

κ/µS/cm
40 °C
and flammability, conduct electric current, circuits, defects in deposition, and corrosion, 50 °C
2
and have selective dissolving properties. reducing yields and increasing manufacturing
60 °C
Modifying the functional groups of the cation costs and waste. IC with automated eluent
70 °C
or the anion allows ILs to be used as tailor- generation provides a convenient method to 0

made solvents, modifiers, and reagents in successfully determine trace anions from high
organic synthesis and homogeneous catalysis. ng/L to low μg/L concentrations in solvents. -2
0 5 10 15 20 24
Automated matrix elimination prevents Minutes
Process monitoring during IL production and column fouling, and analyte concentration 26772

process control during their use require ef- improves detection limits. The method easily Effect of column temperature on retention time
ficient qualitative and quantitative analysis. meets the Semiconductor Equipment and of structurally similar cations in ionic liquids,
The inherent charges of ILs make IC with using an IonPac® CS17 column and suppressed
Materials International (SEMI®) specifications conductivity detection.
suppressed conductivity detection an ideal for solvents.
analytical method for determining these com-
plex electrolyte systems. Chromatographic
3.0 Peaks: 1. Acetate ND* µg/L
separations of ILs can be optimized by 2. Formate ND
varying the acidic or basic eluent concentra- 3. Lactate ND
4. Chloride 3.00
tion, adding an organic modifier, and using an 5. Nitrite ND
elevated separation column temperature. 6. Carbonate ND
7. Sulfate 1.88
µS 4 8. Nitrate 1.34
*ND = not determined
3
5 7
2
6 8
1

0.6
5 10 15 20 25 30 35 40
Minutes 24441

Trace anionic contaminants found in a 2-propanol

sample, after automated concentration and matrix
elimination.

page 4
Contaminants in Acids and Bases overloading, permitting the determination
Peaks µg/L
of μg/L and ng/L concentrations of anions 1. System –
Determination of ions and metals in 2. Lithium 13.8
and cations.
concentrated acids and bases is important 3. Sodium 62.4
to many industries, including chemical, 4. Ammonium ND
Chelation IC is ideal for determination of 4 5. Methylamine ND
electronics, pharmaceutical, food, and 0.75
transition metals in organic solvents and 6. Dimethylamine ND
beverage manufacturing, as well as industrial concentrated acids and bases. 7. Potassium 133.3
8. Magnesium 69.2
plating, environmental monitoring, and 3
9. Trimethylamine ND
mining. Ionic contaminants can combine with Ions and Amines in H2O2 2 10. Calcium 137.3

their respective counterions to form insoluble Hydrogen peroxide is an essential chemical µS

7
compounds that can cause manufacturing used in a number of manufacturing
defects. Calcium in phosphoric acid enhances 8
processes. For polymerization and especially 1 10
the extraction of uranium, but when present for semicunductor manufacturing, purity is 5 6 9

in phosphate fertilizers may contaminate soil. critical. An IonPac CS17 column can be used to
0.35
Excessive amounts of potassium and sodium separate amines without the organic modifiers 1.4 10 20 30 35
in foods can be a health risk. Contaminants required with older cation-exchange columns, Minutes
24533
in concentrated acids and bases must be and an RFIC system with eluent generation
monitored for safety and quality control. permits gradient separation with the ease of Trace cations in 24% sulfuric acid spiked with
the Combined Six Cation Standard-II. The acid
Direct injection of concentrated acid or base use of an isocratic system, using Chromeleon is automatically neutralized using the CSRN II
samples often results in overloading of the software to program the gradient. suppressor, and then injected directly onto an
IonPac CS16 column.
IC column, resulting in poor chromatography Cations or anions can be determined using the
and unreliable quantification. Sample dilution online matrix elimination technique described
can reduce column overloading, but sacrifices in Dionex Application Update 163. However, Peaks: 1. Sodium 0.28 µg/L
detection limits. An AutoNeutralization™ exposure to hydrogen peroxide may reduce the
2. Ammonium (from blank)
3. Potassium 0.13
suppressor eliminates the need to dilute concentrator column lifetime. 4. Unknown —
concentrated acids or bases to avoid column 5. Unknown —
0.5
6. Trimethylamine 0.26
7. Magnesium 0.25
8. Calcium 2.0

8
µS

6
5 7
2 4
1 3

–0.1
0 10 20 30 40 50
Minutes
23233

Trace amines and cations found in a hydrogen

peroxide sample, after treatment with platinum to
remove the H2O2.

page 5
 Consumer Products

Chemical formulations are combinations of compounds that do not react and are designed to yield a mixture with specific desired
characteristics. Many common consumer products, such as adhesives, paints, inks, cosmetics, and detergents, have complex
formulations. They require precise amounts of active ingredients, plus inert ingredients, such as surfactants, to ensure product safety,
consistency, and applicability. The UltiMate 3000 systems with Acclaim® columns, and Dionex IC systems with IonPac columns, are
ideal for separating components of product formulations for process monitoring, quality control, safety, and regulatory compliance.

Surfactant Analysis Allantoin and Urea in Cosmetics 200 1

A
Benzalkonium chloride is a cation surfactant Allantoin is frequently added to preparations n-C12
sometimes used as an antiseptic in consumer used to treat minor cuts, scrapes, burns,
mAU
products. It has applications in products such sunburn, fever blisters, diaper rash and n-C14

as fungicides, antiseptics, and anti-bacterial chapped skin and lips, and to cosmetic 1
consumer products. Cationic surfactants products to support skin protection and –20
0 2 4 6 8 10 12 14 15
have traditionally been difficult to analyze regeneration. Urea is frequently added as an 200
by reversed-phase HPLC. That changed with antiseptic to prevent the growth of bacteria, B
n-C12
the introduction of the Acclaim Surfactant mildew, and yeast in creams, shampoos,
column. While the Acclaim Surfactant lotions, and cosmetics. However, when used mAU

column was designed to analyze all classes in large quantities in the raw materials, they
2
of surfactants, it was especially designed for may adversely affect health and must be –20
cationic surfactants. The Acclaim Surfactant analyzed for quality control purposes. 0 2 4 6 8 10 12 14 15
Minutes
column and an UltiMate 3000 system can 26154
Reversed-phase C18 and polar-embedded
be used to determine both the quantity and Separation of benzalkonium chloride homologs in
reversed-phase columns retain urea and
the polymeric distribution of benzalkonium two eye drop samples using the Acclaim Surfactant
allantoin poorly, and demonstrate limited column.
chloride in a range of consumer products, for
resolution. An Acclaim Mixed-Mode HILIC-1
example disinfectant sprays, eye drops, and
column operated in the HILIC mode is the
sterile elastic bandages. 300 1,2
best choice for retention and resolution of Peaks: 1. Allantoin (25 ppm)
urea and allantion. 2. Urea (200 ppm)

12
mAU C

B
1 2
A

–50
0 1 2 3 4 5 6
Minutes 25504

Comparison of urea and allantoin separation on

A) a polar-embedded reversed-phase column,
B) a C18 reversed-phase column, and C) an
Acclaim Mixed-Mode HILIC-1 column. The Mixed-
Mode column operated in the HILIC mode is the best
choice for retention and resolution of urea
and allantion.

page 6
Glyphosate in Consumer Weed Anions in Toothpaste and APF 800
Killer Formulations Fluoride is well known to play a critical role
Glyphosate is a popular herbicide available in preventing tooth decay. The U.S. Food
in many formulations. Waterproofing agents and Drug Administration has approved the Glyphosate
are often present in the formulation along use of three decay-preventing compounds
with glyphosate, which are hydrophobic, in toothpaste: stannous fluoride, sodium mV

and can interfere with glyphosate retention. fluoride, and sodium monofluorophosphate
The Acclaim Mixed-Mode WAX-1 column (MFP). However, fluoride can react with other
cleanly separates glyphosate from the ingredients in toothpaste to form insoluble
compounds, and MFP can hydrolyze during 0
other ingredients. Glyphosate’s lack of a UV
chromophore and high concentration in the storage, interfering with the efficacy of the
0.0 2.5 5.0 7.5 10.0
sample make detection by evaporative light product. Acidulated phosphate fluoride (APF) Minutes 24553

scattering a convenient option. is approved for use as a topical solution or

gel, and has strict requirements for fluoride Determination of glyphosate in a commercially
available weed killer using the Acclaim Mixed-Mode
Engine Coolant Corrosion Inhibitors concentration. Therefore, the determination WAX-1 column and evaporative light scattering
Corrosion inhibitors are added to ethylene of fluoride and MFP in toothpastes and APF detection (ELSD).
glycol coolant soutions because glycolic is important for quality control purposes.
acid can erode cooling systems, especially if IC provides a convenient and simple method
for the direct determination of fluoride and 25 Peaks: 1. Fluoride 0.018%
copper ions are present. No single inhibitor 2. Acetate –
can protect against all corrosive agents; MFP in a single analysis, with minimal 6 3. Chloride 0.002
therefore, multiple inhibitors are typically sample preparation. 4. Carbonate –
5 5. MFP 0.81
added to antifreeze formulations. The Acclaim 6. Sulfate 0.23
7. Benzoate 0.096
Mixed-Mode WAX-1 column can be used to
µS 8. Phosphate 0.07
separate glycolic acid and numerous corrosion
inhibitors, including benzotriazole (BT), tolyl-
triazole (TT), 2-mercaptobenzothiazole (MBT), 1
8
sebacic acid, 2-ethyl hexanoic acid, and 2 4
3 7
benzoic acid, in a single injection.
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Minutes 19344

Separation of anions in a commercially available

toothpaste using direct injection on an IonPac AS18
column.

page 7
 Industrial Processing

Manufacturing processes are often complex, and depend on steps that use chemicals or produce byproducts that can affect product
quality, worker health, or environmental safety. Dionex IC and HPLC systems and columns are used in a number of industries to help
monitor process intermediates, occupational exposure, and waste streams for process control and regulatory compliance.

Sulfur Speciation Oxalate in Bayer Liquor

Peaks: 1. Sulfide
The measurement and characterization of To isolate alumina, bauxite is dissolved in 25 2. Sulfite
sodium hydroxide, forming a solution referred 3. Sulfate
total sulfur content, as well as identification 65.00
4. Thiosulfate
of sulfur species at trace levels, is important to as Bayer liquor. Aluminum hydroxide 5. Unknown 4
in several industrial processes. Catalytic is precipitated and used in the aluminum
hydrotreating processes, for example smelting process. After precipitation, only µS
40.00

hydrodesulfurization, remove sulfur compounds the large crystals are put into the kiln for
from refinery product streams, and sweetening calcination; small crystals are returned
20.00
processes remove sulfur compounds or to the precipitator. Oxalate can interfere 5
convert them to disulfides, as in the case with crystal formation, and therefore Concentration: 2.00 mM
2 3
1
of mercaptans. This can be a challenging measurement of the oxalate concentration 0
0 5 10 15 20 25
application given their redox chemistry, and helps predict the success of forming large Minutes 26247
the tendency of many sulfur-containing species crystals from a given Bayer liquor. The high
to readily react with each other, decompose ionic strength and metal content typically Determination of sulfur species in calcium polysulfide,
after sample pretreatment using OnGuard H and Na
over time, or oxidize in the presence of air. present in these solutions make it a
cartridges, and separation on an IonPac AS17-C
They are also very sensitive to the solution challenging sample. Dionex has developed a column with an electrolytically generated gradient.
pH, which would affect the distribution of the direct injection method using an RFIC system
species over time. All of these factors make with in-line sample cleanup to accurately and
the determination of these sulfur-containing reproducibly measure oxalate in Bayer liquor.
Peaks: 1. Fluoride 0.1% NaF
anions a very challenging task. Traditional 100 1 2. Chloride 34.47 mg/L
wet chemical methods for sulfur speciation 2
3. Malonate 11.93
4. Sulfate 44.84
are often unreliable for quantification at trace 5. Oxalate 16.77
levels concentrations. However, modern IC
provides an accurate and convenient method 4
µS
for the speciation of sulfur-containing species.
OnGuard® single-use or InGuard™ in-line 5
multiuse sample preparation cartridges can 3

be used to help remove matrix ions and

reduce solution pH to improve –20
0 3 6 9 12 15 18
chromatographic separation. Minutes 25653

Overlay of three consecutive runs of a Bayer liquor

sample separated using an IonPac AS17 column.
The on-line sample preparation and automatically
generated eluent yield highly reproducible results.

page 8
Analysis of Cations in Wastewater Occupational Safety Peaks*:
1. Sodium 26,345 mg/L (ppm)
To prevent nutrient enrichment and the Analysis of Diisocyanates 2. Ammonium 5.6
undesirable ecological impacts associated 3. Potassium 45.3
Toluene diisocyanate (TDI) and 4. Magnesium 11.3
with them, anionic and cationic species 5. Calcium 5.5
hexamethylene diisocyanate (HDI) are
must be closely monitored in chemical *Calc. (in original sample)
commonly used in the manufacture of
wastewater streams. IC has proven to be 0.55
1
urethane polymers, are highly toxic by 3
a useful technique for the determination of
inhalation, and are carcinogenic. OHSA
amines and their breakdown products (usually
method 42 is a standard method for testing
other smaller amines and ammonium),
workplace air for contamination. The Acclaim 2
as well as Group I and Group II metals.
RSLC PolarAdvantage II (PA2) 2.2 μm column µS 4
Alternate techniques using GC and HPLC
not only provides suitable selectivity for
require derivitization for separation and 5
these diisocyanates but also allows an
detection. Typically, these samples are water
acceleration of about eightfold over the
based since amines are used extensively as
conventional method. Elevated temperature
organic bases for pH adjustment in aqueous 0.25
improves resolution while reducing 0 10 20
process streams, for example boiler water,
backpressure to about 210 bar. Minutes
or product formulations (scale inhibitor). 16268

IC with suppressed conductivity detection Analysis of Azodicarbonamide Determination of ammonium and cations in a high-
offers the convenience of analyzing water sodium wastewater sample.
Azodicarbonamide (ADC) is a blowing agent
based systems with excellent sensitivity
used in the production of foamed rubber and
and minimal effort. The IonPac CS16 column
plastics, and as a flour bleaching agent that Peaks 1. 2,6-Toluenediisocyanate
can separate these cations rapidly and
also promotes stable, even baking. But ADC 2. 1,6-Hexamethylenediisocyanate
reliably using electrolytically generated 3. 2,4-Toluenediisocyanate
is also a known respiratory sensitizer, and Samples: A. Reagent blank
methanesulfonic acid (MSA) eluent, and is
occupational exposure is monitored with B. 0.16 µg/mL in matrix
compliant with ASTM Method D6919-03. C. 8.0 µg/mL each in acetonitrile
air filters. The collected ADC is dissolved in
75
EtOAc:DMSO and then quantified by HPLC. 3
As ADC is not retained by reversed-phase or 1
ionic interactions, the Acclaim Mixed-Mode
WAX-1 column can be used in normal-phase 2

mode for analysis of the filter extracts. The mAU

3 µm, 3.0 × 150 mm format column provides
excellent resolution and reduces solvent
consumption as compared to 4 mm columns. C
B
0 A
0 0.5 1.0 1.5 2.0 2.6
Minutes
25773

Fast determination of diisocyanates using the

Acclaim PA2 2.2 µm column.

page 9
 Petroleum Refining and Exploration

The refining and use of fossil fuels in a number of industries poses challenging analytical problems. Raw materials and finished products
must be tested for quality to ensure product yields, protect refinery equipment, increase the lifetimes of engines or equipment using the
fuels, and to protect the environment. Dionex has developed equipment and applications for testing raw material and product quality,
and for compliance with regulations for fuel production and use.

Combustion Ion Chromatography Refinery Heat-Stable Salt Monitoring 3.0

3
Fluorine, chlorine, bromine and sulfur can Alkanolamine is a gas-conditioning scrubbing Peaks:
1
cause corrosion in many industrial processes, solution used in petroleum refineries to 1. Fluoride 1.5 mg/L
2. Chloride 0.019
decreasing the lifetime of many catalysts and remove H2S and CO2 that may be present in 3. S as Sulfate 3.0
the feed gas. Heat stable salts (HSSs) can 4. Phosphate 2.0
causing environmental pollution. Combustion µS
(int. std)
IC permits automated qualitative and form from amines in these solutions. While 4
quantitative analysis of halogens and sulfur H2S and CO2 gasses are retained when they
2
in petrochemicals, coal-based chemicals, are passed through the amine stripper and
0
and construction materials, chemicals, and later removed by heat, inorganic cations such
polymers. Solid or semisolid samples are as formate, acetate, glycolate, sulfite, and –0.5
0 2 4 6 8 10 12
pyrolyzed, absorbed in an aqueous solution, chloride can not be removed. The buildup of Minutes 19007-01
and injected onto an IC system for analysis of HSSs in the amine unit decreases the acid
gas carrying capacity of the amine, increases Determination of sulfur and halogens in a liquified
the combustion products. This system can be
petroleum gas sample, using combustion IC with
used to identify and measure: solution viscosity, and can cause corrosion an IonPac AS11-HC column and suppressed
and foaming problems, thus increasing conductivity detection.
• Sources of contaminants in production
operating costs.
and manufacturing
• Additives for fire retardants and antioxi- IC is the preferred technique for monitoring Peaks: 1. Acetate 4 ppm
dants (plastics, polymers, textiles) several points throughout the amine unit. 2. Glycolate 4
3. Formate 4
• Environmental impact of use (coal), dis- It can unambiguously identify and quantify 4. Chloride 4
4.0
posal and recycling (plastics and solvents) trace-level organic acids such as acetate, 5. Sulfite 4
6. Sulfate 4
• Product specification and QC/QA formate, and glycolate while simultaneously 4
7. Oxalate 4
resolving sulfur species from sulfite and 8. Thiosulfate 4
For example, total halides and sulfur can sulfate to the strongly retained anions such
9. Thiocyanate 4

be determined in an LPG (liquefied petroleum as thiosulfate and thiocyanate, within a single 6

3
gas) sample, to monitor HF alkylation chromatographic run. 7
µS 8
unit effectiveness.
5

1 9
2

-0.50
0 10 20 30 40 50
Minutes 26830

Determination of heat-stable salts in a gas

conditioning scrubbing solution, using an IonPac
AS11-HC column and suppressed conductivity.
Gradient elution accelerates the elution of strongly
retained species, such as thiocyanate.

page 10
Analysis of Polycyclic Aromatic Anticorrosion Amines and Cations Peaks:
1. Lithium 9. Dimethylamine
Hydrocarbons in Fuel Oil Efficient management of process streams in 2. Sodium 10. Triethanolamine
3. Ammonium 11. Methyldiethanolamine
The content of polycyclic aromatic petrochemical production requires frequent 4. Monoethanolamine 12. Dimethylethanolamine
hydrocarbons (PAHs) in diesel fuels is monitoring for contamination prior to use 5. Methylamine 13. Morpholine
6. Diethanolamine 14. 1-methoxypropylamine
regulated, and most often determined or disposal. For example, natural gas liquids 7. Ethylamine 15. Dimethylamino-2-propanol
using standard methods EN-12916 or ASTM (NGL) and liquid petroleum gas (LPG) streams 8. Potassium 16. Magnesium
17. Calcium 16 17
D-6591. Not only does the presence of are typically washed with dilute caustic, 3.00

aromatic hydrocarbons pose a health risk 2–14 wt% NaOH, to remove residual organic 5
because they are known carcinogens, they sulfur compounds, such as mercaptans. 1 2 4 8
Alkanolamine solutions are used to remove 3
also affect fuel quality and performance.
To ensure optimum engine performance acidic components (H2S, CO2) from these 9
µS
and lifetime, the amount of PAHs in diesel hydrocarbon streams. Caustic washing may 6 12
14
7
fuel should be as low as possible. For this also be employed to neutralize the acid 11
13
15

reason it is necessary to be able to detect alkylation process effluents to minimize 10

and quantify trace-level PAHs in complex corrosion of plant piping and mechanical
matrices such as petroleum distillates. The components. IC is an effective analytical 0.20
10 20 30 40 50
UltiMate 3000 x2 dual system facilitates the tool for measuring cation contaminants, for
Minutes 26829
automation of on-line sample preparation example alkali and alkaline earth metals,
and matrix elimination, permitting direct ammonium, and alkanolamines in spent Determination of amines and cations at concentrations
from 0.5–5 ppm in a wastewater sample using an
injection of complex samples using a split caustic washes, alkanolamine solutions, and
IonPac CS16 column and suppressed conductivity
in-line loop injection. other process streams such as wastewater. detection.

Peaks: 1. Parafins
18 2. Toluene
1 3. Napthalene
4. Phenanthrene
5. Pyrene
6. Nitrobenzene

µRIU
3
4
2 5
6

9
0 5 10 13
Minutes 26882

Determination of PAHs in diesel fuel. Split in-line loop

injection using isocratic elution provides baseline
separation of all analytes.

page 11
 Alternative Fuels and Chemicals

Today, biology is transforming the way fuels and chemicals are manufactured, from biodiesel, bioethanol, and cellulosic biofuels to fine
and specialty chemicals. The greatest challenge is to develop analytical methods tailored to deliver fast, accurate results. Dionex has
developed a number of systems and methods for analyzing raw materials, in-process samples, and finished products.

Chloride and Sulfate in Alcohols Cations in Biodiesel 35 Peaks: 1. Fluoride ND

Biodiesel is produced by reacting plant or 2. Chloride 1.0 mg/L
The National Institute of Standards 2 3. Nitrate ND
and Technology (NIST) recently made animal oils with an alcohol in the presence 4. Sulfate 1.0
recommendations for the minimum acceptable of a catalyst to produce the desired methyl
limit for contamination of ethanol with ions esters and the byproduct glycerol. Basic
4
such as sulfate and chloride, with the goal of catalysts, for example, sodium or potassium µS
1 3
harmonizing US, European Union, and Brazilian hydroxide or alkoxides, are most commonly
standards for biofuels quality assurance. used, and leave residual sodium or potassium
Excessive amounts of sulfate and chloride that must be removed. Magnesium and
anions can clog automobile filters and fuel calcium contamination can also occur if hard
injector nozzles, negatively impacting engine water is used in the manufacturing process. 10
0 2.5 5.0 7.5 10.0 12.5 15.0
performance. The NIST study recommends These cations can form soaps and ash that Minutes 24069
IC as the preferred analytical method for can clog engines.
determining sulfate and chloride in ethanol, Determination of 1 mg/L chloride and sulfate in reagent-
ASTM D6751 and EN 14214 standards set a grade ethanol containing 5% gasoline. Anions were
and also recommends lowering the regulated
limit for these cations at <5 ppm combined separated using an IonPac AS22 column and carbonate/
limit of chloride ion to more closely match the bicarbonate eluent after concentration on a TAC-ULP1
sodium and potassium, and <5 ppm combined
specification of 1 ppm adopted by Brazil. concentrator column.
magnesium and calcium, and limits as
Using the matrix elimination method with the low as <0.3 ppm total combined cations
Dionex ICS-3000 ICS system, chloride and are required for a 6% biodiesel blend. The Peaks: mg/L
1. Sodium 0.0472 0.956
sulfate ions are concentrated automatically methods specified in these standards, ICP- 0.5 1 2. Unknown — —
A
for detection limits as low as 1 ppm chloride OES and atomic absorption spectroscopy, 3. Potassium 0.0046 0.0053
4. Magnesium 0.0065 0.0362
and sulfate in denatured ethanol, methanol, suffer from matrix interference, difficulty in 2 5. Calcium 0.0314 0.183
µS
and alcohol-gasoline blends, without arduous simultaneously determining contaminants, and
sample preparation. Dionex IonPac AS24 and complicated sample preparation. IC can quickly 5
and specifically determine multiple cations 3 4
AS22 columns provide accurate, reproducible
0.2
results in even the most complex matrices. simultaneously, with direct injection after 0.7 1
B
Choose between hydroxide (IonPac AS24) or only a simple water extraction and filtration.
5
carbonate (AS22) electrolytically generated Detection limits are routinely at the 1 ppm
eluents for fast run times (<12 min) with level, far below the 5 ppm concentrations µS
specified in the standard methods. 4
excellent retention time stability.
3
0.2
0 5 Minutes 10 15
25494

Determination of cations in A) B20 and B) B99 biodiesel

mixtures using an IonPac CS12-5µm column, MSA
eluent, and suppressed conductivity detection.

page 12
Carbohydrate Profiling of Biomass Lipid Profiling of Algal Biomass
Peaks: 1. Glycerol
Accurate, precise compositional analysis Many plants, including algae, store large 2. Galactose
3. Sucrose
of biomass is critical for understanding and amounts of oil as carbon storage reserves, 4. Xylose
250
assessing biomass conversion technology. making them an important biomass feedstock 4
5. Mannose
6. Fructose
Analytical methods that provide a high for the development of alternative fuels.
3 7. Cellobiose
degree of confidence are required for Profiling of the mixture of microalgal lipids 1
B
accurate yield and mass balance calculations, is critical in order to screen out polar nC 2 7
which in turn are necessary for sound cost phospholipids from the feedstock because 5 6

estimates for biofuels production. The they can contaminate the precious metal A
Dionex method for monosaccharide analysis refining catalysts used in the production of a
-100
permits determination of carbohydrates form of biodiesel known as renewable diesel. 0 12.5 25 37.5 50
by direct injection using high-performance Minutes
® ™ 26006
The Corona ultra with Charged Aerosol
anion-exchange chromatography with pulsed
Detection® (CAD®) technology permits Determination of carbohydrates in corn stover
amperometric detection (HPAE-PAD). Our hydrolysate using a CarboPac PA1 column and pulsed
detection of multiple lipid classes including
new low-volume microloop injection valve amperometric detection. Direct injection without dilution
neutral lipids such as triacylglycerols and still shows excellent resolution and sensitivity, saving
technology provides baseline-resolved
sterols, polar lipids such as phospholipids, as time and labor.
separation of highly concentrated samples
well as the non-acyl lipids including phytols
(up to 100 g/L) without dilution, thus
in a single analytical run. Methods based
eliminating dilution errors while delivering
on charged aerosol detection are more Peaks: 1. Dextrin 7. Lactic acid
stellar performance. 2. Maltotriose 8. Glycerol
convenient than older, ELSD technology, 3. Maltose 9. Acetic acid
Fermentation Process Monitoring often requiring multiple different chemistries, 4. Glucose 10. Methanol
5. Fructose 11. Ethanol
from normal-phase to reversed-phase 6. Succinic acid
Precise monitoring of fermentation in
chromatography, employing more complex 5.00
ethanol production is critical for maximizing
method development, and the need for
fermentation rates and minimizing
multiple analytical runs.
organic acid inhibitors. A key requirement µRIU
t=0
is determining three different analyte
classes—carbohydrates, organic acids, and
alcohols—in a single analytical run. For –0.50
0 2 4 6 8 10 12 14 16 18 20 22 25
this application, the Dionex Integral Process 200
Minutes
11
Analytical System using HPLC with refractive
index detection, permits simultaneous
t = 2 hr
monitoring of multiple analyte classes. µRIU 4
23 8
1 5 6 7 9
10
–5
0 2 4 6 8 10 12 14 16 18 20 22 25
Minutes 26033

Simultaneous separation and determination of

carbohydrates, organic acids, and alcohols at time zero
and after 2 hours.
page 13
 Plastics and Polymers

Plastics and polymers are widely used in the manufacture of industrial products. Monomers, oligomers, plasticizers, and stabilizers must
all meet strict quality standards to ensure product quality. Because of health hazards associated with plasticizers and other additives,
their presence must be limited in products which come into contact with food, for example water bottles, or human skin, as in consumer
products, for example absorbent diapers and cosmetics.

Bisphenol-A Diglycidyl Ether Acrylic Acid and Oligomers Peaks: 1. BADGE + 2H2O 50 µg/mL
2. Bisphenol-A 50
(BADGE) and Related Impurities Acrylic acid is an important monomer that can 3. BADGE + H2O 50

BADGE is a widely used epoxy monomer be polymerized to yield a class of hydrophilic 4. BADGE + HCl + H2O 50
5. BADGE 50
derived from bisphenol-A. It is used to polymers with high absorption capacity for 6. BADGE + HCl 50
manufacture many kinds of coatings, such aqueous solutions. Hydrophilic polymers are 7. BADGE + 2HCl 50
125
as powder coatings, solid coatings, solvent- capable of absorbing several times their own R1,2 = 2.69

based coatings, and anti-corrosive coatings. weight in water, transforming them into gels 2
R1,2 = 4.55
The purity of the monomer is critical because with agricultural, horticultural, and sanitary
it is used to produce polycarbonate, epoxy, applications. Ensuring the purity of the acrylic
phenolic, polyester, and other resins. Residues acid monomer is critically important to meet mAU 1
3 4 5
67
of epoxies used in food-contact applications the demanding market requirements for
are of great health concern, because they are products which come into contact with food,
B
endocrine disruptors. For these reasons, it beverages, or human skin. Acrylic acid can
A
has become necessary to examine the quality also spontaneously form oligomers that can 0

of the raw materials of such monomers prior affect the properties of the finished product. 0 5 10 15 20
to polymerization and scale-up for industrial The Acclaim Organic Acid (OA) column is well Minutes 25398

purposes. Most analytical methods for these suited for quality-control assays related to
this important class of polymers. Separation of BADGE and related impurities using
contaminants use organic solvent-water A) conventional LC: Acclaim 120 C18, 5 µm. 4.6 × 150 mm
gradients and a relatively long column to column and B) UHPLC: Acclaim RSLC C18, 2.2 µm, 2.1 ×
provide acceptable selectivity. The high 150 mm column, and detected using UV absorbance.
flow-pressure footprint of the UltiMate 3000
RSLC system permits use of Acclaim RSLC Peaks: 1. Monomer
columns with smaller particle sizes, such 2. Dimer
3. Trimer
as the 2.2 µm Acclaim RSLC C18 2.1 mm i.d. 4. Tetramer
5. Pentamer
column. This column can resolve BADGE and 6. Hexamer
related impurities in less than 5 min, a quarter 7. Heptamer
1
of the time necessary to resolve the same Acrylic acid (Aldrich)
components using the conventional 5 µm OH
Acclaim 120 C18 column.
2 O
A 3
AU 2 Acrylic acid oligomers
(Aldrich)
1 O O
3
HO O
4 n
B 567

0 5 10 15 20
Minutes 20029

Separation of acrylic acid monomers and oligomers

using an Acclaim OA column, with detection by UV
absorbance. A) Acrylic acid sample showing dimer
page 14 and trimer impurities. B) Separation of a mixture of
acrylic acid oligomers.
Extraction of Plasticizers from PVC Analysis of Polymer Additives 40 Peaks: 1. Irganox 245
2
2. Tinuvin 234
Poly(vinyl chloride) PVC is a popular, versatile Polymer additives are used as processing 3. Irganox 259
polymer used in many different products and long-term thermal stabilizers to protect 4. Irganox 1010
including water pipes, toys, and shower the polymer from breakdown caused by UV
curtains. PVC is typically composed of light or oxidation. These antioxidant additives
mAU
resins, stabilizers, pigments, and plasticizers. are critical to maintain the lifetime of 1
3
Platsicizers soften the polymer, aid in the consumer products containing thermoplastic
4
manufacturing process, and provide form polyurethane (TPU) resins. Tinuvin® is a
and function to various PVC materials. common UV stabilizer used as an additive (for
Plasticizers may account for 30-35% of the example, in polyethylene and polypropylene). 0
PVC formulation. Extraction and determination Irganox® is the trade name for a class of 0 Minutes 70
of plasticizers in a PVC material are critical phenolic-based antioxidants used widely as 24205

steps in evaluating a polymer for an intended additives (for example, in PET and polyolefins). Determination of a polymer UV stabilizer and three
use. Traditionally, plasticizers are extracted In order to control formulation levels and to antioxidant additives, after extraction using an ASE
from PVC using a 6 h Soxhlet method and conduct stability studies, the additive content system. Additives were separated using an
Acclaim 120 C18 column and detected using
identified using infrared spectrometry or of the polymer must be determined. UV absorption.
gas chromatography. Compared to Soxhlet
extractions, ASE methods decrease extraction An ASE system is ideal for extraction of
times from hours to minutes and reduce additives from in-process materials and final Peaks: 1. Irganox 1010 488 m/z
solvent consumption while delivering products. The additives can be separated 2. Irganox 259 471
3. Irganox 245 587
equivalent recoveries. using an UltiMate 3000 HPLC system with the 4. Tinuvin 234 448
Acclaim 120 C18 column using a CH3CN/H2O
1.0 × 106
gradient. For identification and analysis of
decomposition products of polymer additives,
MS detection by positive ion APCI is required.

Counts
Irganox 1010
Irganox 259
Irganox 245
Tinuvin 234

0
0 Minutes 70
24210

Overlay of selected ion mode (SIM) chromatograms

of a polymer UV stabilizer and three antioxidant
additives.

page 15
CHROMATO G RAPHY MANAG EMENT SYSTEM
 THE WORLD’S MOST COMPLETE CHROMATOGRAPHY

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Chromeleon looks and works like software
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MANAGEMENT S OLUTION

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MULTI-VENDOR AUTOMATION

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Chromeleon’s integrated, Excel-compatible 3-D Data Analysis
spreadsheet makes it easy to analyze your data, Want more information about the composition of
and to present results in the ways your customers your samples? Take advantage of Chromeleon’s
want to see them. Report workbooks can have as powerful diode-array and mass spectrometry
many worksheets as you like, and can include options to confirm peak purity, positively identify
result tables, chromatograms, calibration plots, compounds, and develop optimum analytical
spectra, audit trails—even custom equations and methods. View, compare, and manipulate
charts. Every cell, table, and chart updates instantly multiple chromatograms and spectra quickly,
if any of the source data change, so you never have easily, and interactively. Store any number of
6
to worry about consistency. spectra in your own custom libraries, then use
You get the custom reports you need without intelligent spectral library matching algorithms to
the hassles and validation issues of exporting to determine the composition of unknown samples.
external software, so you save time, effort, Never before has the power of 3-D data analysis
and frustration. been so accessible to chromatographers!

Find and collate Produce the Get more information

results quickly custom reports from your samples
and easily with you need with Chromeleon’s
Chromeleon’s quickly, easily, diode-array and
powerful data and automatically mass-spectrometry
mining tools. with Chromeleon’s features.
built-in spreadsheet.
ACHIEVE FULL R EGULATORY

Good analytical work alone is

no longer sufficient. You have to
be ready to defend your results,
not only with sound science, but
also with compelling evidence
that the results are authentic and
fully traceable. Access control,
validation records, audit trails,
chain of custody—they’re all
required. You know you need to
comply with the rules, but how
can you make your existing
chromatography systems and
operations compliant? How can
you cope with an increasing
workload of analyses while still
ensuring that you’re always
ready to pass an audit? You
need a solution that meets
compliance requirements AND
increases your productivity.
You need Chromeleon.

Chromeleon provides
the controls you
need for a secure
and productive
laboratory,
whatever your
environment.
COMPLIANCE WHILE I NCREASING P RODUCTIVITY
Security that Fits Your Workflow
Chromeleon’s comprehensive security system versatile System Suitability Testing and statistical
provides fine-grained control over specific analysis features. In addition to standard USP and
chromatography objects and operations. Over EP tests like efficiency, resolution, tailing, and
100 different chromatography privileges can be reproducibility, you can define any number of tests
allocated as appropriate to any number of to automatically check any reportable variable
Privilege Groups, so you can control who can across any set of samples in your sequences.
run samples, who can modify methods, who can Detailed Audit Trails
change peak baselines, and so on. You can also With Chromeleon’s comprehensive audit trails, you
control access to specific instruments and data always have the documentation you need to defend
folders by defining any number of Access Groups. your results. Every event related to instrument
Administration tools let you set rules for login control and data acquisition is automatically
and signature passwords, and automatically lock captured in event logs. Every change to a baseline,
inactive stations and accounts that have too many a quantitation method, or a calibration is tracked in
login failures. All security-related events are the modification history. And all security-related
automatically tracked in a detailed security log. events—logins, password changes, privilege
Fully Automated Validation changes, and electronic signature events—are Seamless Electronic Signatures
Qualifying your analytical systems no longer recorded in a detailed security system log. The vision of the paperless laboratory is now a
has to be a tedious chore. Chromeleon’s AutoQ™ But Chromeleon does more than just capture reality within your reach. Chromeleon has all the
qualification suite fully automates the Installation the audit trails. It also helps you extract the tools you need to implement an efficient electronic
Qualification, Operational Qualification, and information you need out of them, quickly and record-keeping system that complies with 21 CFR
Performance Qualification procedures for your easily. View the events pertaining to a single Part 11 requirements. All of the processes for
instruments and software. Simply select the tests sample, a sequence, or an entire data warehouse. document creation, review and approval fit right
you want to perform, load your autosampler, and Filter the display down to records matching a into the natural laboratory workflow, so you can
select <Start> — Chromeleon does all the rest specific user, time period, or event type, and sort eliminate the paper chase without having to
while you go work on other tasks. When you the filtered list simply by clicking on column titles. completely restructure your operations. Result?
return, detailed reports are waiting for you. Method Print the filtered and sorted list directly, or embed You achieve both compliance and improvements
validation is easy, too, thanks to Chromeleon’s the audit trails directly in your analytical reports. in productivity.

With AutoQTM All instrument All the functions you

automated events and user need to implement
qualification, actions are paperless record-
validating your automatically keeping are
chromatography documented seamlessly
systems becomes in detailed integrated into
almost effortless. audit trails. Chromeleon.
 Window

SQ
Serv

SATISFY NEEDS T HROUGHOUT YOUR LABORATORIES WITH

Flexible, Scalable Client/Server Architecture SQL Server for a robust, multi-user enterprise
Because the user interface and instrument control implementation. Mix platforms as you wish, and
Chromeleon is more functions of Chromeleon are handled by separate migrate with ease.
than your best choice in a server and client software modules, you can Convenient System Administration
chromatography workstation— implement the software in any configuration that Satisfying diverse needs is much easier when
it’s also a complete multi-user suits your needs. Operate client and server modules you have the right tools. From a single console,
chromatography management on the same computer for a complete, stand-alone a Chromeleon administrator can allocate software
solution that adapts to diverse instrument workstation. Connect workstations and licenses, configure workstations, manage user
needs throughout your
client stations with a network to centralize data privileges, and perform system maintenance.
enterprise. You c an start with a
storage and system administration, and to gain Set up and monitor remote stations from the
small installation and scale it up
remote access to instruments and data. Start your convenience of your office. Enforce limits on
at your own pace, using the
implementation with a single workstation and what users can see and do, while granting them
operating system and database
expand it to a laboratory, department, or your flexibility to adapt the software to their own
platforms that best suit your
needs. Get the benefits of
whole enterprise—without changing the needs. Define global and user-specific operating
centralized data storage and software, without any downtime, and without policies, and delegate routine administrative
system administration without retraining the users! tasks to laboratory managers. Automate data
giving up the flexibility of Cross-Platform Compatibility backup and archiving processes without
distributed workstations. Unlike other data systems, Chromeleon gives you a affecting system users.
Standardize or customize user choice of operating systems and database platforms. Industry-Leading Reliability
interfaces, methods, and reports It works transparently with any combination of Nobody can afford downtime, especially with a
as appropriate to address Windows 2000, Windows XP, and Windows Vista multi-user installation. Chromeleon’s distributed
different users and different computers, so you can migrate from one platform to client/server architecture and intelligent
applications. Integrate another at your own pace. Use the default database instrument control provide more inherent
Chromeleon with other software
format (compatible with Microsoft Access) for an reliability than other data systems do, but
products to further improve
economical workstation solution with no database Chromeleon takes reliability a step further. Its
overall laboratory productivity
license fees or administration requirements, or exclusive Network Failure Protection feature
and efficiency.
deploy Chromeleon on Oracle® or Microsoft keeps your automated instruments running in

Expand your Choose the Administer your

Chromeleon platforms that Chromeleon
network easily fit your needs, systems from
and economically. and migrate anywhere in
with ease. the network
 Windows Vista

ws XP Windows 2000

QL Access
ver

Oracle

C HROMELEON—THE MASTER OF ADAPTABILITY

the event of sudden or scheduled network
server unavailability, without any loss of data
or productivity. Even transient power failures
don’t stop Chromeleon. It simply brings your
instruments back up to operating conditions,
then resumes analyses in accordance with your
pre-defined instructions. All contingency actions
are documented in detailed audit trails. With
Chromeleon, you get the high availability and fault
tolerance you need for optimum productivity.
Easy Integration with Other Applications
Chromeleon works seamlessly with other
software products in your laboratory. Copy
chromatograms, spectra, and other graphics to
office applications to create journal publications
and custom presentations. Import sample worklists
from your LIMS, and post results back to it upon
completion of analyses.
Export Chromeleon reports in popular formats,
such as Microsoft Excel, Adobe PDF, and HTML,
for convenient distribution to people outside your
laboratory environment. Develop highly customized
applications in popular programming languages
like C++ and Visual Basic, and make direct
function calls to the chromatography software with
the optional Chromeleon Software Developer’s Kit.

Chromeleon Integrate
keeps working Chromeleon
productively even seamlessly with
in the event of your favorite
problems that office applications
paralyze ordinary and your LIMS.
data systems.
 Quality Products from a Global Leader
in Separation Science
Dionex offers an extensive array of innovative,
high-quality instruments, software, consumables,
and associated products that solve problems for
laboratories. All Dionex products are designed,
developed, tested, and manufactured in
accordance with life cycle processes modeled
after ISO 9001. Corporate Headquarters
Software with an Open Future
Dionex Corporation
For over 20 years, Dionex has continually brought 1228 Titan Way
new and exciting chromatography software to P.O. Box 3603
W hen you inves t in market, while always providing our customers Sunnyvale, CA 94088-3603
C hromeleon, y ou get more with a smooth upgrade path. Our ongoing Tel: (408) 737-0700
investments in software development and testing Fax: (408) 730-9403
than jus t a p roduct—
continue to broaden Chromeleon’s instrument Worldwide Sales and Service
you ge t a c omplete s olution
support, add data analysis capabilities, strengthen
with an op en future, a nd a North America
security and robustness, and simplify usage. With
U.S. (847) 295-7500
partner committed Chromeleon, your software investment does not
Canada (905) 844-9650
to your s ucces s . depreciate – it actually grows in value!
South America
Services that Streamline Your Deployment Brazil (55) 11 3731 5140
At Dionex, we know that our success depends on
Europe
your success. To ensure both, we provide a complete
Austria (43) 1 616 51 25
suite of support services, including consultation,
Benelux (31) 20 683 9768
installation, validation, training, technical support,
(32) 3 353 42 94
and on-site repair. And to protect your investment, Denmark (45) 36 36 90 90
we offer extended warranties and software France (33) 1 39 30 01 10
subscriptions that keep your Chromeleon systems Germany (49) 6126 991 0
up to date with the newest software technologies Ireland (353) 1 644 0064
and capabilities. Italy (39) 02 51 62 1267
Sweden (46) 8 473 3380
Whether your s cope is Find Out More
Switzerland (41) 62 205 9966
jus t one laboratory s tation For more information, demonstrations, and United Kingdom (44) 1276 691722
or a s ite-wide initiative, no-obligation quotations, contact your local
Dionex representative. Asia Pacific
Dionex is ready to provide Australia (61) 2 9420 5233
the solutions and ser vices China (852) 2428 3282
India (91) 22 2764 2735
you need to reap a r apid
Japan (81) 6 6885 1213
return on your investment. Korea (82) 2 2653 2580
And our ongoing sof tware Singapore (65) 6289 1190
Taiwan (886) 2 8751 6655
development program
protects that investment by
ensuring that Chrome leon
continues to be the best www.dionex.com

solution to your needs, Dionex products are designed,

far into the future. developed, and manufactured
under an ISO 9001 Quality System.

© 2008 Dionex Corporation

Chromeleon is a registered trademark of Dionex Corporation.
Oracle, Microsoft, Windows XP, and Windows 2000 are
registered trademarks of Microsoft Corporation.
LPN 1172-07 10K 11/08 Printed in U.S.A.

Passion. Power. Productivity.

 Discover Chromeleon 7
Go From Samples to Results Quickly and Easily

The Chromeleon® 7 Chromatography Data System delivers rich, intelligent functionality with Operational Simplicity™. Everything you need
is within easy reach, whether you have only a single workstation or on an enterprise-scaled installation. The intuitive, easy-to-navigate user
interface guides you effectively towards your goals. Innovative eWorkflows enable anyone to start chromatographic analyses and get good
results with just a few clicks. Powerful data analysis tools ensure that any required data manipulation can be done efficiently and accurately.
Chromeleon 7 gets you from samples to results quickly and easily—boosting your overall lab productivity.

Begin
Automate Take advantage of Operational Simplicity
Use eWorkflows to streamline your lab's daily routines, whether with the easy to understand Chromeleon
they involve quality control, research, method development, software user interface.
or other applications of chromatography.

Control
Gain complete Instrument Control throughout the
entire network for fully automated and documented
analytical processes.
Analyze
Get the Data Treatment you want in seconds, without tedious
parameter tweaking.

page 2
 Improve your productivity, increase accuracy,
and train new users quickly. With its intelligent
features and operational simplicity, Chromeleon 7
is simply intelligent.

Deliver
Secure Get Final Results instantly, in the exact format you want them.
Ensure Compliance by controlling and managing your workflow.
View results on screen, print custom reports, and export raw
data and results to other programs.

page 3
 Advantages of Operational
Find Everything You Need Within Easy Reach

The Chromeleon 7 user interface combines intelligent functionality with operational

simplicity. Features you need most often are kept within view, while specialized
options are kept out of the way, yet easy to find. The Chromeleon Console's
category bars guide you to your area of interest, and give immediate access to
instruments, data, and eWorkflows.

Thumbnail images give

you an instant visual
summary of your data.

page 4
Simplicity

Instantly access everything related

to your sequence in the
Chromatography Studio:

• Chromatograms
• Processing Parameters
• Calibration Plots
• Reports
• Spectral Libraries
• And more…

Spectral Library

All functions are easily accessible. For instance,

just drag the integration parameter lines…

… and the updated results are

displayed immediately.
Initial Peak Selection

Extended Peak Selection

page 5
Streamline Your Workflow
Automate Your Process at a Higher Level

The Challenge: Every lab has its own procedures to get from samples
to results. These procedures are constructed from a complex collection
of protocols, documents, and references. Due to this complexity, prepar-
ing to run an analysis can be slow, tedious, and error-prone.

Solutions: The Chromeleon eWorkflow framework provides an easy way

to automate all of your chromatography analyses. This eliminates unnecessary
steps, speeds up the execution, and prevents errors.

Advantages: This revolutionary Chromeleon eWorkflow frame-

work minimizes operator tasks between sample receipt and genera-
tion of the final result, dramatically boosting laboratory productivity.

page 6
 Simply choose an eWorkflow template, enter the number of samples, and start your analysis.

The eWorkflow creates the sequence, starts the run, and ensures that data are
processed and reported correctly—minimizing user error and reducing review time.

page 7
 Gain Complete Control
Simplify Management of All Your Instruments and Data

Intuitive ePanels give you full control of

instruments from Dionex and other manu-
facturers. Get immediate access to all main
instrument functions (such as flow rate, tem-
perature, detector settings), plus easy access
to advanced features. Instrument control has
DP
never been easier!

ePanel for Instrument 1

DC EG

ePanel for Instrument 2

ePanel for Instrument 3

All instruments are automatically displayed in the software and can be

controlled from anywhere in your network. Simply click on the instrument you
want to use, and start working with it.

page 8
 Intelligent database functionality provides
immediate access to all data in your laboratory,
across all laboratories on site, or even
throughout your organization worldwide.

Intelligent Network Failure Protection

based on the Chromeleon software's secure
XVault™ infrastructure keeps instruments
running and data accessible for processing
during network outages.

Server

ASE 350

ASE

One central Administration Console

accessible throughout your network
provides all the tools needed to efficiently
manage your Chromeleon domain, such as
license and user management.

page 9
 Speed Up Data Analysis
Get the Peak Treatment You Want Quickly and Reproducibly

A major decision area in chromatography is Peak Detection. Where do

the peaks really start and end, how should baselines be drawn, and which
detection variables will give the desired integration?

The new Cobra™ detection algorithm

reliably detects peak start and end times,
and correctly assigns peak baselines
without requiring you to enter a long list
of detection parameters.

page 10
 For the challenge of unresolved peaks, the unique SmartPeaks™
integration assistant gives you the baselines you want quickly, easily,
and intuitively. No training is required, so even novices can immediately
integrate complex chromatograms appropriately and reproducibly.

Simply select a region of the chromatogram,

and then choose the integration treatment
you want.

The detection parameters are automatically

created. SmartPeaks saves time and effort,
and gets you quickly to your integration goals.

page 11
 Ensure Compliance
Control and Manage Your Laboratory Operations

Control what users can access and change with comprehensive user management tools.

Validate software and hardware automatically.

Trace all actions performed in the software, quickly

and easily, using Chromeleon's built-in Audit Trails.

page 12
Track changes with Audit Trails and
Version Comparison tools, and easily
revert to a prior version.

Implement an efficient, paperless approval process

that fully complies with 21 CFR Part 11, using electronic
reports with up to three levels of signatures.

Include versatile System Suitability Tests in your

methods, to automatically ensure chromatography
quality as your samples are analyzed..

page 13
 Finalize and Deliver Results
Get the Most Out of Your Data Quickly and Easily

Find and collate results quickly and easily using

powerful data mining tools .

Use queries to quickly gather, display, and report related data, even from
several independent sequences.

page 14
Take advantage of extensive 3D data analysis capabilities to confirm peak
purity, positively identify compounds, and optimize methods.

Produce the reports you need–quickly and easily–using

built-in spreadsheets.

page 15
Discover Chromeleon 7 Enjoy Industry-Leading Support Corporate Headquarters
Chromeleon 7 is the next-generation Dionex Customer Support Centers are Dionex Corporation
1228 Titan Way
Chromatography Data System that located in the United States, Europe, and
P.O. Box 3603
adapts to your needs with its simplified Asia. These state-of-the-art laboratories Sunnyvale, CA 94088-3603
user interface, innovative eWorkflows, are equipped with the full line of Dionex Tel: (408) 737-0700
powerful data mining and analysis tools, LC instrumentation and software Fax: (408) 730-9403
and unrivaled reporting capabilities. capabilities. Support Centers provide
accessible locations for advanced training Worldwide Sales and Service
Chromeleon 7 software is simply
and enhanced application development North America
intelligent, from samples to results. U.S./Canada (847) 295 7500
capabilities. Users can attend these
For more information or to place an laboratories to learn new skills in South America
order, call (800) 346-6390 within the addressing challenging applications, Brazil (55) 11 3731 5140
U.S., or contact the Dionex Regional receive training and support, and discover Europe
Office nearest you. Outside of the U.S., new, innovative HPLC and IC solutions. Austria (43) 1 616 51 25 
order through your local Dionex office Benelux (31) 20 683 9768
(32) 3 353 42 94 
or distributor. Denmark (45) 36 36 90 90 
France (33) 1 39 30 01 10 
Germany (49) 6126 991 0 
Ireland (353) 1 644 0064
Italy (39) 02 51 62 1267
Sweden (46) 8 473 3380
Switzerland (41) 62 205 9966 
United Kingdom (44) 1276 691722
Asia Pacific
Australia (61) 2 9420 5233 
China (852) 2428 3282 
India (91) 22 2764 2735 
Japan (81) 6 6885 1213 
Korea (82) 2 2653 2580
Singapore (65) 6289 1190
Taiwan (886) 2 8751 6655
www.dionex.com

Dionex products are designed,

developed, and manufactured
under an ISO 9001 Quality System.

Cobra, SmartPeaks, Operational Simplicity, and XVault are

trademarks and Chromeleon and UltiMate are registered
trademarks of Dionex Corporation.

© 2011 Dionex Corporation

LPN 2200-01 10M 01/11 Printed in U.S.A.

Passion. Power. Productivity.

 Corona ultra RS Charged Aerosol Detector
See the Complete Picture

Now sold under the

Thermo Scientific brand
Corona ultra RS Charged Aerosol
The Next Generation of Charged Aerosol Detection

The Problem—Detection
No HPLC or UHPLC detector is perfect. UV detection is the most widely used
technique, but it fails to detect compounds without chromophores. Other universal
detectors do not combine application versatility with reliability. This results in
detection gaps.

The Solution—the Corona ultra RS Detector

The Corona® ultra RS™ Charged Aerosol Detector (CAD®) is the solution. Charged
aerosol detection delivers performance that refractive index (RI), low-wavelength
UV, and evaporative light scattering (ELS) detectors simply cannot match. It
measures analytes that other technologies fail to detect. This is
because charged aerosol detection has greater sensitivity, wider Comparison of Charged Aerosol Detection to UV and MS
dynamic range, and more consistent response. Virtually every
pharmaceutical company has adopted charged aerosol detection.
The Corona ultra RS detector goes beyond simple HPLC. It UV at 220 nm
combines all the benefits of charged aerosol detection with the
high speed and increased resolution of UHPLC.

Corona ultra RS Detector Benefits UV at 254 nm

• Consistent response independent of chemical structure
• Compatibility with UHPLC
• Simple and easy to use Propanolol
Verapamil
Void + Cl- Ketoprofen

The Corona ultra RS Detector Sees the Complete Picture

The Corona ultra RS detector can be used with the most up-to-date Charged Aerosol
Detection
UHPLC technology to measure analytes that cannot be seen by
UV and may not be readily detected by mass spectrometry.
With the flexibility and performance for analytical R&D, and the
MS TIC
simplicity and reproducibility needed for manufacturing QC/QA, (+ ion scan 150-500AMU)
the Corona ultra RS detector can be used for the analysis of
pharmaceuticals (large and small molecule), biofuels, foods and
beverages, specialty chemicals, and counterions. The detector 0 0.5 1 1.5 2
can also be used in a range of applications from basic research Minutes
27612
to quality control.

2
De tector for UHPLC

Charged Aerosol Detection

What makes any detector useful is its ability to accurately measure a wide range
of analytes with consistent response. However, most detectors exhibit limitations.
Often, one analyte responds more strongly than another, or may not respond at all.
The Corona ultra RS detector measures charge that is imparted to analyte particles,
with the charge being in direct proportion to the amount of the analyte in the sample.
Measuring this charge is accurate and consistent, regardless of the analyte. The
result is that the Corona ultra RS detector can quantify any nonvolatile analyte—this
includes those without chromophores or those that cannot ionize—thus providing a
consistent response that is independent of chemical structure. With charged aerosol
detection, you can even measure many semivolatile analytes.

Nebulizer and Impactor

HPLC Column Eluent Signal Out

Analyte is
nebulized and
dried to produce
particles

Gas Inlet

Ion Trap Collector

Analyte particles transfer
their charge to a
sensitive electrometer

Largest droplets
are eliminated Positive charge added
to gas stream

Positive charge is transferred

to analyte particles

Simplicity in Operation
Step One Step Two Step Three
Charged aerosol detection begins by A stream of positively charged gas The particles are transferred to a
nebulizing the eluent into droplets, collides with the analyte particles. collector where the charge is measured
which are subsequently dried into The charge is then transferred to the by a highly sensitive electrometer. This
particles. The particle size increases particles–the larger the particles, the generates a signal in direct proportion to
with the amount of analyte. greater the charge. the quantity of analyte present.

3
Consistent Response and a
Wide Dynamic Range
Consistent Response Detector Response for Nonvolatiles
The magnitude of response obtained 10.7% RSD variation in CAD response
among 1.0 µg samples by flow injection
for nonvolatile analytes using charged 250,000
aerosol detection is independent of
200,000
chemical structure. This is demonstrated
150,000
in the figure at right, where response by
flow injection analysis is very similar for 100,000

equivalent amounts of a wide variety of 50,000

analytes. The detector response does 0
not depend on analyte optical properties
Nortriptyline
Salicylic acid
Fructose
Homocysteine
Sucrose
Dextrin
Umbelliferone
Glutathione
Amitriptyline
Methionine
Lactose
Glucose
Propranolol
Progestrone
Dibucaine
Cyclodextrin
Adrenosterone
Glucosamine
Tartaric acid
Primidone
Gentisic acid
Papaverine
4 Aminoquinaldine
Warfarin
as with ultraviolet (UV) absorbance,
or the ability to ionize as with mass Analyte
spectrometry (MS). This is critical for 27613
studies dependent on mass balance
assessment or where UV response varies
greatly. The response with charged
aerosol detection is predictable.

Forced Degradation
Wide Dynamic Range
The Corona ultra RS detector is unique
140
among universal detectors in that it 20 minutes
heating
allows quantitation across a range that
exceeds four orders of magnitude. This 600
10 minutes
wide dynamic range provides significant heating
mV

Amikacin
advantages for the simultaneous
measurement of an analyte and low- no
heating
level impurities in a single run. In the 0
mV

forced degradation example shown at 0.4 0.6 0.8

right, trace levels of degradant are found Degradant Impurities

Impurities
at the 0.19% level—well above the limit
of detection. The wide dynamic range
0
means that the low-level impurity and
0 0.5 1 1.5 2 2.5 3 3.5 4
the active pharmaceutical ingredient Minutes
27677
(API) can be measured in the same run.
With response independent of structure,
relative levels can be assessed even
without knowing the identity of the peak.

4
Enhanced Performance

Enhanced Sensitivity
The Corona ultra RS detector provides an enhancement in sensitivity when used with
UHPLC. Sub-nanogram levels of detection can be readily achieved, but there is more
than just greater sensitivity. More importantly, the level of sensitivity is consistent
across analyte types.

Complementary to UV and MS Detection

Achieving New Levels of Sensitivity
The Corona ultra RS is an ideal primary detector. However,
when combined with UV, diode array detection (DAD), or
MS, it provides an orthogonal and complementary detection
mechanism, allowing you to obtain additional analytical data. 500 pg of each sugar on column

Even More with UHPLC

The high acquisition rate and low-peak dispersion of the Glucose Sucrose Lactose
Corona ultra RS detector allows you to exploit the increased
separation speed and resolution of UHPLC, giving results that
˜50 ng of each sugar on column
are 5–10 times faster than standard HPLC.
When used with the UltiMate® 3000 RSLC x2 Dual System
employing inverse gradient capability, relative peak response
becomes a practical reality, providing the benefit of consistent 27678
response across the entire gradient range. Now, you can
determine relative purity or level of degradation even
without standards.
The Corona ultra RS incorporates capabilities to
make applications easier, including:
• An internal valve to send unwanted analytes to waste
For Ultimate Performance...
• An optional flow splitter for easier interfacing with MS
Combine the Corona ultra RS
• A flow diversion system to eliminate waste overfilling with the UltiMate 3000 x2
• A unique algorithm for data processing pump for cost effective gradient
• All the filter settings of the Corona CAD compensation, providing an ideal
tool for approximation of relative
concentrations when standards are
not available.

5
Intuitive, Simple to Operate, and

Ease of Use
The Corona ultra RS detector is virtually plug-and-play. It uses
touchscreen technology that is easy to navigate. There are
few parameters to set, and the few options are displayed on
a single screen. Instrumental parameters are explained on the
screen to allow easy choice of settings. This simple, intuitive
operation means the detector can be quickly installed, enabling
you to begin generating data.

Diagnostics
A diagnostic screen provides all the information necessary to
assure that the detector is working correctly. In addition, values
are easy to interpret. This function provides information to help
with instrument and method validation and troubleshooting.
The detector even prompts you when it is time for
preventive maintenance. Reproducibility and Reliability
Charged aerosol detection is highly reproducible. The figure
Solvent Compatibility and Conservation below shows the overlay of five runs. The peak shapes and
Virtually any volatile solvent can be used with the Corona responses are so reproducible that it is difficult to distinguish
ultra RS detector without concern for compatibility or UV the individual runs. RSDs of less than 1% are typical.
cutoff. When used with UHPLC, run times can be reduced by a
factor of 5–10 times. This saves solvent and reduces waste. Data from 150 randomly selected units over a six-month period
show an inter-unit variability of approximately 2.5%. This
means that you can expect the same results independent of
the location of the detector.

Comparison of UHPLC to HPLC

400

200 PEG 400

Overlay of five injections
400

0
0 1 2 3 4 5

200 HPLC
UHPLC

0
0 2.5 5 7.5 10 12.5 15 17.5
Minutes
27679

6
Easy to Integrate

Easily Integrates with any HPLC System

The Corona ultra RS detector can be used with any standard HPLC system, but is also
ready for UHPLC without any modification.
The detector is designed to integrate into any liquid chromatographic system, HPLC or
UHPLC, from any manufacturer. Software drivers are available for the Chromeleon® 7
Chromatography Data System, ChemStation®, EZChrom®, and Empower2®. The
stackable design and rugged construction allow it to be placed anywhere in a system.
There is no concern about moving or tipping the detector.

Makes Using the System Simple Flow Diversion

4
An on-board switching valve allows flow diversion of high-salt
samples during the analysis to simplify the chromatogram. The Overlay of multiple injections
same valve can be used to divert flow to a different detector 3

without having to disassemble the system. There is nothing to

obtain separately, nothing that needs to be installed. It is all pA

built into the detector. An optional variable-ratio flow splitter

pA
is available to integrate with MS or other parallel detectors 0
-.50
0.3 0.6 0.9 1.2 1.5
without loss of resolution. Minutes

2 Original chromatogram

1 Chromatogram with flow diversion

0

-0.5
0.3 0.5 1 1.5 2
Minutes
28628

7
Application Solutions Available Wi

Lipids Algal Oil Lipid Profile

180
HPLC is a powerful tool for lipid analysis.
With UV detection, quantitation is made Fatty Acids

difficult by differing relative response Monoglycerides

due to the wide variety of structures. Diglycerides

Phospholipids Triglycerides
Other universal detectors demonstrate Fatty Alcohols

Steroids Sterols
a lack of response consistency making
relative quantification difficult. With
response independent of chemical
structure, charged aerosol detection
delivers consistent, predictable results -5
0 5 10 15 20 25 30 35 40 45 50 55 60 65 72.7
with no optimization.
Minutes
27680

The same detector can be used to assess

purity or to perform in-process quality Total Glycerides of Biodiesel
550 20.3
control of biodiesel. Triolein 9.75
Glycerol
pA
pA
56 ng
1.4 6.93
5.95 7.5 8.75 9.95 28.28 29 29.33
300 Minutes Minutes
1,3-Diolein

1-Oleylglycerol

0
-50
0 5 10 15 20 25 30 35 39.4

Minutes
27681

Carbohydrates Carbohydrates
300
Sugar analysis using HPLC is often
performed using RI detection. But RI 2
1. Fucose
detection is limited by poor sensitivity 2. Fructose
3. Glucose
and the inability to use gradients. The 4. Sucrose
3
Corona ultra RS provides gradient mV
4 5. Lactose
1
separations and sensitivity to low ng. It
also offers the ability to use reversed-
5
phase and HILIC modes of separation,
making it an ideal complement to
analysis with HPAE-PAD. 0

0 2 4 6 8 10

Minutes
28629

8
th the Corona ultra RS Detector

Surfactants Surfactants
Surfactants such as Triton® X-100, Corona ultra RS (n = 5)
900
which lacks a chromophore, can be
9
easily measured with the Corona
ultra RS detector. Unlike some other 500
universal detectors, the wide dynamic 0

-3
range of charged aerosol detection 0 1 2 3 4 5 6 7 8 9 10 11 12 13

technology lets you assay a full range

of concentrations without overloading 0

the detector. 0 2 4 6 8 10 12 14 16 18 20 22 24 26

Other nebulizer-based detector (n = 1)

10
1000

50

0
19.20 20 21.20
500
1.1
1 6 112
Detector
overload
0

0 2 4 6 8 10 12 14 16 18 20 22 24 26
Minutes
27682

Unique Data Algorithm Effect of Power Function

The Corona ultra RS includes firmware
1.5
to apply a power function to the data
Progesterone
Hydrocortisone
Primidone

Warfarin
Ketoprofen

output, often decreasing baseline noise 3

and linearizing the data. With a few

Response (peak area)

pA
injections the ideal setting can 1.5

be determined. 0 1

0
0 500 1000 1500 2000 2500
Mass on Column (ng)
-0.5

0.70
Hydrocortisone
Primidone

Progesterone
Warfarin
Ketoprofen

2
Response (peak area)

pA
1

2
0
0
0 500 1000 1500 2000 2500
-0.25 Mass on Column (ng)
0.17 0.5 1 1.4

Minutes
28630

9
Application Solutions

Pharmaceutical Analysis
Typical Pharmaceutical Agents
5 overlaid concentrations from 11–170 ng

Dodecylsulfate Na
28
Product Characterization

Theophylline
The Corona ultra RS detector makes it

Diclofenac Na
Erythromycin
Acetaminophen

Naproxen Na
DL-Leucine

Progestrone
easy to determine the concentration and

D-Phenylalanine
purity of any non- and many semivolatile
active pharmaceutical ingredients
(APIs) and excipients. With response
independent of the chemical nature of
the analyte, almost any API or compound
used in the formulation can be measured Diclofenac sodium salt (5.7 µg on column) spiked with 0.1% and 0.15% (w / w) chloride.
with predictable response. -2.8
0.12 1 2 3 3.95
Minutes
27140

Using the unique Acclaim® Trinity™ P1

column and the Corona ultra RS detector, API and Counterions
10.5
the API, counterion, and impurity ions Sodium Diclofenac
can all be quantified, simultaneously. Chloride
This speeds up selection of counterions
0.15% Chloride Spike
in the development process.
0.1% Chloride Spike

No Spike

Diclofenac sodium salt (5.7 µg on column) spiked with 0.1% and 0.15% (w / w) chloride.
-5

0 1 2 3 4 5
Minutes
27683

Cleaning Validation
110 Typical Cleaning Agent
Cleaning validation can be both
difficult and time consuming. Using the
Corona ultra RS detector, it is possible
to measure the API and cleaning agents,
either separately or together, as well as
estimate their relative amounts. A profile
representing the relative amounts of
cleaning agent can be generated even
without knowing the identity of
the components. –10

0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0

Minutes
27142

10
Biopharmaceutical Analysis Pegylated Protein
Analysis of biopharmaceuticals present
new challenges. Complex mixtures of PEG-MAb 50 µg o.c.

large and small molecules, many without

chromophores, can be analyzed using
charged aerosol detection. 3.0 0.1% PEG

PEGylated MAb
PEGylation helps the biological stability 2.0 0.3% PEG
0.2% PEG
of therapeutic monoclonal antibodies 0.1% PEG
(MAbs) but measuring the residual PEG
can be difficult. With predictable response
1.0
irrespective of the analyte, charged
0 10 20
aerosol detection can be used to measure Minutes
27685
residual PEG in PEGylated proteins. With
its sensitivity and dynamic range, both
low levels of the PEG impurity and the
Cationic Cell-Penetrating Peptides
high levels of PEGylated protein can be
50
measured together, without the need to
reanalyze the sample.
Transportan
MPG
siRNA Delivery Vehicles Syn B1

The use of siRNA as a therapeutic agent

requires optimized delivery vehicles. The
detector lets you measure the purity and
stability of both lipids and peptides used
0
in these delivery systems. -5
0 1 2 3 4 5 6 7 8.2
Minutes 27686
Final Product Purity
Charged aerosol detection is ideal for
peptide and protein analysis. With no Fast-Acting Insulin
50
restriction on solvents, as with UV,
6.09
the CAD gives you a greater range of Insulin
Unknowns
selectivity. And you never need worry
pA
about acetonitrile shortages again!
From raw material qualification to 0.53
10.23 Minutes 12.96
final product purity of large or small
molecules, the Corona ultra RS detector 0
lets you see things as you have never -5
0 2 4 6 8 10 12 14 16.1
seen them before.
Minutes
27687

11
Discover the Corona ultra RS For more information or to place an order, Corporate Headquarters
Detector–Giving You More for contact the Dionex office nearest you or Dionex Corporation
your local distributor. Phone numbers and 1228 Titan Way
HPLC and UHPLC P.O. Box 3603
addresses for worldwide subsidiaries can Sunnyvale, CA 94088-3603
In the world of HPLC/UHPLC, one be found in the About Dionex section of Tel: (408) 737-0700
detection technology stands out. www.dionex.com. Fax: (408) 730-9403
Charged aerosol detection has response
independent of analyte structure, Worldwide Sales and Service
provides consistent responses across a Enjoy Industry-Leading Support
North America
range of non- and semivolatile analytes, Dionex Customer Support Centers are U.S./Canada (847) 295 7500
has a wide dynamic range and broad located in the Americas, Europe, and
South America
applicability, and is as easy to use as UV. Asia, and provide accessible locations Brazil (55) 11 3731 5140
for advanced training and enhanced
The Corona ultra RS detector combines Europe
application development capabilities.
the benefits of charged aerosol detection Austria (43) 1 616 51 25 
Users can visit these laboratories to learn Benelux (31) 20 683 9768
with the speed and resolution of UHPLC
new skills in addressing challenging (32) 3 353 42 94 
for faster, high-resolution separations Denmark (45) 36 36 90 90 
applications, receive training and support,
while still giving superior performance France (33) 1 39 30 01 10 
and discover new, innovative LC and Germany (49) 6126 991 0 
with HPLC. This detector provides
sample preparation solutions. Ireland (353) 1 644 0064
the flexibility and performance for Italy (39) 02 51 62 1267
analytical R&D, and the simplicity and Sweden (46) 8 473 3380
reproducibility needed for manufacturing Switzerland (41) 62 205 9966 
QC/QA. It can be used for almost any United Kingdom (44) 1276 691722
analysis in pharmaceuticals (large and Asia Pacific
small molecule), biofuels, foods and Australia (61) 2 9420 5233 
China (852) 2428 3282 
beverages, specialty chemicals, and ions India (91) 22 2764 2735 
including a wide range of applications— Japan (81) 6 6885 1213 
from basic research to quality control. Korea (82) 2 2653 2580
Singapore (65) 6289 1190
This makes the Corona ultra RS the
Taiwan (886) 2 8751 6655
detector of choice for your application.
www.thermoscientific.com/dionex

Dionex products are designed,

developed, and manufactured
under an ISO 9001 Quality System.

Specifications subject to change without notice.

Corona, CAD, UltiMate, Acclaim, and Chromeleon are registered

trademarks and Trinity and ultra RS are trademarks of Dionex Corporation.

Chemstation and EZChrom are registered trademarks of Agilent Technologies.

Empower2 is a registered trademark of Waters Corporation.
Triton is a registered trademark of Rohm & Haas.

LPN 2542-02 PDF 06/11

© 2011 Dionex Corporation.

Speed • Simplicity • Solutions

 Corporate Headquarters
Dionex Corporation
1228 Titan Way
P.O. Box 3603
Sunnyvale, CA 94088-3603
Tel: (408) 737-0700
Fax: (408) 730-9403

Worldwide Sales and Service

North America
U.S./Canada (847) 295-7500

South America
Brazil (55) 11 3731 5140

Europe
Austria (43) 1 616 51 25
Benelux (31) 20 683 9768
(32) 3 353 42 94
Denmark (45) 36 36 90 90
France (33) 1 39 30 01 10
Germany (49) 6126 991 0
Ireland (353) 1 644 0064
Italy (39) 02 51 62 1267
Sweden (46) 8 473 3380
Switzerland (41) 62 205 9966
United Kingdom (44) 1276 691722

Asia Pacific
Australia (61) 2 9420 5233
China (852) 2428 3282
India (91) 22 2764 2735
Japan (81) 6 6885 1213
Korea (82) 2 2653 2580
Singapore (65) 6289 1190
Taiwan (886) 2 8751 6655

www.dionex.com

Dionex products are designed, developed, and

manufactured under an ISO 9001 Quality System.

© 2010 Dionex Corporation

PEEK is a trademark of Victrex PLC.
Analyst is a registered trademark of Applied Biosystems/MDS Sciex.
Hystar is a trademark of Bruker Daltonics.
Xcalibur is a registered trademark of Thermo Fisher Scientific.
All other trademarks and registered trademarks are the property of Dionex Corporation.

LPN 1820-05 10M 08/10 Printed in U.S.A.

UltiMate 3000 Liquid
Chromatography Systems

Giving you more.

Contents
UltiMate 3000 Systems and Solutions 4

Rapid Separation LC 6

Best In Class HPLC Systems 8

Pumps 10

Samplers and Column Compartments 11

Detectors 12

Viper Fitting System 13

Chromeleon Software 14

D-Library 16

Mass Spectrometry 17

LC Columns 18
UHPLC delivers major benefits, we all know this—
faster runs, better resolution, and lower running
costs. The products available to serve UHPLC
have become more sophisticated over the past
five years and yet remain mostly expensive and
therefore limited in their accessibility to the wider
HPLC user community.

Thanks to Dionex this is no longer true. Dionex now

offers UHPLC performance at standard HPLC costs. In
fact Dionex is so focused on UHPLC for all
HPLC users that it now only provides ‘U’
compatible instruments.

Dionex has not only added UHPLC compatibility to all

of its standard and basic automated HPLC systems but
it has also extended the UHPLC performance of the
UltiMate® 3000 RSLC systems – giving you more
opportunities to get the answers you are looking for;
today, tomorrow and with confidence of future
proofness too.

• Dionex offers the exceptional industry leading

Chromeleon® software
• Dionex Corona® Charged Aerosol Detectors
(CAD®), plus other detector options
• Dionex Applications D-Library. Never before has so
much power and information been available in a
single package and for such a low initial investment
Now everyone can have the confidence to move to
UHPLC technology + everyone can have access to
UHPLC technology for nearly all methods + everyone
has access to the unique strength of Dionex UltiMate
in combination with Chromeleon Software and
Corona detectors.

Dionex – the only HPLC company uniquely

focused on making UHPLC technology available to
all users, all laboratories, and for all analytes.
 UltiMate 3000 Systems and Solutions
The Most Versatile Choice for Your Analytical Needs

The UltiMate 3000 HPLC system offers UHPLC compatibility across

all modules, ensuring maximum performance for all users and all
laboratories. Covering flows from 20 nL/min to 10 mL/min and offering a
wide range of pumping, sampling, and detection modules, the UltiMate
3000 series provides solutions for all your chromatography needs.

• UHPLC design philosophy • x2 Dual Systems form a unique • Offered with Viper™ and
throughout the whole range of platform for routine analysis, nanoViper™ fitting systems—
nano, standard analytical, increased productivity solutions, fingertight, zero-dead-volume
and RSLC and advanced chromatographic connections even at UHPLC
• 620 bar maximum pressure for techniques pressures
basic and standard analytical • Controlled using Chromeleon
systems sets a new benchmark Chromatography Data System
in HPLC software—providing Intelligent
Functionality and Operational
Simplicity™

RSLCnano Systems RSLC Systems Standard Systems

• Widest nano/cap/micro flow range • Binary or quaternary systems for • Optimal performance and reliability for
from 20 nL/min up to 50 μL/min both UHPLC and conventional conventional LC applications
HPLC applications
• Column pressure up to 800 bar • 620 bar maximum pressure and 100 Hz
• Extensive flow-pressure-footprint for data rate for UHPLC compatibility
• Continuous direct flow delivery
ultrafast, ultrahigh-resolution
• Widest range of system configurations
• Loading pump provides flows from separations
for maximum application flexibility
10 μL/min to 2.5 mL/min
• Column pressure up to 1000 bar
• Flow rates up to 10 mL/min covering
• x 2 Dual RSLC systems for ultimate all application needs
productivity solutions

4
 1,000

Pressure (bar)
500

The UltiMate 3000 systems are fully

modular, allowing you to choose
common system configurations or
design the most suitable system for 100
your application needs.
0.01 0.1 1.0 10 100 1,000 10,000
Flow rate (µL/min)

The extensive flow-pressure-footprint of the

UltiMate 3000 family allows you to quickly identify
the most suitable system for your needs. RSLCnano Standard and Basic RSLC

Basic Systems Detection Options Software and Consumables

• Cost-effective system for • Wide range of optical detectors— • Chromeleon software for intuitive
routine applications UV-vis, fluorescence, and instrument control and streamlined
refractive index data handling
• Robust operation for consistent, reliable
results • Unique Corona universal Charged • D-Library for application searching
Aerosol Detection
• 620 bar maximum pressure and 100 Hz • Viper and nanoViper fingertight fitting
data rate for UHPLC compatibility • High sensitivity electrochemical systems for easy configuration
detectors
• Optional Autosampler Column • Extensive range of columns including
Compartment with integrated sampling • Supported by MS detectors from monolithic technology and advanced
and column temperature control AB/MDS Sciex, Bruker Daltonics, multi-mode phases
and Thermo Fisher Scientific

5
 Rapid Separation LC
Widest Flow-Pressure Footprint for Ultimate Performance and Versatility

UltiMate 3000 RSLCnano Systems

The RSLCnano system was developed with throughput
in mind. The robust, continuous direct-flow delivery is 5
designed for interruption-free analysis. The wide flow-
pressure-footprint enables the application of UHPLC to 4

Intens. × 107
the nano scale, allowing analysts to tune for the highest 3
resolution or the fastest analysis time, even for tryptic
peptide samples of utmost complexity. Configurable for 2

speed, separation power, or sensitivity, the UltiMate 3000 1

RSLCnano is the only system to deliver all. 0
0 20 40 60 80 100 120 140
Minutes
Operation at nano, capillary, and micro flow rates and the
powerful dual-gradient availability provide application Column: Acclaim® PepMap™ RSLC nano column, 75 µm × 15 cm
flexibility. The UltiMate 3000 RSLCnano supports Peak
Capacity: ~300
advanced workflows, for example automated off-line 26260

2D-LC, with ease. Using both pumps, two switching

Base peak chromatogram of a complex tryptic peptide sample.
valves, and the microfractionation option of the
autosampler, this set-up demonstrates the full flexibility of
the system.

Sample protein extraction Separate proteins 1D

Fractionate protein sample Separate proteins 2D In-well digestion

Separate peptides LC-MS/MS Database search, protein ID

6
UltiMate 3000 RSLC Systems Ten Peaks in Ten Seconds

The RSLC system accelerates HPLC for unrivaled

performance and flexibility. Precision-engineered
instrumentation, advanced data processing, and highly
optimized chemistries meet all chromatographic
performance challenges. With binary, quaternary, or
dual-gradient pumps, the RSLC system offers industry-
leading versatility covering the maximum range of HPLC
applications, including conventional and ultrafast LC.

The UltiMate 3000 x2 Dual RSLC systems offer

unprecedented sample throughput and easy automation
of advanced procedures. They provide highest
selectivity and resolution with multidimensional LC and 10
increased instrument use time by automatically Seconds
0
switching between applications: UHPLC, HPLC, or both.
Separation of uracil and 9 alkylphenones in 10 s, with a full 100 Hz DAD
Seamless integration of UHPLC with x2 Dual RSLC
spectral scan. The run was performed using a flow rate of 3.7 mL/min,
technology and powerful Chromeleon software brings a back pressure of 730 bar, and an oven temperature of 100 °C on a
new possibilities to laboratories. 30 × 2.1 mm C18 column with 1.8 µm particle size.

Application #1 Automated Switchover Application #2

4:00 pm 6:00 pm 8:00 pm 10:00 pm 12:00 pm 2:00 am 4:00 am 6:00 am 8:00 am

Acclaim TrinityTM P1: Unique

4
selectivity for API/counter ion analysis
Cations Anions Acclaim C18 RSLC: Unique
1. Procaine 7. Mesylate speed for analysis of statins
2. Choline 8. Maleate
3. Tromethamine 9. Chloride
4. Sodium 10. Bromide
5. Potassium 11. Iodide 1
1 - Mevastatin
6. Meglumine 12. Phosphate 2 - Lovastatin
13. Malate 3 3 - Simvastatin
14. Tartrate 2
15. Citrate
10 16. Oxalate
2 9
5 7
3 8 15
1 6 11 12 13 14
16

0 3 6 9 12 15 0 12 24 36 48
Minutes Seconds
26165

Automate two applications to run unattended overnight or over the weekend. The system will automatically flush columns and lines, switch to the other
column, re-equilibrate the system with the new mobile phase, and start the second application. Applications can be standard HPLC, UHPLC, or both, as
shown above. The Corona CAD detector enables simultaneous detection of API and counterions, and the 5 µL semi-analytical flow cell in the DAD ensures
full compatibility with a wide variety of methods.

7
 Best In Class HPLC Systems
Fully UHPLC Compatible – Covering All Application Needs

Standard Systems
HPLC is a primary tool for many chromatography laboratories, but specific
system requirements vary greatly from one application to another. The
UltiMate 3000 Standard Analytical systems are designed to meet today's
requirements and future challenges. They offer full support of all HPLC
applications and provide UHPLC compatibility – allowing you to move to
UHPLC whenever you are ready.

If you need even more throughput or are looking to run automated techniques
such as on-line sample preparation, the UltiMate 3000 x2 Dual LC systems
provide the perfect solution.

1 – Acesulfame K
Standard mixture WVL:210 nm
2 – Saccharin
3 – Caffeine
4 – Aspartame
5 – Vanillin
1,000 6 – Benzoate
7 – Sorbate
8 – Benzaldehyde

50.0

mAU
3 5
6 7
1 2
4 26.0
7.0 % CH3CN
0.06% Formate
8
7.0

Fast analysis of soft drink additives at 515 bar.

Flow:
1.200 mlL/min

0 1 2 3 4 5 6 The standard mixture shows baseline separation

Minutes of all analytes in only 6 minutes. The overlay
Overlay of 10 energy drink
samples
3
of 10 consecutive injections of the sugar free
RT Precision
1 – Acesulfame K 0.052% RSD energy drink shows excellent retention time and
1,000
3 – Caffeine 0.048% RSD area precision.
4 – Aspartame 0.034% RSD
5 – Vanillin 0.024% RSD

50.0

mAU

7.0 % CH3CN 26.0

0.06% Formate
1
4
5
7.0 Peaks:
Flow:
1.200 mL/min
1. Norepinephrine
2. Epinephrine
0 1 2 3 4 5 6
3. Internal Standard
Minutes 27703
4. Dopamine
1
4

3
UltiMate 3000 x2 Dual LC systems offer unprecedented levels of
flexibility, significantly increased sample throughput, and automation
2
of advanced procedures:

• Double sample throughput with Parallel and Tandem LC

• Switch between two applications easily and unattended
• Automate sample preparation and analysis of complex samples
0 2 4 6 8 10 12 14 16 18
by on-line SPE-LC Minutes
27541
• Full UHPLC compatibility for decreased run times, and On-line SPE-LC analysis of drugs in
better separations biological fluids.

8
Basic Automated Systems
The Basic Automated LC system is optimized for 3
Peaks:
reliability and ease-of-use with routine LC applications, 2
1. Retinol
2. -Tocopherol
and also offers full UHPLC compatibility. The ACC-3000 1
3. -Tocopherol
4. -Tocopherol
Autosampler Column Compartment is at the heart of the 5. -Tocopherol acetate

system. Its unique instrument design combines a rugged

sample injection principle with a powerful column oven.

counts
• Future proof yet cost-effective alternative to
4
standard systems
• 620 bar maximum pressure and 100 Hz data rate
5

for UHPLC compatibility

• Minimum number of wear parts and critical 0 0.5 1 1.5 2 2.5 3 3.5
connections
Minutes 27704

• Industry leading range of detectors Fast isocratic separation of tocopherols in less than 4 min
at 460 bar (6700 psi) using fluorescence detection.
• Modular versatility at compact system size

The Basic Automated system provides extremely

reliable and robust chromatography. This ensures that
you get precise and accurate results, every day, and
Peak Area of Acesulfame K
every application. 3.6

• Optimized design for routine analysis

3.5

• High levels of precision for consistent results 3.4

• Integrated sampler and column compartment

3.3

3.2

3.1
0 10 20 30 40 50 60 70
No. of Injection

27705

Peak areas for Acesulfame K over 68

consecutive injections of a soft drinks analysis,
all ran under UHPLC conditions. The RSD is
only 0.151% demonstrating excellent precision.

9
 Pumps
Delivering Powerful and Precise Flows

The UltiMate 3000 pump family offers the most complete choice in the industry.
From nano LC to rapid separation applications, from conventional applications
to UHPLC, the UltiMate 3000 pumps always provide industry-leading flow,
pressure, and precision.

Isocratic Binary Quaternary Dual-Gradient

Analytical RSLCnano Micro Micro
Analytical Analytical Analytical
RSLC RSLC RSLC

• Extensive mixer portfolio to

cover all application needs
• Adapt the mixing volume within
seconds

Longitudinal Mixing

Solvent A
Flow Direction

Unique SpinFlow™ mixing design for exceptional mixing performance

Solvent B Radial Mixing Capillary

• Clean and intuitive fluidic design

• Easy access and smart software support for
effortless maintenance
• Ultra precise and robust pump drives with
near-zero acoustic noise
 martFlow® technology automatically ensures optimal
• S
chromatographic performance for any operating conditions

10
Samplers and Column Compartments
Advanced Automation Techniques

The UltiMate 3000 autosamplers ensure reliable, precise, and accurate

injections of nL to mL sample volumes, with extremely low carryovers. Fraction
collectors range from simple collection to advanced collection-reinjection
workflows such as automated off-line 2D-LC.

Autosamplers Fraction Collectors Column Compartments

Split-Loop, Fraction Collector Integrated Switching Valves
Pulled-Loop Autosampler/Fraction Collector
With integrated column compartment MALDI Spotter

• High robustness injection valves for maximum reliability

• Easily change syringe for maximum injection volume flexibility

• Choose from a wide range of sample storage formats

• F
 reely configurable and user-interchangeable high-pressure
switching valves
• Precise temperature control, even under varying ambient conditions

11
 Detectors
Detection Options to Fit Your Application

Optical Detectors
The UltiMate 3000 optical detectors are available for UV-vis
absorbance, fluorescence, and refractive-index detection of
a wide variety of analytes. High data collection rates,
multiple wavelength detection, and 3D UV spectra provide
the UHPLC compatibility and the versatility to match your
analyte and separation technique.

Detector Type Maximum Data Rate Details

DAD-3000 Diode Array 3D UV spectra, accurate library searches, peak purity analysis, multiple
200 Hz or 100 Hz
Detector channel acquisition
MWD-3000 Multiple
200 Hz or 100 Hz Simultaneous data acquisition of up to 8 data channels, upgradable to DAD
Wavelength Detector
VWD-3000 Variable
200 Hz or 100 Hz Single-channel detection with widest linear range and lowest noise
Wavelength Detector
FLD-3000 Fluorescence Trace analysis of fluorescent compounds, ultrafast switching between different
200 Hz or 100 Hz
Detector excitation / emission wavelengths

RI-101 10 Hz Universal detection of main compounds under isocratic elution conditions

Charged Aerosol Detectors Electrochemical Detectors Mass Spectrometry

Corona charged aerosol detectors The Coulochem® III is an The MSQ Plus™ is one of the
(CAD) provide universal detection electrochemical detector (ECD) for smallest and most sensitive
for any non-volatile and some easily oxidized or reduced analytes. quadrupole mass spectrometers on
semi-volatile analytes and are It offers outstanding performance for the market and is fully supported by
compatible with gradients. The amperometric, coulometric, and the Chromeleon software. In addition,
Corona ultra™ offers up to 100 Hz pulsed amperometric detection. The fully integrated, single-point control
output rate for full compatibility with coulometric dual electrode 6011 for any Dionex LC system through
UHPLC applications. The Corona ultra Analytical cell offers full Xcalibur ® (Thermo Fisher Scientific),
CAD is ideal for conventional UHPLC compatibility in coulometric Analyst® (AB/MDS Sciex), and
HPLC applications. mode along with the high sensitivity HyStar™ (Bruker Daltonics) is
and selectivity of ECD. provided by DCMSLink™, a free
control plug-in.

12
Viper Fitting System
Connect to the Future

The First Universal Fitting System • Compatible with virtually every type of valve and
The Viper and nanoViper fitting systems do away with column hardware
problems experienced with conventional fitting systems. • Flexible stainless steel or fused silica capillaries
They provide a perfect fit each time and ensure superior All RSLC and x2 Dual system tubing kits feature the
chromatographic performance. unique Viper design, making the best HPLC systems even
easier to use. The nanoViper connections are standard on
• Zero-dead-volume UHPLC fingertight fittings for
RSLCnano accessories and Acclaim PepMap RSLC nano
nano/cap, micro, and analytical LC
columns.

Injection #4 Injection #1 Uracil

600 Injection #101 600 Injection #50
Uracil
Injection #100
mAU

Acetanilide Acetophenone
Acetophenone
Acetanilide

300 300

0 0
Slipping capillaries cause deteriorated
0 9 18 0 9 18 peak shapes (left). Viper capillaries
Seconds Seconds provide robust performance at UHPLC
26166 pressures (right).

The Viper connector tightens at the

tip of the capillary, and does not use
ferrules that may cause incompatibility
with the opposite holder.

13
 Chromeleon Software
Bring the Power of Operational Simplicity to Your Laboratory

The Dionex Chromeleon 7 Chromatography Data System delivers rich,

intelligent functionality with Operational Simplicity, providing everything you
need within easy reach. The features you need most often are kept in view, and
the thoughtfully designed user interface guides you effectively through
instrument setup, sequence programming, and data analysis. Innovative
eWorkflows enable anyone to start chromatographic analyses and get good
results with just a few selections. Powerful data analysis tools ensure that data
can be displayed, interpreted, and reported efficiently and accurately.
Chromeleon 7 software gets you from samples to results quickly and easily—
boosting your overall lab productivity.

Intuitive ePanels provide direct access to all main instrument control Take advantage of the Chromeleon software’s extensive diode-array
features (for example, flow rate, temperature, detector wavelengths), options to confirm peak purity, positively identify compounds, and
plus easy access to advanced options. Instrument control has never optimize methods.
been easier.

14
eWorkflow Takes You from Samples to SmartPeaks for Fast Integration
Results, Quickly and Easily

The eWorkflows feature provides the simplest possible operation of The unique data processing tools
instruments. They automatically create sequences containing the correct SmartPeaks™ and Cobra™ make peak
instrument conditions, data processing parameters, and reporting detection and integration easy and
templates. Samples are run automatically, immediately processed, and the straightforward.
resultant data instantly reported, ensuring the fastest possible time from
samples to results.

15
 D-Library
The Easiest Way to Find Applications

D-Library is an exciting application library that uses web 2.0 technologies to

achieve maximum accessibility. With its web user interface, customers can
quickly find LC and IC applications based on various search criteria such as
analyte, matrix, market, column type, instrument type, or run time.

Users get instantly notified about new applications by the built-in RSS feed.

All details of the application are available in a rich interactive view.

Once a suitable application has been found D-Library can provide users with
ready-to-run Chromeleon software files

Quickly search for applications, easily review

them, and download for immediate use.

16
Mass Spectrometry
DCMSLink Software for LC and Mass Spectrometer Control

DCMSLink, a free control-only software package, provides fully integrated,

single-point control of any Dionex LC system through Xcalibur (Thermo Fisher
Scientific), Analyst (AB/MDS Sciex), and HyStar (Bruker Daltonics). The MS
workstation sets up the sample sequences, processes the mass spectrometry
data, and uses DCMSLink to control all LC modules. Learning new software is
held to a minimum, but the full benefit of the UltiMate 3000 single and dual
pump systems are ready to support your MS applications.

Unique and intuitive instrument control through comprehensive and

customizable graphical user interface within the MS workstation.

Link
DCMS software provides single-point
LC-MS control for Applied Biosystems, Bruker
Daltonics, and Thermo Fisher MS instruments.

17
 LC Columns
Wide Selection of Flow Rate Ranges and Selectivities

Nano Columns
A wide range of dedicated Acclaim PepMap nano columns
with particle sizes down to 2 μm, lengths up to 50 cm, and
zero-dead-volume connections.

Standard Columns 3
4 6
5
2 Acclaim C18
The Acclaim column family features innovative bonding 1

technology for a wide range of different selectivities, and AU

4
5
highly efficient packings in all common column formats. 12
3 6
Acclaim PA2

0 2 4 6 8 10
Minutes

Rapid Separation LC Columns

The Acclaim RSLC columns provide ultimate speed
and resolution at low back pressures for straightforward
method speed-up.

Bio Columns
Numerous column chemistries including a unique monolith
technology for ultrafast high resolution separations of
biomolecules.

Specialty Columns 1
2
3 4
Acclaim Explosives E1
5 6 7 8 9 10 11
12 13 14
Customized selectivities for dedicated application
requirements. The Acclaim specialty columns are the AU 3 4
Acclaim Explosives E2
5 7 11
best choice for separating surfactants, organic acids, 1 2 610 12
13 14 8 9
or explosives.
0 9 18 27 36 45
Minutes

Mixed-Mode Columns
Innovative columns with tunable selectivities, allowing
simultaneous separations of ionic and hydrophobic
and/or hydrophilic organic analytes.

18
Dionex Training & Support Facilities

Dionex measures success by enabling customers to

succeed. This level of success relies on training and
support that empower our customers worldwide.
To reach customers in every region, Dionex manages
service and application facilities or "Customer Support
Centers," located in the United States, Europe, and Asia.

These state-of-the-art laboratories are equipped with the

full line of Dionex HPLC and ion chromatography
instrumentation, as well as updated software capabilities.

Dionex Customer Support Centers provide the worldwide

scientific community with accessible locations for
advanced training and enhanced application development
capabilities. Chemists can attend these laboratories to
learn new skills in addressing challenging applications,
receive training and support, and discover new, innovative
HPLC and IC solutions.

19
UltiMate 3000 RSLC System
Giving you more.

Passion. Power. Productivity.

 Rapid Separation LC
Ultimate Speed, Ultimate Resolution, Ultimate Performance

Accelerate LC with the UltiMate 3000 RSLC System

The UltiMate® 3000 Rapid Separation LC (RSLC) system accelerates HPLC for unrivaled
performance and flexibility. Precision-engineered instrumentation, advanced data processing,
and highly optimized chemistries meet all chromatographic performance challenges. With
its binary, quaternary, and dual-gradient pumps, the RSLC offers industry-leading system
versatility covering the maximum range of HPLC, including conventional and ultrafast LC.
• Up to 50 times faster than conventional LC
• High resolution for maximum peak capacity
• Instant results with Chromeleon® software UltiMate 3000 RSLC Flow-Pressure Footprint
• Universal method transfer and speed-up Zone 1 Zone 2 Zone 3 Zone 4 Zone 5
• Viper™ fingertight fitting system RSLC
• Dual-gradient pumps and switching valve options Resolution Ultrahigh Conventional Very High High High
for advanced chromatographic techniques
Speed High Conventional Very High Ultrahigh Ultrahigh
Together, these characteristics make RSLC the only
available choice for maximum resolution to Typical Flow (mL/min) 0.2–1.5 0.75–2.0 1.0–3.0 2.5–5.0 5.0–8.0
maximum speed LC. Column Length (mm) ≥100 ≥150
≥50
≤50 ≥100
≤100
Column i.d. (mm) ≤3 ≥4 ≤3 ≤3 ≥4

Particle Size (µm) ≤3 ≥3 ≤3 ≤3 ≥2

1000
Pressure Range (bar)

0
0
1
2
3
4
Flow r
a te (mL 5
/min) 6
With its extensive flow-pressure footprint, RSLC fully 7
meets your chromatographic goals. Simply work with your 8
column of choice and in the appropriate zone for your application.

2
The Right Combination of Components and
Data Management
The UltiMate 3000 RSLC system delivers superior performance, excelling at
pressures of 100 MPa (15,000 psi) and flow rates up to 8 mL/min. Detector
and autosampler technologies, powerful software, and small particle columns
contribute to RSLC capabilities.

Solution Component Key Feature

Pressure up to 100 MPa
Binary, Quaternary, or Dual
(15,000 psi) at flow rates up to
Gradient RS Pump
8 mL/min
In-line Split Loop Cycle times of 15 s
Well Plate Sampler Injection volumes of up to 500 µL

Thermostatted RS Temperature range of 5–110 °C

Column Compartment Up to 12 columns
Data collection rates up to
Diode Array, Variable Wave-
200 Hz, even when running full
length, and Fluorescence RS
wavelength scans on the
Detectors
DAD-3000RS
Particle sizes of 2.2 and 3 µm
Acclaim® RSLC Columns with up to 1000 bar pressure
ratings
Zero dead-volume connections,
Viper Fingertight Fitting System robust performance, and unpar-
alleled ease-of-use
Chromeleon Chromatography Dynamic data processing that
Data System produces results instantly

UltiMate 3000 RSLC system

3
 Ingredients for Speed
High-Performance UHPLC Over a Wide Range
of Flow Rates and Pressures

A Complete Package for Performance Leadership

Engineered to the highest level of performance, the UltiMate 3000 RSLC system delivers
the key flow rate and pressure capabilities to meet all LC challenges. With its extensive
flow-pressure footprint, short sampler cycle times, high column temperatures, ultrafast data
collection and processing, and high-resolution Acclaim RSLC 2.2 µm columns, the system
maximizes LC flexibility. This combination of features delivers optimal separations at ultrahigh
speed while maintaining good resolution.

100

10 µm particles
5 µm particles
3 µm particles
2.2 µm particles

H [µm]

70 0
0 5 10
Linear Velocity u [mm/s]

According to the van Deemter curve, the lower the H value,

the higher the separation efficiency. Smaller particle sizes
give low H values, ideal for fast separations on short columns.

H [µm] 3× Faster. 90% of

Original Resolution
2× Faster. 96% of
Original Resolution
Optimum Flow for
Maximum Resolution

0
0

u [mm/s]
Using columns with small
particle sizes, you can accelerate LC at
higher flow rates and achieve almost identical resolution to
conventional LC at lower flow rates. For example, with 2.2 µm particles you
can double the speed of your method and still have 96% of your original resolution.
20

4
High Flow and Efficiency RSLC Provides Unrivaled Speed: 10 Peaks in 10 Seconds
The Dionex UltiMate 3000 RSLC system is
designed to deliver robust operation at high
flow rates and high pressure.
• Backpressures up to 100 MPa
(15,000 psi)
• Flow rates up to 8 mL/min
• Oven temperatures up to 110 °C
• Data collection rates up to 200 Hz

Seconds
10
0
Separation of uracil and nine alkylphenones in 10 s, with a full 100 Hz DAD spectral scan.
The run was performed using a flow rate of 3.7 mL/min, a backpressure of 730 bar,
an oven temperature of 100 °C, and a 30 × 2.1 mm C18 column with 1.8 µm particle size.

700
Benzoate
Sorbate
Caffeine
Acesulfame K
mAU

Benzaldehyde
Saccharin

Aspartame

-100
0 Seconds 40
25332

Analysis of seven key compounds in soft drinks in less than 40 s on the Acclaim RSLC 2.2 µm column.

5
 High-Resolution RSLC
Acclaim Columns and RSLC Ensure Efficiency, Speed, and Resolution

Resolution and Speed: Meeting the Challenge for Superior LC

Achieving the highest resolution in the fastest run times takes more than a great LC system.
High resolution also requires low extracolumn volumes and long columns with small particle sizes.
The way to achieve high-resolution UHPLC is to improve efficiency and reduce band broadening.
Small particle sizes significantly increase efficiency; lower extracolumn volumes ensure that this
increase is not lost again due to band broadening.

700

Separation on 5 µm material
mAU

Reducing particle size increases separation of compounds,

0
1.7 2 3 provides optimal peak resolution, and increases peak
Minutes
25333a height for improved sensitivity.

700
Separation on 2.2 µm material
mAU

0
1.7 2 3
Minutes
25333b

6
Small Particle Size Columns Provide the Highest Efficiency
To optimize your fast separations, the Dionex UltiMate 3000 RSLC system delivers the
lowest extracolumn volumes and highest efficiency with small particle size columns.
• Unique, easily scalable Hurricane mixer technology
• Comprehensive mixer portfolio from 35 µL to 1500 µL for a wide application range
• Revolutionary Viper fingertight fitting system for performance without compromise
• Dedicated range of 1, 2.1, and 3 mm i.d. high-resolution columns
• Compatible with all commercially available stationary phases

Conventional reversed-phase LC separation of

16 19 amino acids in 31.5 min (60 min total run time).
1 Asp

2 Glu

19 Lys
3 Asn
4 Ser

9 Arg
10 Ala

11 Tyr
6 His
5 Gln
7 Gly
8 Thr

14 Met
13 Val

18 Leu 30.22
15 Trp
16 Phe
mAU

17 Ile
12 Cys-Cys

-6
0 7.5 Minutes 31.5
27459

2 × 10 6
49.2
5 Ser 1.71

8 Gly#1 2.1

38.0
12 Ala 2.61
11 Arg 2.51
4 Asn 1.61
6 Gln 1.89

9 Thr 2.17
7 His 1.99

Counts
2 Asp 0.58

14 Tyr 3.13

18 Impurity 4.53

24 Leu 6.18
3 Glu 1.01

16 Val 4.23
17 Met 4.4

20.0
21 Phe 5.47
13 Impurity 2.68

22 Ile 5.63
20 Trp 5.19

25 Lys 7.06
15 Cys#2 3.78

10.0

%ACN/MeOH (45/45/10): 2.0 %

Flow : 0.700 mL/min
0.2 × 10 6
0 Minutes 7.5
27460

Baseline separation of 19 amino acids in less than 7.5 min

(16 min total run time).
7
 Beyond UHPLC
Unique Solutions for Ultimate Throughput and Ease-of-Use

UHPLC Reaches a New Level of Flexibility and Performance

UltiMate 3000 ×2 Dual RSLC systems offer laboratories unprecedented sample throughput
and easy automation of advanced procedures. Seamless integration of UHPLC with ×2 Dual
RSLC technology and powerful Chromeleon software brings laboratories new possibilities:
• Doubled sample throughput with parallel and tandem LC
• Increased use time by automatically switching
between applications
• Highest selectivity and resolution with
multidimensional LC

The DGP-3600RS combines two UHPLC pumps in a single housing.

Application #1 Automated Switchover Application #2

4:00 pm 6:00 pm 8:00 pm 10:00 pm 12:00 pm 2:00 am 4:00 am 6:00 am 8:00 am

Acclaim® Trinity™ P1:

4
Unique selectivity for API/
counterion analysis
1. Procaine 9. Chloride Acclaim C18 RSLC:
2. Choline 10. Bromide Unique speed for analysis of statins
3. Tromethamine 11. Iodide
4. Sodium 12. Phosphate 1
1 - Mevastatin
5. Potassium 13. Malate
2 - Lovastatin
6. Meglumine 14. Tartrate 3
3 - Simvastatin
7. Mesylate 15. Citrate 2
8. Maleate 16. Oxalate
10
2 9
5 7
3 8 11 12 13 15
1 6 14
16

0 3 6 9 12 15 0 12 24 36 48
Minutes Seconds 26165

Automatic switching between a conventional and a UHPLC method.

During the week Typical Analysis Workflows with and without AAS
Tuesday Wednesday
Without AAS
9:00 17:00 1:00 9:00 17:00 1:00
With AAS
9:00 17:00 1:00 9:00 17:00 1:00
Over the weekend
Friday Saturday Sunday Monday Tuesday
Without AAS
9:00 17:00 1:00 9:00 17:00 1:00 9:00 17:00 1:00 9:00 17:00 1:00 9:00 17:00 1:00
With AAS
9:00 17:00 1:00 9:00 17:00 1:00 9:00 17:00 1:00 9:00 17:00 1:00 9:00 17:00 1:00

Manual Smart Sequence Shutdown Application #1, Shutdown/ System System is ready and
labor Startup is active Switchover, Equilibration Application #2 flush idle time can be used
23925

Automated application switching increases productivity by using nights and weekends, when the instrument would otherwise sit idle.

8
Connect to the Future
The revolutionary Viper fitting system does away with all problems experienced with
conventional fitting systems. It provides a perfect fit each time it is used and ensures superior
chromatographic performance.
• Straightforward, zero-dead-volume UHPLC fingertight fittings
• Compatible with virtually every type of valve and column hardware
• Tool-free, Viper-based kits for advanced LC solutions
All RSLC and ×2 Dual RSLC system tubing kits
feature the unique Viper design, making
the best UHPLC system even easier to use.
Injection #4 Injection #1 Uracil
600 Injection #101 600 Injection #50
Uracil
Injection #100

mAU Acetophenone
Acetophenone Acetanilide
Acetanilide

300 300

0 0

0 9 18 0 9 18
Seconds Seconds 26166

Slipping capillaries cause deteriorated peak shapes (left). Viper capillaries provide robust performance at
UHPLC pressures (right).

The Viper connector tightens at the tip of the capillary, and does not use ferrules
that may cause incompatibility with the opposite holder.

9
 Instant Results
Seamless Integration of Data Processing Tools

Chromeleon Software Ensures that RSLC is Ultrafast

The Dionex RSLC solution comes with Chromeleon Chromatography Data System software
that produces results instantly. The following features ensure increased productivity and
a laboratory focus on results, not just data.
• Data processing times reduced by as much as 90%
• Instant calculation of results
120
• Dedicated reports for method validation, related substances, EPA statistics,
dissolution testing, content uniformity, and more

Method Speed-Up
A method speed-up calculator is provided for quick and easy conversion of conventional

% Release
LC methods to RSLC methods.

0
0

To use the calculator, simply enter your old

method details and your new column details.
The calculator immediately identifies your new
method parameters without the need for
time-consuming lab work associated
with method development. In addition,
it automatically calculates how much time
and solvent your new method will save.

10
 RSLC Data Processing
UHPLC requires ultrafast data handling. Recognizing the necessity of data processing tools
for RSLC, Dionex engineered the Chromeleon software to deliver seamless data handling
that ensures instant results.

140
Dissolution Profile Acetylsalicylic acid

mAU

Minutes 35 -10
0 Seconds 12
25336a 25336b

Using a fast dissolution method with a total run time of 18 s allows the
analysis of all dissolution samples in less than 30 min. After that, the
dynamic processing tools of the Chromeleon software instantly calculate
the dissolution profile and assess the results against specifications.

Using Chromeleon software, a 40 page

validation report (based on ICH guidelines)
is created in only 1 min.

11
 Ultrafast Method Development
Automated UHPLC Method Scouting with RSLC

Dedicated UHPLC Hardware Setup and the Intelligent

Simplicity of Chromeleon Software
RSLC facilitates the acceleration of your existing methods and also speeds the development
of new methods. The RSLC-based Automated Method Scouting solution enables you to screen
a defined set of UHPLC columns combined with a set of buffers, solvents, and temperatures
in the shortest possible time. This solution works with both quaternary and binary gradient
pump types.
• Optimized hardware design with integrated multi-position buffer selector and
ultrahigh-pressure, multi-position column selector valves
• Intuitive instrument control and method setup with Chromeleon software
• Powerful queries and automated processing to find all methods that meet user criteria
• Visualization tools make the best separations obvious at a glance

Best resolution. Quickest separation.

160 160
mAU

mAU

0 0

-17 -17
3 5 7.5 10 12.5 15 3 6 8 11
Minutes Minutes

The bubble chart makes finding the most promising separations easy. The lower the bubble on the y-axis, the faster the separation.
The size of the bubble represents the resolution between the critical peak pairs.

12
Method Scouting Workflow
RSLC-based Automated Method Scouting enables screening with ultrafast generic gradient
methods, easily set up in the Chromeleon software. Execution, evaluation, and validation are
fully automated.

System Setup Scouting Methods

Identifying Peaks through 3-D Spectra Selecting Criteria

Identifying Best Results Fine-Tuning and Validating

13
 Flexibility and Reliability
RSLC Ensures Ultrahigh Performance for All Your Applications

A Powerful Combination of Capabilities and Specifications

Flexible and reliable ultrafast LC has three main requirements: the ability to run ultrafast
and conventional LC on one system; superior robustness to maximize productivity; and full
compatibility with your detector of choice for optimal front-end separations. The data below
demonstrate the UltiMate 3000 RSLC system’s flexibility, handling both conventional LC and
UHPLC with excellent performance.

Reliability—a Must for Liquid Chromatography

Capable of running both conventional and ultrafast LC methods, the UltiMate 3000 RSLC
system delivers unrivaled reliability. RSLC ensures reproducibility and uptime for injections day
after day, week after week, and year after year.

DCMSLink™ provides seamless operation of the

Area RSD: 0.22%

UltiMate 3000 RSLC system from

RT RSD: 0.03%

third-party MS software.
Area RSD: 0.30%

300
RT RSD: 0.04%

Column specifications:
75 x 3 mm id
Area RSD: 0.28%

3 µm particles
RT RSD: 0.06%

Area RSD: 0.23%

RT RSD: 0.03%

Area RSD: 0.12%

RT RSD: 0.04%
Area RSD: 0.11%
mAU

Excellent retention time and area

RT RSD: 0.04%

precision in U-HPLC mode 700 Column specifications:

(10 injections)
Area RSD: 0.11%

250 x 4.6 mm id
RT RSD: 0.04%

5 µm particle
Area RSD: 0.12%
RT RSD: 0.04%

0
0 Seconds 90
mAU

25337a Excellent retention time and

area
precision in conventional mode
The chromatograms show a UHPLC method (above) and a conventional LC method (right), demonstrating the (10 injections)
UltiMate 3000 RSLC system's industry-leading performance in both operational modes.

0
0 Minutes

14
 Maximum Flexibility
• Run conventional LC and UHPLC methods on the same instrument
• Take advantage of the powerful alternative quaternary and dual-gradient RSLC pumps for
highest flexibility in analysis and method development at the UHPLC level
• Industry-leading range of detectors, including MS detectors from ABI/Sciex, Bruker,
and Thermo Fisher Scientific

Maximum Reliability
• Precision engineered instrument provides robust operation and maximum uptime,
even with advanced ×2 Dual configurations
• Patented injection valve design ensures long-term operation at 100 MPa and
up to 500 µL injection volume
• Easy-to-use diagnostic tests allow immediate assessment of instrument performance

8 1. Pararosaniline
55.0 2. Pararosaniline degradation 1
3. Patent Blue VF degradation 1
4. Patent Blue VF
45.0 5. Victoria Blue degradation 1
6. Victoria Blue degradation 2
7. Crystal Violett degradation 1
8. Crystal Violett
10 9. Victoria Blue degradation 3
10. Victoria Blue
11. Victoria Blue degradation 4
12. Victoria Blue degradation 5
mAU 25.0
CH3OH: 25.0 % 4

1
10.0
CH3CN: 10.0 % 7 9
3
6 11 12
2 5

0 4
Minutes 27461

UHPLC analysis of ink using ternary gradient elution.

The UltiMate 3000 Quaternary RSLC pump can easily
accelerate non-binary gradient methods.

12
25337b

Easy-to-use tests provide instant assessment of instrument performance.

15
 Enjoy Industry-Leading Corporate Headquarters
Support Dionex Corporation
1228 Titan Way
Dionex Customer Support Centers are P.O. Box 3603
located in the United States, Europe, and Sunnyvale, CA 94088-3603
Tel: (408) 737-0700
Asia. These state-of-the-art laboratories
Fax: (408) 730-9403
are equipped with the full line of Dionex LC
instrumentation and software capabilities.
Support Centers provide accessible locations Worldwide Sales and Service
for advanced training and enhanced North America
U.S./Canada (847) 295 7500
application development capabilities. Users
can visit these laboratories or sign up to South America
learn new skills in addressing challenging Brazil (55) 11 3731 5140

applications, receive training and support, Europe

and discover new, innovative HPLC and Austria (43) 1 616 51 25 
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IC solutions. (32) 3 353 42 94 
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www.dionex.com

Dionex products are designed,

developed, and manufactured
under an ISO 9001 Quality System.

© 2010 Dionex Corporation

All trademarks and registered trademarks are
the property of Dionex Corporation.
Passion. Power. Productivity. LPN 2064-06 7M 11/10 Printed in U.S.A.

16
Dionex as your Continual Source for Water Analysis
As a leader in providing chromatography solutions for water analysis we understand the needs of providing
the best solutions for water analysis. Not only does water analysis require good chromatorgraphy, but also
an understanding of regulations. Finally, there are the practical implications for water analysis. These include
workflow, cost, time, reducing complexity, reporting, system maintenance and upkeep, quality service, and
many other factors. Learn how Dionex innovations like Reagent-Free Ion Chromatography, Eluent Generation,
Corporate Headquarters
Eluent Regeneration, and Chromeleon Chromatography Data System software make IC Easy!
Dionex Corporation
1228 Titan Way
P.O. Box 3603
Important Literature
Sunnyvale, CA 94088-3603
Analyte Application or Poster Tel: (408) 737-0700
Fax: (408) 730-9403
AN 154: Determination of Inorganic Anions in Environmental Waters Using a Hydroxide-
Anions Worldwide Sales and Service
Selective Column
North America
Anions AN 140: Fast Analysis of Anions in Drinking Water by Ion Chromatography U.S./Canada (847) 295-7500

South America
Anions AN 135: Determination of Inorganic Anions in Wastewater by Ion Chromatography Brazil (55) 11 3731 5140

Europe
AN 141: Determination of Inorganic Cations and Ammonium in Environmental Waters by
Cations Austria (43) 1 616 51 25
Ion Chromatography Using the IonPac CS1
Benelux (31) 20 683 9768
AN 187: Determination of Sub-μg/L Bromate in Municipal and Natural Mineral Waters (32) 3 353 42 94
Bromate Using Preconcentration with Two-Dimensional Ion Chromatography and Suppressed Denmark (45) 36 36 90 90
Conductivity Detection France (33) 1 39 30 01 10
Germany (49) 6126 991 0
AN 208: Determination of Bromate in Bottled Mineral Water Using the CRD 300 Ireland (353) 1 644 0064
Bromate
Carbonate Removal Device Italy (39) 02 51 62 1267
Sweden (46) 8 473 3380
AN 184: Determination of Trace Concentrations of Chlorite, Bromate, and Chlorate in Switzerland (41) 62 205 9966
Bromate
Bottled Natural Mineral Waters United Kingdom (44) 1276 691722

AN 236: Determination of Iodide and Iodate in Seawater and Iodized Table Salt by HPLC Asia Pacific
Iodine, Iodate
with UV Detection Australia (61) 2 9420 5233
China (852) 2428 3282
India (91) 22 2764 2735
Haloacetic Acids AN 217: Determination of Haloacetic Acids in Water Using IC-ESI-MS/MS
Japan (81) 6 6885 1213
Korea (82) 2 2653 2580
AU 144: Determination of Hexavalent Chromium in Drinking Water Singapore (65) 6289 1190
Chromate
Using Ion Chromatography Taiwan (886) 2 8751 6655

AN 227: Determination of Total Cyanide in Municipal Wastewater and Drinking Water

Cyanide
Using Ion-Exclusion Chromatography with Pulsed Amperometric Detection (ICE-PAD)
www.dionex.com
Carbamates AN 96 Determination of N-Methylcarbamates by Reversed-Phase HPLC

Poster: High-Efficiency Separation of Anionic Surfactants Using Reversed-Phase HPLC and

CEC: Surfactants
Suppressed Conductivity Detection
Dionex products are designed,
developed, and manufactured
CEC: Surfactants New Development in Surfactant Analysis by HPLC under an ISO 9001 Quality System.

Link
DCMS , Reagent-Free, and RFIC are trademarks and Acclaim, ASE, ASRS,
AutoSuppression, AutoTrace, CarboPac, Chromeleon, CSRS, IonPac,
and UltiMate are registered trademarks of Dionex Corporation.

© 2010 Dionex Corporation

LPN 2527 5M 05/10 Printed in U.S.A.

Passion. Power. Productivity.

 Water Quality
Global Supply, Local Concern

Currently, less than 1% of the planet’s water is available for human consumption. Resources for potable water are under increased
demand due to global population growth and global warming. With surface water contamination and groundwater resources
overexploited, regulation and enforcement levels are increasing along with new regulatory and conservation policies. Coupled with
the increased scrutiny of emerging contaminants, the need for effective water analysis and monitoring has never been higher.

Ground and surface water are a vital treatment process, drinking water is treated separation and detection of analytes, often
resource for a healthy environment. They are with disinfectants to remove potentially resulting in low recoveries, false positives,
also the largest source of fresh water. These harmful bacteria. These disinfectants also or false negatives. To overcome these
waters often represent complex matrices react with ions and residual organic matter interference effects, wastewaters often
that interfere with detection for the analytes resulting in the formation of DBPs. DBPs require matrix elimination strategies, such as
of interest. Dionex has overcome many of are highly toxic, are regulated, and require sample preparation, solid phase extraction,
the challenges of surface and groundwater mitigation of their concentrations prior to or other techniques in order to accurately
analysis through unique and innovative distribution. Dionex has developed innovative determine the contaminant of interest.
chromatography and sample preparation techniques for the complete analysis of
Dionex offers the most comprehensive
techniques. drinking water contaminants and DBPs.
capabilities for inorganic and organic
Drinking water is analyzed for both primary Wastewaters discharged by industries contaminant extraction and analysis for
and secondary contaminants. Primary and municipalities discharge a variety of drinking, ground, surface, and even complex
contaminants are typically toxic compounds pollutants, including heavy metals, toxins, contaminated wastewaters. As your partner
that cause health issues while secondary oils, nutrients, and solids, all of which for water analysis, Dionex can provide you
contaminants affect taste and overall endanger ecosystems and pose a threat with the technology, experience, and support
water quality. Another major challenge for to human health. Contaminant analysis is to help you monitor your water quality,
drinking water is the analysis of disinfection complicated because waste waters contain provide safe drinking water, and protect
byproducts (DBP). During the drinking water a multitude of compounds that interfere with the environment.

page 2
System Solutions

Ion Chromatography Liquid Chromatography Solid-Phase Extraction

Since the development of ion chromatography The Dionex UltiMate® 3000 series of liquid Liquid–liquid extractions for organic contami-
(IC) analysis over 30 years ago, Dionex has chromatography systems allows you to nant extraction that normally take hours can
pioneered the development of IC systems, choose from a variety of modules and configu- be automated using the AutoTrace® 280 SPE
including Reagent-Free™ ion chromatography rations to adapt the instrument to workstation. AutoTrace significantly reduces
(RFIC™) systems. Whether you are running your applications. sampling handling compared to separatory
a few samples or high throughput, whether funnel or vacuum manifold extraction. The
• Dual systems for multidimensional tedious and time-consuming steps of liquid-
your analytical task is simple or complex,
separations of organic compounds liquid extraction can be greatly decreased by
Dionex has the right system for you.
• Rapid Separation LC (RSLC) systems for automating the SPE steps of condition, load,
• Starter line and basic systems for straight
fast, high-flow-rate, ultrahigh-pressure and rinse and elute. AutoTrace offers
forward and repetitive analyses of ions in
applications a lower sample preparation cost per sample
water samples
by reducing solvent and labor costs by as
• Reagent-Free systems reduce eluent and • Reversed-phase (mixed-mode HILIC much as 90%.
regenerant preparation and ion-exchange) and monolith
• Dual systems allow double throughput, columns for fast analysis • Extractions for liquid sample sizes from
simultaneous analysis, and complex 20 mL to 4 L
methods • Diode array, multi-wavelength, fluorescence, • Dramatic solvent reduction and reduced
Corona® CAD®, and MS detectors sample handling
• Suppressed conductivity, pulsed
amperometry, UV-vis, and MS detectors Columns • Wide range of applications
• Ion-exchange, ion-exclusion, concentrator, • Automation
The Acclaim® column family meets the high
and trap columns • Handles acidic and alkaline matrices
standards set by modern HPLC, UHPLC, and LC/
Columns MS methods. • Approved for use by many government
The IonPac® column family offers a wide vari- agencies
ety of ion exchange capabilities for hydroxide Columns
and carbonate based eluents.
Dionex has several columns for offline SPE
that are ideally suited for AutoTrace. These
columns are designed for extraction based on
EPA approved methods.

ICS Series UltiMate Series AutoTrace SPE workstation

IC/RFIC Analytical Systems HPLC/RSLC Analytical Systems

page 3
 Ion Analysis

The determination of common inorganic anions in drinking water is one of the most important applications for water analysis. Fluoride,
nitrite, and nitrate are regulated as primary contaminants in many countries around the globe. In the United States, organic anions such
as chloride and sulfate are regulated under the U.S. National Secondary Drinking Water Standards, which are guidelines regarding taste,
color, and odor.

Anions acetate, and formate, and is an excellent For example, EPA 300.0 recommends diluting
choice for EPA 300.0 and 300.1 for the the sample if sulfate exceeds 95 mg/L. The
Dionex has a wide variety of carbonate-
determination of anions in drinking water. IonPac AS18 column allows a large linear
and hydroxide-selective columns for the
range from 0.2 to 200 mg/L, eliminating the
determination of anions in a broad range of The IonPac AS18 represents the culmination of
need to dilute and re-analysis of high-ionic-
water matrices. The high capacity IonPac® over 20 years of column development, and is a
strength samples.
AS22 is a carbonate-selective column that key part of our RFIC system. The AS18 allows
allows the separation of common inorganic improved resolution between closely eluting Iodide and Iodate in Seawater
anions in a variety of sample matrices, peak pairs chloride:nitrite and sulfate:nitrate.
including drinking water, wastewater, process Iodine is an essential element naturally
It also elevates the ability to tolerate higher
streams, and scrubber solutions. The AS22 present in seawater and seafood. The most
ionic strength matrices without column
selectivity provides retention of fluoride common forms of natural iodine in the
overloading. Hydroxide-selective stationary
from the water dip and resolution of fluoride, diet are iodide and iodate. Dietary iodine
phases also give a better retention of weakly
deficiency affects thyroid function and leads
retained anions like fluoride and acetate, and
to developmental diseases, neurological
only moderate retention of divalent anions
Peaks: 1. Fluoride 0.84 mg/L (ppm) damage, goiter, and paralysis. Because iodine
2. Formate 0.03
such as sulfate. Additional advantages of
is found in high concentrations in seawater, it
3. Chloride 15.59 hydroxide eluents are improved linearity,
4. Nitrite 0.01 may be possible to generate iodated drinking
5. Unknown NQ lower background conductivity, and improved
water from seawater, thereby eliminating the
6. Chlorate 0.18 MDLs compared to carbonate-based eluents.
7. Bromide 0.02 need to iodize salt.
8. Nitrate 0.89
9. Carbonate NQ
10. Phosphate 0.22 A Amount Added
11. Sulfate 20.45 Peaks: 1. Fluoride 0.14 1 mg/L 5
NQ: Not Quantified 2. Chloride 28.8 30 Peaks: 1. Unknown —
3. Nitrite-N — 2 2. Iodate 559 µg/L (ppb)
1 3 11
0.5 8 4. Carbonate — — 3. Unknown —
5. Bromide 0.03 2 4. Bromide —
6. Sulfate 99.5 80 1 5. Iodide 12.3
7. Nitrate-N 0.89 5
8. Phosphate-P — 0
µS
6 mAU
2 45 7 9 10 4
Fortified Surface Water
30 2 3
2 6
–0.2 5
0 5 10 14 7
Minutes
22941 µS
3 8
Figure 1. Determination of inorganic anions in a 1 4
-1
5 0 5 10 15 20
municipal drinking water sample on an IonPac
0 Minutes
AS22 column. 0 4 8 12 16 26219

Minutes 19120
Figure 3. HPLC using a mixed-mode column can
Figure 2. Determination of inorganic anions determine iodide and iodate in seawater, synthetic
in fortified surface water using the IonPac sea salt, and table salt. This method is specific,
AS18 column. sensitive, and rapid which minimizes the need for
sample pretreatment or dilution.

page 4 4
Calcium and magnesium are also routinely measured to determine water hardness, an important parameter for corrosion control.
Ammonium cation is routinely measured in the U.S. for wastewater discharge compliance monitoring, and in the EU and Japan in both
wastewater and drinking water.

Cations and Ammonia Fast IC for Anions and Cations Peaks: 1. Fluoride 6. Bromide
The IonPac CS16 column is a high-capacity To meet increasing customer demands for 2. Formate 7. Nitrate
3. Chloride 8. Carbonate
cation-exchange column that replaces the productivity and higher throughput, faster 4. Nitrite 9. Phosphate
5. Chlorate 10. Sulfate
CS15 column for disparate concentration analysis of anions and cations is becoming
ratios of ammonium and sodium in diverse 100 3
more and more important. The standard
sample matrices. The CS16 is ideal for approach for achieving fast analysis times is
the determination of low concentrations simply to increase the flow rate. Limitations µS
of ammonium in environmental waters. It to this approach are high backpressure and 10
1
provides improved resolution of sodium from increased waste. Another approach is to 2 45 6 7 8 9
–10
ammonium and alkanolamines, even for shorten the column length. However, this
samples high in ionic strength. sacrifices resolution of critical peak pairs. To 1 3 10
0.81
overcome these two limitations, IonPac ion-
7
exchange resins with high capacity maintain µS 5
Peaks: 1. Lithium <0.2 mg/L (ppm)
2. Sodium 200 resolution in a shorter column format. 2 4
6 8
9
3. Ammonium 0.03
4. Potassium 0.5 The IonPac AS22 and IonPac CS12A columns
5. Magnesium 8.0 0.56
6. Calcium 20 have been specifically designed to have 0 1 2 3 4 5
enough capacity to maintain resolution even Minutes
0.150 26269

in a short column format and for use on any Figure 5. Analysis of municipal drinking water
2 4 5 6 Dionex system. This permits the determination sample is shown using a new prototype Fast
of anions and cations with high resolution even IonPac AS22 column (4 x 150 mm) at 2.0 mL/min.
Run time is well under 5 min for this application.
in drinking, ground, surface, and wastewater The bottom trace shows an enlarged image of
matrices in under 5 min, allowing laboratories the separation.
µS
to achieve higher productivity and increased
throughput to get faster results.
3

0.095
0 10 20 30
Minutes
16521

Figure 4. Resolution of trace ammonium from

high sodium with the IonPac CS16 column.

page 55
 Disinfection Byproducts

th
Disinfection of drinking water is considered to be one of the major public health advances of the 20 century. Water is often disinfected
before it enters the distribution system to ensure that dangerous microbial contaminants are killed. Chlorine, chlorates, chlorites,
chloramines, or chlorine dioxides are most often used because they are very effective disinfectants, and residual concentrations can be
maintained in the water system. Drinking and bottled waters are commonly disinfected with ozone. These processes can leave behind
dangerous byproducts that must be monitored for safety.

Oxyhalides and Bromate present in most municipal drinking water Another strategy for bromate determination
samples. Finally, postcolumn methods using in high-ionic-strength drinking waters is the
Over the last two decades, Dionex has
visible detection either generate carcinogenic use of a two-dimensional (2-D) IC system.
led the effort in developing sensitive and
by products and/or have not been shown Figure 7 shows the results of a 2-D IC
robust ion chromatography (IC) methods
to be robust enough under normal use. The separation of bromate in a high-ionic-strength
for the determination of bromate and other
high-capacity IonPac AS19 and IonPac AS23 matrix. The bromate that was undetectable
oxyhalides (e.g., chlorite and chlorate).
columns allow conductivity detection to be in the first dimension is fully resolved in the
Determination of bromate in natural mineral
used for bromate determination in many second. Using this 2-D IC technique, an MDL
waters has been demonstrated using IonPac
drinking waters of 0.036 μg/L can be achieved in high-ionic-
AS19 column with a hydroxide eluent, or
strength matrices without the need for
the IonPac AS23 column and a carbonate/
postcolumn derivatization. This method is
bicarbonate eluent using direct injection and A B
Peaks: 1. Fluoride approved for compliance monitoring as
suppressed conductivity detection. 2. Chlorite 8.8 11.3 µg/L
3. Bromate 4.7 5.1 EPA 302.
Determining low concentrations of 4. Chloride
5. Nitrite
bromate in high-ionic-strength matrices 6. Chlorate 13.5 9.5 Peak: 1. Bromate 0.5 µg/L
using suppressed conductivity detection 7. Bromide
8. Nitrate
is subject to potential interferences and 9. Carbonate 0.6
1st Dimension
A
loss of sensitivity. Dionex products were 10. Sulfate
11. Phosphate
instrumental in the development of the
postcolumn derivation techniques in U.S. 0.5 A 1 4 8 910 11
µS

EPA Methods 317.0 and 326.0, and ISO-1428. 7 1

6
Although postcolumn reaction methods do µS 2
3 5
0.3
not generally suffer from interferences by 0 10 20 30 35
0.2
common anions, column overloading with 1
2nd Dimension
B
high-ionic-strength samples can still cause 0.7 B
1 4 8 10 11
peak broadening and loss of response.
µS 9
Natural mineral waters typically contain 3
µS
elevated levels of common anions that can
2 567
1
-0.1
significantly exceed the concentrations 0 5 10 15 20 25 30
Minutes
24075 0.6
0 10 20 30 35
Figure 6. Comparison of the A) IonPac AS19 and Minutes
24221-01
B) IonPac AS23 columns for the separation of trace
concentrations of common anions and DPB anions
Figure 7. Results of a 2-D IC separation of bromate
spiked in mineral water A.
in a high-ionic-strength matrix.

page 6 6
Haloacetic Acids, Bromate, Nitrosamines We present an alternative using HPLC. The
and Dalapon Nitrosamines represent a nitrogen based substances in this list differ greatly in hydro-
class of DBPs that are formed from natural or- phobicity, and a very wide gradient program is
Haloacetic acids (HAAs) are disinfection
ganic matter present in water. Their presence needed to resolve them all. The Acclaim® PA
byproducts produced during chlorination of
is heavily monitored through the U.S. EPA column, unlike most C18 columns, can be used
water containing natural organic matter and
Contaminant Candidate List and Unregulated reliably with highly aqueous mobile phases
bromide. The current EPA Methods 552.1,
Contaminant Monitoring Rule. The analysis without risk of dewetting.
552.2, and 552.3 require derivatization and
multiple extraction steps followed by gas of nitrosamines by GC, as in U.S. EPA Method
chromatography (GC) with electron capture 8270, is problematic due to the thermal Column: Acclaim PA, 5 µm
Dimensions: 4.6 × 250 mm
detection (ECD). Ion chromatography-mass instability and reactivity of these substances. Mobile Phase: (A) DI H2O
spectrometry (IC-MS/MS) offers a sensitive (B) Acetonitrile
Temperature: 30 °C
and selective direct injection technique that Column: IonPac AS24 250 × 2 mm i.d., Flow Rate: 1 mL/min
does not require sample pretreatment. The AG24 50 × 2 mm Inj. Volume: 15 µL
KOH Gradient: 7 mM for 0–18 min then Detection: UV, 230 nm
separation of all nine HAAs and bromate 18 mM for 18–36.5 min, Peaks (20 mg/mL): 1. N-Nitroso-dimethylamine
2. N-Nitroso-morpholine
is achieved using the IonPac AS24 anion- then 60 mM for 36.6–52 min
3. N-Nitroso-methylethylamine
Eluent Flow Rate: 0.30 mL/min
exchange column with a hydroxide gradient. Postcolumn Solvent: 100% Acetonitrile at 0.3 mL/min 4. N-Nitroso-pyrrolidine
® 5. N-Nitroso-diethylamine
The unique selectivity of this column allows Suppressor: ASRS 300, 2 mm,
6. N-Nitroso-piperidine
external water mode
separation of these analytes from common Matrix Diversion: 17–22 and 33–41 7. N-Nitroso-dipropylamine
Sample Volume: 100 µL Loop; 0.8 µL injection valve 8. N-Nitroso-dibutylamine
inorganic matrix ions so that the chloride, dead volume 9. N-Nitroso-diphenylamine
sulfate, nitrate, and bicarbonate are diverted Column Compartment
Temperature: 15 ºC 1
to waste during the analytical run, thereby Period 3
avoiding signal suppression of the ESI-MS/ 4 56
Period 2 Re-equilibration
MS instrument. Using RFIC technology KOH Period 1
7
gradient
along with the IonPac AS24 column, all nine AU
1. MCAA 3
haloacetic acids, bromate, and Dalapon can 2. MBAA 2 89
3 3. DCAA
be separated and quantified in low ppb levels 7500
4. BCAA
Cl– Br–
using IC-MS/MS detection. Dionex developed 6500 5. DBAA
NO3–
5500 CO32– 6. TCAA
this technique specifically for the U.S. EPA
Intensity, cps

4500 SO42– 7. BDCAA

for compliance monitoring of disinfection by 3500 5 6 8. CDBAA
4 7 9. TBAA
products in drinking water. This IC-MS/MS 2500
2
0 10 20 30
8 9 Minutes
Br
based method has been approved by the U.S.
–
1500 1
Note: This gradient will also resolve benzidines. 20109a
500
EPA Office of Ground Water and Drinking
0 5 10 15 20 25 30 35 40 45 50 55 60 Figure 9. Separation of nitrosamines using an
Water as EPA 557. This strategy is also used Minutes
25675 Acclaim PA column.
in Japan and the United Kingdom.
Figure 8. Complete chromatographic resolution of
HAA9 in high high-ionic-strength matrix waters
using an IonPac AS24 column.

page 7
 Pesticides and Herbicides

Pesticides play a large role in agriculture, industry, home/garden maintenance, and public health by supporting the ability to produce
many crops, commodities, and services around the world.

Carbamates Atrazine Glyphosate

N-methylcarbamates and N-methylcarbamo- Atrazine is the most widely used herbicide Glyphosate is the most commonly used
yloximes are among the most widely used in conservation tillage systems, which are agricultural herbicide and second most
pesticides in the world, and due to their designed to prevent soil erosion. As a result commonly used herbicide around homes and
toxicity are regulated in water and soil. of widespread and long-term usage, Atrazine gardens. Although the bacteria in soil break
HPLC is the preferred method for separating and other triazine herbicide metabolites can down glyphosate into the aminomethyl phos-
carbamates. Gas chromatographic analysis be found in the environment and groundwa- phonic acid (AMPA), wastewater discharge
has proven unsuccessful due to degradation ter at low levels. Monitoring of atrazine, its samples and drinking water samples in United
of the analyte compounds during vaporiza- metabolites desethyl-atrazine and desiso- States and Europe have tested positive for
tion. HPLC with UV detection does not offer propylazine, as well as the other triazine glyphosate.Typical methods for glyphosate
the sensitivity or specificity required for herbicides, is important for environmental use precolumn derivatization or solid-phase
the sample matrices of interest. HPLC with protection and food safety control since it has extraction (SPE) followed by postcolumn de-
postcolumn derivatization using fluorescence been linked to endocrine and possible carcino- rivatization. Silica-based C18 columns which
detection or positive mode electrospray genic effects. use cation-exchange mechanisms experience
ionization mass spectrometry have been difficulty with the retention of such polar
shown to be effective for optimal detection. Peaks:
compounds. Using IonPac AS24 direct injec-
1. Atrazine-desisopropyl SIM1: 173.9–174.3 (0–5 min) tion RFIC systems, along with MS Detection,
2. Atrazine-desethyl SIM2: 187.9-188.3 (2–8 min) it is possible achieve chromatographic resolu-
3. Simazine SIM3: 202.0-202.4 (8–15 min)
Peaks: (10 µg/L each) tion with ppt level detection.
4. Atrazine SIM4: 216.1-216.5 (12–18 min)
1. Aldicarb sulfoxide
5,6 Terbuthylazine/Propazine SIM5: 230.1-230.5 (17–22 min)
2. Aldicarb sulfone
3. Oxamyl 5
5
4. Methomyl 2.5×10
4 6 5
60 5. 3-Hydroxy carbofuran Glyphosate
6. Aldicarb
7. Propoxur 3 AMPA
µs
4 8. Carbofuran
9. Carbaryl Counts 2
10. 1-Naphthol 1 0
2 11. Methiocarb 230 m/z –0.3 5.0 10.0 15.0 20.0 25.0 30.0 35.4
3 216 m/z
12. BDMC 9 202 m/z 14,000
mV 10 188 m/z
1 174 m/z
6 AMPA 20.439
5 0
78 0 Minutes 22
counts
1112 24194

Figure 11.Triazine pesticides can be separated by

HPLC using an Acclaim 120 C18 column and an 0
acetonitrile/water gradient. Triazine herbicides are 19.66 21.00 22.00 23.00 23.76
-5.9
detected using ESI in positive mode. 60,000
5 10 15 20 23.8
Minutes Glyphosate 32.065
24088
counts
Figure 10. The Acclaim 120 C8 column provides the ideal
chromatographic resolution of most carbamates in a Figure 12. Anion-exchange separation using an
single injection. IonPac AS24 column and IC-MS/MS detection. IonPac –5
AS24 is a high-capacity column created for dealing 31.26 32.00 33.00 33.26
with high-ionic-strength samples which are known to Minutes 26012
contribute to signal suppression within MS/MS detectors.
page 8
Toxic Ions

Dionex has developed a wide variety of analytical techniques for the quantitative determination of toxic ions and metals. These
applications involve a thorough understanding of analytical separation as well as matrix interferences that can result in poor recoveries,
false negatives and false positives, and affect overall method robustness.

Cyanide interferences during analytical separation. ICE the United States, where most of the contam-
is preferred because strong acid anions such ination appears to be confined to the western
Cyanide is regulated as an environmental
as chloride and sulfate are excluded from the and southwestern regions. It is estimated
contaminant in drinking water, surface water,
column, and cyanide is resolved from sulfide. that over 11 million people have perchlorate in
and wastewater. The typical sources of
their drinking water supplies. Recent evidence
cyanide contamination are industrial waste Perchlorate suggests that perchlorate is also becoming a
from plating and mining industries, burn-
Perchlorate inhibits the normal uptake of global problem. EPA Methods 314.0 and 314.1
ing coal, plastics, and effluent from pub-
iodide by the thyroid gland which results in were developed to determine trace concen-
licly owned treatment works (POTW). Total
reduced thyroid hormone production. Ammo- trations of perchlorate in drinking water. The
cyanide can be determined spectrophoto-
nium perchlorate is manufactured for use as determination of trace perchlorate in high-
metrically, amperometrically, or by titration.
the primary component in solid propellant for ionic-strength matrices is still a challenging
These methods are complicated, often
rockets, missiles, and fireworks. Perchlorate problem. Alternatively, a two-dimensional ion
requiring multiple distillation apparatuses,
has been detected in nearly 400 sites across chromatographic approach can be used to
and are subject to interference from high-pH
resolve perchlorate from high concentrations
solutions, oxidizers, and sulfur-containing
of common matrix ions. This 2-D strategy has
compounds. Chromatographic methods, such Peaks: 1. Perchlorate 0.5 µg/L
resulted in a new method, EPA 314.2, which
as ion-exchange (IE) and ion-exclusion chro- 1st Dimension
3 A provides an MRL of 0.06 ppb.
matography (ICE) can eliminate some of these

µS
Peaks: A B

1. Cyanide 5.1 5.0 µg/L

1
2. Unknown — —
2nd Dimension
3. Sulfide — 19,000 1.0 B
120
3
µS
1
nC

B 2 0.3
1 0 10 20 30 40 45
A
Minutes 23320
97
0 10 20 30
Minutes Figure 14. 2-D IC provides several advantages:
27368
injection of large sample volumes, the ability to
Figure 13. Low-level CN- determinations in the trap perchlorate that is partially resolved in the
presence of high sulfide. Using ICE, interfering first dimension onto a concentrator column and
anions, such as Cl- and SO42-, are quickly eluted from separating it in the second dimension, and the
the column, while sulfide is well resolved downstream ability to combine two different column chemistries
of CN-. The detection step has also been improved to enhance selectivity and reduce the possibility of
with the development of platinum electrodes. a false positive.

page 9
 Metals and Chemical Contaminan

Naturally occurring and man-made contaminants are prevalent in environmental waters. Metal and chemical precursors often
contaminate groundwater sources to impact drinking water supplies. Recent toxicological information indicates that contaminant
groundwater species and/or breakdown products must also be considered when making regulatory decisions.

Metals metals in samples including seawater, brines, exchange column, postcolumn reaction
estuarine waters, and a variety of biological with diphenlycarbazide, and UV detection
Metals, amalgams, and alloys play an instru-
samples. Chelation ion chromatography at 530 nm.
mental role in almost every facet of our lives.
Yet when used or disposed of incorrectly, removes alkali and alkaline earth metals while
concentrating the sample, then determining Arsenic
metals can become serious contaminants
the analytes of interest. Arsenic is ubiquitous in the environment and
in soil and water. Dionex has developed
speciates into toxic and nontoxic forms. In
columns that can accurately assay groups of
Selenium and Chromium general, methylated and other organoarseni-
metals at very low levels in a variety of ma-
Hexavalent chromium, Cr(VI), is the most toxic cals are less toxic than inorganic arsenic, and
trices, as well as speciate certain elements
form of the metal chromium and frequently pentavalent arsenic is considerably less toxic
such as Fe2+ and Fe3+. Chelation ion chroma-
requires regulatory monitoring as a primary than the trivalent state. The inorganic forms
tography facilitates the determination of low
drinking water contaminant at levels as of arsenic—arsenite and arsenate—are the
concentrations (µg/L and lower) of transition
low as 0.2 µg/L. Ion chromatography can usual forms found in drinking water, and the
determine dissolved Cr(VI) in drinking water, U.S. EPA has set a maximum contamination
Sea Water Spike Amt. Spiked Sea Water groundwater, industrial wastewater, and level (MCL) for total arsenic at 10 μg/L. Some
1. Iron 10.8 µg/L 10.0 µg/L 21.6 µg/L
2. Copper – 2.50 2.42 solid waste extracts. U.S. EPA Method 218.6 foods, such as fish and seaweed, can contain
3. Nickel 1.60 5.00 5.85
specifies the use of the IonPac AS7 anion- organic forms of arsenic resulting from con-
4. Zinc 1.88 5.00 6.78
5. Cobalt _ 2.50 2.57 tamination and biological processes.
6. Cadmium _ 12.5 12.5
7. Manganese 7.16 7.50 14.3
0.02
0.002 Anion Exchange Mixed Mode
Spiked Sea Water A 90
4 µs
A Conductivity
7 –10
1 5 18,000
AU 2 3 6 Chromate Counts +SIM 23, Sodium
B
0
70,000
Counts C –SIM 35, Chloride
–10,000
9000
-0.01 Counts –SIM 107, Arsenite
0 D
AU B 450,000
0.02 Chromate +SIM 139, Dimethylarsinate
Counts
Sea Water 0 E
40,000
Counts F –SIM 141, Arsenate
1 7 0
AU 4
70,000
3 Counts G +SIM 141, Methane Arsonate
0
550,000
Counts +SIM 179, Arsenobetaine
0 H
-0.01 0 0 6 12 18 24 30
0 2 4 6 8 10 12 14 0 2 4 6 8 Minutes
23390
Minutes 12427 Minutes 17328_29
Figure 17. The above shows dual analysis of the
Figure 15. Demonstrates the trace level detection Figure 16. Determination of chromate in drinking anionic and cationic forms of arsenic and an
of metals in high-matrix-strength samples such as water (A) and drinking water spiked with 0.2 µg/L example of a complex speciation experiment. Using
seawater. The spiked sample shows the potential to chromate (B); peaks, (A) chromate (0.055 µg/L) and IC coupled with MS detection, structural forms of
find additional metals resulting from pollution runoff (B) chromate (0.245 µg/L). each species are also obtained.
into seawater.

page 10
ants

Acrylamide Aldehydes and Ketones as Explosives

Acrylamide has been found in certain DNPH Derivatives Gas chromatography has traditionally been
prepared foods, and has recently been Large quantities of carbonyl compounds are used to detect and quantify explosive com-
included among the substances to be moni- used worldwide, primarily in the chemical pounds. Because some are thermally unstable
tored in drinking water according to the last and plastics industries. Potentially carcino- or nonvolatile, using this method can result
European Community Directive on potable genic, these compounds have been found in inexact determinations. HPLC with UV
water. A new method for determination of in industrial waste which can contaminate detection, however, is ideally suited for low-
this compound based on the combination of groundwater and drinking waters. Aldehydes level determination because it is not subject
ion-exclusion chromatographic separation and and ketones in air or vehicle exhaust are to these limitations. U.S. EPA Method 8330
MS detection has been developed according- converted to their dinitrophenylhydrazine describes an HPLC method with UV detection
ly. A sample of drinking water can be directly derivatives before analysis. for determination of 14 priority explosives and
injected onto the IonPac ICE-AS1 column and related substances. The method recommends
detected in SIM mode by a single quadrupole the use of a C18 reversed-phase column as
system with electrospray ionization. This Peaks: 1.
2.
Formaldehyde-DNPH
Acetaldehyde-DNPH
the primary column for separation, and a CN-
method was evaluated for analysis of drinking 3. Acetone-DNPH secondary column for confirmation. Acclaim
4. Acrolein-DNPH
water samples. 5. Propionaldehyde-DNPH Explosives E1 and E2 columns have equivalent
6. Crotonaldehyde-DNPH selectivities to these phases, respectively,
7. Butanone-DNPH
8. Methacrolein-DNPH with improved resolution.
3000
9. Butyraldehyde-DNPH
10. Benzaldehyde-DNPH
Peaks: 1. Acrylamide 0.5 µg/L 11. Valeraldehyde-DNPH A. EPA 8330 standards mix
12. m-Tolualdehyde-DNPH B. ISO22478 additions
Note: Resolution may be fine-tuned by temperature adjustment
13. Hexaldehyde-DNPH
1 700
55 1 20

Counts 5
B
mAU 13
2 11 1
34 10 12 4 12 14 17
A 5 89 78 9 13 16
67 mAU 11 18
15
0 A 21

1500 0 5 10 15 20 25 30 23 19
B 6 10
0 Minutes 25 Minutes
12427 0
24192

Figure 19. Conventional C18 columns do not have 0 10 20 30 40 45

Figure 18. Chromatogram of a water sample spiked favorable selectivity, but the unique selectivity Minutes
with 0.5 μg/L acrylamide using IonPac ICE-AS1 and of the Acclaim Explosives E2 column for 26910
MS (SIM) Detection. nitroaromatic molecules makes it the superior Figure 20. Acclaim Explosives E1 and E2 columns
choice for this application. can provide baseline resolution for 14 compounds
with complementary selectivity under identical
chromatographic conditions while reducing
analysis time.

page 11
 Metals and Chemical Contamina

Chemicals from the manufacturing industry often contaminate the environment. The very aspects that make chemicals viable for
manufacturing, such as reactivity, solubility and persistence, make them potentially toxic to living things. Chemical toxicity and release
12 14 into the environment leads to problems for future generations.
12427

Benzidine Phenols pollutants list. The method typically used for

determining phenols is gas chromatography
Benzidine (4,4'-diaminobiphenyl) is an Phenolic compounds are subject to regulation
(GC) combined with flame ionization detec-
aromatic amine. A mutagen and a proven as water pollutants due to their toxicity. The
tion (FID) or mass spectrometric detection
human carcinogen, its primary site of European Community (EC) Directive speci-
(GC-MS). However, liquid chromatography (LC)
tumor induction is the bladder. Water fies a legal tolerance level of 0.5 μg/L for
methods combined with UV/DAD electro-
can be contaminated with benzidine, its each phenol in water intended for human
chemical, and fluorescence detection are find-
derivatives, and dyes if plant water is consumption and Japan’s Ministry of Health,
ing increased application, particularly due to
discharged into water supplies serv- Labor, and Welfare specifies a maximum con-
nonvolatiles in many samples that can poison
ing a residential community. As with taminant level (MCL) of 5 μg/L for phenols in
GC columns. Method detection limits (MDLs)
some other aromatic amines such as drinking water. The U.S. EPA specifies a MCL
of LC techniques employing direct injection
2-aminonaphthalene, benzidine has been of 1 μg/L for pentachlorophenol and eleven
of samples are too high for the detection of
significantly withdrawn from use in most common phenols are on the U.S. EPA priority
the low levels allowed in natural waters.
industries because it is so carcinogenic.
Therefore, water samples require precon-
Column: Acclaim 120 C18, 3 µm Peaks: 1. Phenol 7. 4,6-Dinitro-2-methylphenol centration before analysis. The new Acclaim
2. 2,4-Dinitrophenol 8. 4-Chloro-3-methylphenol
Dimensions: 2.1 × 100 mm
3. 4-Nitrophenol 9. 2,4-Dichlorophenol
RSLC PA column shows fast separation of a
Mobile Phase: 50/50 (v/v) acetonitrile/
100 mM NaOAc, pH 4.7 4. 2-Chlorophenol 10. 2,4,6-Trichlorophenol wide variety of phenols.
Flow Rate: 0.17 mL/min 5. 2-Nitrophenol 11.Pentachlorophenol
Inj. Volume: 1 µL 6. 2,4-Dimethylphenol
Detection: Glassy carbon electrode, 800 mV 60.0
(1 µg/mL)
Peaks 1. Benzidine 7
2. 3.3'-Dimethylbenzidine
3. 3.3'-Dichlorobenzidine mAU
Note: Scaling down the column size and flow
rate produces a superproportional increase in 5
sensitivity in the electrochemical detector.
3
2
0.0010
1
6

1 4 89
2 10
µA
3 11
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Minutes
27360
–0.0001
0 2.5 5.0 7.5 10.0 Figure 22. The Acclaim RSLC PolarAdvantage
Minutes
20400
20400 column shows fast separation of phenolic
compounds.
Figure 21. The Acclaim 120 C18 column is
used with electrochemicaI detection for the
determination of benzidines.

page 12
ants

Pthalate Because phthalates do not chemically bind although the ranges of concentrations and
to the PVC, they can easily leach into ground frequencies of detection of PFCs among coun-
Phthalates, or phthalate esters, are esters of
water, evaporate into the air, or be absorbed tries and even within the same region may
phthalic acid and are mainly used as plasticiz-
through direct contact. Phthalates can be vary. No clear association between human
ers (substances added to plastics to increase
analyzed by GC-MS. However, online SPE exposure to PFCs and adverse health effects
their flexibility, transparency, durability, and
using HPLC with UV detection is a cost- has been established. However, based on
longevity). They are primarily used to soften
effective alternative for trace analysis of results from animal studies, a potential risk
polyvinyl chloride (PVC) plastic. Most plastic
these environmental contaminants. exists for developmental and other adverse
shoes are made from softened PVC. Phthal-
effects associated with exposures to PFCs in
ates are being phased out of many products Perflourinated Organic Compounds humans. For this reason PFOS and PFOA are
in the United States and European Union over
Perflourinated organic compounds have been placed on the EU priority pollutant list and the
health concerns. The phthalate DEPH (diethyl-
used for decades. The chemical structure of US Contaminant Candidate List 3.
hexyl phthalate) is classified as harmful to the
the alkyl fluorine bond imparts unique and
reproductive system.
useful properties for both consumer and
industrial applications. Some of these
properties result in these compounds Sample: Perfluorocarboxylic acids
mix in water, 0.20 ng/µL
Peaks: 1. Dimethyl Phtalate being widely dispersed in the environment 0.4
C7
2. Diethyl Phthalate and bioaccumulating, yielding highly per-
3. Di-n-butyl Phthalate C8
4. Bis(2-ethylhexyl)phthalate sistent and potentially toxic degradation C5
C9
C6
884 products. Perfluorochemicals (PFCs) have C10
1 - DMP

WVL:224 nm C11
been used extensively since the 1950s in µS
C12
3 - DBP

multiple commercial applications includ- C13

500 C14
4 - DEHP

ing surfactants, paper and textile coatings,

2 - DEP

mAU
lubricants, paints, polishes, food packaging,
0
0 4 and fire-retarding foams. Several PFCs are
3 persistent in the environment and humans,
2 and are ubiquitous contaminants in wildlife. 0 2 4 6 8 10 12 14
1 Human exposure to PFCs is also widespread Minutes
(Baseline correction by blank subtraction.) 24555
-667
1.0 4.0 6.0 8.0 10.0 12.0 15.0 Figure 24. The Acclaim PA 2 column shows excellent
Minutes resolution of PFC’s from C5 to C14 for both the
26912 acidic and sulfonated forms
Figure 23. Using the NG1 trap column with the
Acclaim C18 120A in combination with the dual
UltiMate HPLC system, you can easily determine
trace analysis with direct injection. Online SPE takes
the labor out of sample preconcentration.

page 13
 Contaminants of Emerging Concer

Contaminants of Emerging Concern (CEC) is a general term that attempts to describe a broad group of compounds including pesticides,
pharmaceuticals, personal care products (PCPs), and surfactants that have adverse effects on the environment. Although present in low
levels, the long term exposure effects are not well known.

Online Sample Preparation for fast chromatography up to 10 peaks in Surfactants

Pharmaceuticals and Personal 10 seconds. Using online SPE, the separation Alkylphenol polyethoxylates (APEO), one
and quantitation of over 100 pesticides and of the most commonly used classes of
Care Products
PCPs from a single injection is possible from nonionic surfactants, are known endocrine
Due to the large number of man-made complex water samples with reproducibility disruptors that affect estrogenic function.
chemicals manufactured and their equivalent to EPA 1694. APEO breakdown products are also important
environmental persistence, there is a growing for understanding the overall environmental
This table shows the recoveries from an
need for multi-target screening techniques. impact in surface waters, sediments, and
The figure below shows an example of online SPE method using a dual UltiMate
sludge, and are regulated by the U.S. EPA
pesticide screening of 100 pesticides and HPLC system with MS/MS detection from
and EU commission. Determination and
PPCP's using the UltiMate® 3000 Rapid drinking, raw, surface, and wastewaters.
quantification of the breakdown profile
Separation LC (RSLC) system optimized for Note the wide variety of compounds classes of APEOs is hindered by the inability to
that can be readily determined from chromatographically resolve the numerous
these matrices. breakdown products, resulting in ion
suppression from co-eluting species. The
160%
Dionex Acclaim Surfactant column solves this
problem by chromatographically separating
140%
the majority to APEOs.
120%

100%
NPEO2
80% NPEO3
60%
n-NP
Intensifty (counts)

OPEO2
40%
OPEO3 NPEO1 OP
20%
t-OP
0% OPEO1
Lopressor
Meclofenamic Acid
Tylosine Tartrate
Sulfamethoxazole
Amoxicillin
Lidocaine
Erythromycin
Norgestimate
Miconazole
Norfloxacin
Ranitidine
Roxithromycin
Ofloxacin
Testosterone
4-aminoantipyrine
Oxolinic acid
Meprobamate
Chlorotoluron
Metolachlor
DIA
Hexazinone
Cotinine
Sarafloxacin
Codeine
Erythromycin Anhydrate
Pentoxifylline
Ormetoprim
Bezafibrate
Metazachlor
Thiabendazole
Flumeqine
DEA

2 4 6 8 10 12 14 17
Minutes
25432

Figure 26. Chromatographic resolution minimizes

signal suppression and in turn lower detection
limits are achieved when using the Dionex Acclaim
Surfactant column.
Figure 25. Online SPE of surface water samples spiked with over 100 pharmaceuticals, pesticides,
and personal care products shows excellent recovers from a wastewater effluent, drinking, and
surface waters.

page 14
ern

Dionex also offers the AutoTrace® instrument as an alternative to labor intensive, solvent wasting liquid-liquid extraction for a variety of
regulated methods. The AutoTrace instrument is an automated solid-phase extraction (SPE) system designed for use with SPE adsor-
bents (cartridge or disk format).. Strong solvents are then used to generate an extract ready for analysis.

Offline Sample Preparation Matrix often interferes with analysis when

Peaks: 1. Chloride – mg/L
Dionex also offers the AutoTrace instrument present in disparate amounts from the 2. Nitrite 2
analyte of interest. For example, seawater 3. Carbonate –
as an alternative to labor intensive, solvent 4. Nitrate 2
wasting liquid-liquid extraction for a variety or brine samples typically contain very high 5. Sulfate 2
concentrations of chloride that interfere with 7
of regulated methods. SPE requirements
vary depending on the amount of sample determination of nitrite. To overcome this, one
1
required and correspondingly SPE systems strategy may be to use an analytical column
are separated into large and small volumes. with a higher capacity. When this still does
The AutoTrace 280 workstation automates not solve the problem, matrix elimination can
cartridge or disk conditioning, sample loading, be a more viable choice. The OnGuard® and
rinsing, drying, and eluting steps for large InGuard™ line of disposable sample pretreat- µS

volume liquid extractions (20 to 4000 mL). ment cartridges remove matrix interferences 1.6% NaCl w/o
InGuard
2
such as phenols, metals, cations, anions, and
3 4
hydrophobic substances such as humic acids 5
commonly found in surface waters. 4
3
0 2
1

Performance 0 5 10 15 20 25 30 35
Pesticide Recovery Study N=6 AutoTrace 280 SPE Vacuum Manifold SPE Minutes 26913

Compound Recovery % %RSD Recovery % %RSD Figure 27. The most common application for this
cartridge is the removal of chloride from high
Atrazine 88 1.8 54 12.2
chloride samples for the determination of ppm
Propazine 91 1.5 80 7.3 concentrations of other anions. In this application,
a 1.6% NaCl solution results in a very large chloride
Alachlor 99 3.4 96 4.1
peak that obscures nitrate. Using the InGuard or
Metachlor 99 4.3 96 2.9 OnGuard Ag in tandem with an InGuard Na or H
cartridge, the chloride is effectively removed for
Table 1 shows a pesticide recovery study comparing the AutoTrace 280 SPE with a quantitation of low levels of nitrate.
vacuum manifold technique. The improvements in recovery and reproducibility are
attributed to the microprocessor control of all the liquid flow rates—both sample and
SPE reagents.

page 15
systems ICS-2100 Ion Chromatography System

The ICS-2100 system is the first Versatility • The dual-piston pump design
Reagent-Free™ ion chromatogra- • This integrated system performs reduces pulsations, allowing high-
phy system with electrolytic sample all types of IC separations using sensitivity detection and excellent
preparation (RFIC-ESP™ system) and conductivity detection. flow-rate accuracy and precision.
eluent generation (RFIC-EG™ sys- • Flexible flow rates support 2, 3, 4,
• RFIC-EG system technology
tem) capabilities designed to perform
converts deionized water into high- and 5 mm column formats.
all types of electrolytically generated
purity eluents on-line. • The streamlined design with its
isocratic and gradient IC separations
using conductivity detection. Microbore • RFIC-ESP system accessories en- small footprint occupies minimal
2 mm columns as well as standard bore able control of electrolytic sample bench space.
4 mm columns are fully supported. preparation devices such as water • An LCD touch-pad front panel
The ICS-2100 RFIC-ESP system now purifiers and sample conditioners. provides clear identification of key
provides automation for many sample • Sample preparation capabilities operating parameters permitting at-
preparation techniques with multiple extend the range of the instrument instrument control and monitoring.
valving configurations and support for into areas, such as on-line filtration,
electrolytic sample preparation devices. matrix elimination, neutralization,
The ICS-2100 system provides high per- and ultratrace analysis.
formance with unequalled ease-of-use
when coupled with an AutoSuppression®
device, such as the SRS® 300 suppressor.
Chromeleon® software provides full
control and digital data collection from
a PC using simple USB connectivity.

Passion. Power. Productivity.

Reagent-Free IC Systems Simple and Precise Control High Performance
• Electrolytic eluent and regenerant • Control for the SRS and Atlas® • For improved reproducibility, the
production minimizes time, labor, electrolytic suppressors is built in. heated and thermostated high-
operation costs, and eluent prepara- These AutoSuppression devices performance conductivity detection
tion errors. eliminate the need to manually cell permits measurements that are
• The eluent generator delivers prepare acid or base regenerants. unaffected by temperature variation.
sodium, potassium, and lithium Electrolytic suppression reduces • Advanced single-range digital out-
hydroxide and carbonate/bicarbon- background conductivity and pro- put provides an operating range to
ate eluents for anion separations vides high signal-to-noise ratios. 15,000 µS full scale with autorang-
and methanesulfonic acid eluents • Full control and digital data col- ing to provide accurate detection
for cation separations. lection are available with the of major and minor constituents in
• Eluent generation provides the Windows®-based Chromeleon a single run. Single-range analog
utmost in reliable, reproducible Chromatography Data System signal output is also standard.
eluent concentrations from a supply software using a USB high-speed • Column heater provides day-to-day
of deionized water. Gradient elution communication protocol. consistency, ensuring reproducibil-
becomes routine. EG-produced • Chromeleon eWorkflows preload all ity and stability. Preheating of the
hydroxide eluents offer the lowest instrument parameters for fast and eluent prior to the column maintains
conductivity backgrounds possible. easy operation and data analysis. the column temperature set by the
• Chromeleon software includes an user. A transparent cover allows
Automated Sample Preparation electronic logbook of nearly un- viewing of the column without
• Optional valving supports matrix limited user-selectable operational temperature disruption.
elimination, sample concentration, parameters. • Optional built-in vacuum degas pro-
and on-line filtration. vides in-line degassing of eluents,
• Auxiliary power port supports ensuring reproducibility and protec-
electrolytic sample preparation tion of eluents from contamination
devices. and decomposition. Control of the
degas operation can be automated
to sense when degassing is required.
• Inert, metal-free PEEK™ compo-
12 nents throughout the system ensure
Column: IonPac® AG15, AS15, 4 mm
Eluent: 8 mM KOH from 0–6 min, compatibility and metal contamina-
5 8-55 mM from 6–18 min
tion-free chromatography.
Eluent Flow Rate: 1.6 mL/min
3 6 7 8 Eluent Source: ICS-2000 with CR-ATC
2 Inj. Volume: 25 µL
Temperature: 30 ˚C
µS 1
Detection: ASRS® ULTRA (4 mm)
AutoSuppression recycle mode
Power Setting – 220 mA
4
C
Peaks: 1. Fluoride 2 mg/L
B 2. Chloride 5
3. Nitrite 10
A 4. Carbonate -
-2.0
0 10 20 30 5. Sulfate 10
Minutes
6. Bromide 20
7. Nitrate 20
Component System A System B System C 8. Phosphate 30
retention Time retention Time retention Time
(%rSd) (%rSd) (%rSd)

Fluoride 5.367 (0.062) 5.361 (0.031) 5.384 (0.017)

Chloride 12.166 (0.026) 12.163 (0.022) 12.165 (0.020)
Nitrite 13.908 (0.022) 13.906 (0.019) 13.904 (0.021)
Sulfate 17.685 (0.027) 17.623 (0.025) 17.618 (0.021)
Bromide 19.382 (0.031) 19.404 (0.018) 19.385 (0.018)
Nitrate 20.759 (0.036) 20.783 (0.014) 20.760 (0.017)
Phosphate 22.219 (0.049) 22.089 (0.043) 22.066 (0.018)

(%RSD) n = 20 19133

Reagent-Free IC systems produce consistent lab-to-lab eluent concentrations for highly

reproducible retention times and peak areas. Results are the same day to day, system to
system, and lab to lab.

2
Convenient Key Features
• Versatile eluent organizer tray. • Automated eluent generation
Accommodates 1-, 2-, or 4-L eluent • LCD front panel control
bottles. • Dual-piston pump
• Electrically actuated six-port • Column heater
Rheodyne PEEK injection valve. • Electrolytic suppression
• Ergonomically placed injection port • Digital conductivity detection
for easy manual sampling. • Vacuum degas (option)
• Eluent valve for positive shut-off • Optional 6- or 10-port valve
of eluent flow prior to the pump for • Optional RFIC-ESP water purifier
easy servicing. • USB connectivity, plug-n-play
• Easy-access door to chromatogra- • Electronic logbook and trending
phy components. through virtual channels
• Leak detection and management
for fast response to system leaks.
• TTL controls for external pump,
injection valve, range selection,
and signal offset for stand-alone
operation. Small Loop
Waste
Detector

6 CRD
7 5
Waste
Standard
8 4
9 Pump Suppressor
3
10 1
2
4
5 Analytical
Column
6
3

Large Loop Guard

1 2
Concentrator Column

Sample

Waste

In RFIC-ESP systems, the optional water polisher also serves as a

sample preparation pump, facilitating preconcentration or matrix
elimination applications.

All components are easily accessed

through the front chromatography panel.

3
 ICS-2100 IC SYSTEM SPECIFICATIONS

Analytical Pump and Fluidics Column Heater (Standard) Continuous Operation (4 L of Eluent):
Type: Operating Temperature Range: Up to 28 days or 2000 samples
Serial dual-reciprocating pistons, 30 to 60 °C (86 to 140 °F); minimum Always On, Always Ready Capable:
microprocessor-controlled constant 5 °C above ambient; settable range is Standard feature
stroke, variable speed equal to working range
Remains Fully Calibrated for Extended
Construction: Temperature Accuracy: Periods (<28 days):
Chemically inert, metal-free ±0.5 °C at sensor, at 40 °C Standard feature. results are traceable
PEEK pump heads and flow paths Eluent Generation (Standard) to a single calibration
compatible with aqueous eluents of System Wellness:
pH 0–14 and reversed-phase solvents Eluent Types Consumables usage monitoring for
KOH, LiOH, NaOH predictive maintenance
Pump Operating Pressure K2CO3, K2CO3/KHCO3
0–35 MPa (0–5000 psi) MSA Maximum Operating Pressure:
Flow Rate Range: 21 MPa (3000 psi)
Gradient Profiles
0.00–5.00 mL/min without Option; combination of unlimited Operating Temperature Range:
changing pump heads number of linear, convex and 4–40 °C
Flow Precision: concave positive and negative Suppressors and Control
<0.1%, typically gradient profiles
Chemical Suppression:
Flow Accuracy: Concentration Increments 2 mm and 4 mm anion and cation,
<0.1%, typically 0.01 mM membrane suppression bed types
Pressure Ripple: Concentration Range Displacement Chemical Regeneration:
<1% at 13.8 MPa (2000 psi) 0.1–100 mM (depending on 2 mm and 4 mm anion and cation,
and 1.0 mL/min eluent used) membrane suppression bed types
Eluent On-Off Valve: Flow Rate Electrolytic Suppression—Self-
Standard 0.1–3.0 mL/min Regenerating:
Piston Seal Wash: Maximum Operating Pressure 2 mm and 4 mm anion and cation,
Dual-pump head, wash can be 21 MPa (3000 psi) membrane and MonoDisk™
continuous when connected to rinse suppression bed types available
Maximum Solvent Concentration
solution supply Anions: 25% methanol Electrolytic Suppression—Self-
Pressure Alarm Limits: Cations: no solvents Regenerating with External Water
Upper limit 0–35 MPa or 0–5000 psi Mode:
Auxiliary Power Supply (Standard) 2 mm and 4 mm anion and cation.
in one unit (MPa or psi) increments;
lower limit can be set up to one unit Current: Both membrane and MonoDisk
lower than upper limit Constant, 0–200 mA at up to 35 V suppression bed types available

Vacuum Degas: Alarms: Current Control Range:

Optional, automatic control Overvoltage and overcurrent alarms; SRS:
linked to pump flow to protect 4 mm, 0–300 mA in 1 mA increments
Eluent Bottles: devices from power on at zero flow 2 mm, 0–100 mA in 1 mA increments
Polypropylene, up to 4 L volume ®
AES : 0–150 mA in 1 mA increments
Auxiliary Valve (Optional)
Eluent Bottle Pressure: CMD: 0–500 mA in 1 mA increments
Available Valves: SRN: 0–500 mA in 1 mA increments
Not required
6- or 10- port, 2-position high-
Injection Valve: pressure rheodyne valves, fully inert Salt Converter:
6-port, 2-position Rheodyne valve, PEEK construction, electrically Available in 2 and 4 mm versions
electrically activated activated AMMS-ICE:
Columns Supported: Eluent Regeneration (Optional) Available in 2 and 4 mm versions
2, 3, 4, and 5 mm i.d., maximum Carbonic Acid Removal for Anions:
Eluent Regeneration Support:
length 250 mm analytical column ®
ASRS 300 with CRD 200 for
With optional kit
with 50 mm guard column hydroxide eluents
Eluents: ASRS 300 with CRD 300 for
Carbonate and carbonate/bicarbonate carbonate eluents
up to 20 mM
MSA up to 34 mM Non-Suppressed Chromatography:
Supported
Flow Rates:
0.01–2.00 mL/min

4
 ICS-2100 IC SYSTEM SPECIFICATIONS (Cont'd)

Suppressor Wear Parts: Cell Electrodes: 3-D software for photodiode

None; peristaltic pump and inline Passivated 316 stainless steel. arrays, mass spectrometers,
filters not required Compatible with MSA and electrochemical detectors
Suppression Capacity: Cell Body: (optional)
Anion SRS 300 (4 mm): 200 µeq/min Chemically inert polymeric material • Customizable system control
Cation SRS 300 (4 mm): 110 µeq/min panels
Cell Volume: • System status Virtual Channels
Anion SRS 300 (2 mm): 50 µeq/min
<1 µL • Power failure protection
Cation SRS 300 (2 mm): 37.5 µeq/min
Anion MMS™ 300 (4 mm): 150 µeq/min Heat Exchanger: • Sequential injection
Cation MMS 300 (4 mm): 150 µeq/min Inert, tortuous path for low axial • System trigger commands and
Anion MMS 300 (2 mm): 37.5 µeq/min dispersion conditionals
Cation MMS 300 (2 mm): 37.5 µeq/min • Daily audit trail
Maximum Cell Operating Pressure:
Anion AES: 25 µeq/min • Sample audit trail
10 MPa (1500 psi)
Cation AES: 25 µeq/min • Multiple network control and
Autosampler network failure protection
Void Volumes:
Automation Using Autosampler: (optional)
SRS 300 (4 mm): <50 µL
Dionex AS40, AS-DV, AS, AS-HV, • System calibration storage
SRS 300 (2 mm): <15 µL
or third-party autosamplers (factory, present, and previous;
MMS 300 (4 mm): <50 µL
completely user selectable)
MMS 300 (2 mm): <15 µL Sequential/Simultaneous Injection • Customized reporting (unlimited
AMMS-ICE 300 (4 mm): <50 µL Depending on autosampler report workbooks)
AMMS-ICE 300 (2 mm): <15 µL capabilities
Anion AES: <35 µL • Automated system qualification
Automated Dilution: (detailed, comprehensive
Cation AES: <35 µL
Available with AS Autosampler qualification reports)
Conductivity Detector Electronics
Dilution Factor, AS Autosampler: Physical Specifications
and Flow Cell
1:1 to 1:1000
Type: Power Requirements:
Dilution Time, AS Autosampler: 100–240 V ac, 50-60 Hz autoranging
Microprocessor-controlled digital
15 seconds with sample overlap
signal processor Operating Temperature:
Inline Sample Degassing: 4–40 °C (40–104 °F); cold-room-
Cell Drive:
Optional with CRD 300/200 compatible (4 °C) as long as system
8 kHz square wave
Inline Filtration: power remains on
Linearity:
Yes, AS40 and AS-DV Autosamplers Operating Humidity Range:
1% up to 1 mS
or inline filter 5–95% relative, noncondensing
Resolution:
High Automation Flexibility: Control Modes:
0.00238 nS/cm
Conditionals using Chromeleon and Full control through front panel and
Full-Scale Output Ranges: Autodilution License Chromeleon software; alternative
Digital signal range 0–15000 µS
System Software control through TTL or relay
Analog signal range 0–15000 µS
Chromeleon Chromatography closures; two relay outputs, two TTL
Electronic Noise: Data System software, supports outputs, four programmable inputs
±0.1 nS when background Windows XP or Vista USB Communication Protocol:
conductivity is 0–150 µS/cm • Automated ProcedureWizards One USB input; one built-in two-
±2 nS when background conductivity • System Wellness and Predictive output USB hub
is 151–3200 µS Performance
• Data trending plots (numerical Leak Detection:
Filter:
device parameters) Built-in, optical sensor
Rise times from 0 to 10 s, user
selectable • Virtual Column Simulator Dimensions (h × w × d):
(evaluation mode standard, 56.1 cm × 22.4 cm × 53.3 cm
Temperature Compensation: isocratic and gradient optional) (22.1 in × 8.8 in × 21 in)
Fixed at 1.7% per 1 °C at cell • Application templates
temperature Weight:
• Multivendor automation support
24.5 kg (54 lb)
Temperature Range: of 3rd party instruments (fully
Ambient +7 °C, 30 to 55 °C controls over 300 instruments
from more than 30 manufacturers,
including GC, HPLC, and MS)

5
 Orderin g Information
In the U.S., call (800) 346-6390 or contact the Dionex office nearest you.
Outside the U.S., order through your local Dionex office or distributor. Refer
to the following part numbers.

ICS-2100 Ion Chromatography System with Software and PC

An ICS-2100/Chromeleon Windows Workstation bundled package
includes: an ICS-2100 with isocratic dual-piston pump, eluent generator to run
Full EG, injection valve, column heater, heated conductivity cell, LCD touch-
pad front panel, USB cable, Chromeleon Computer (with Windows XP), and
USB dongle. Comes with two Class 1 Timebases controlling one Dionex IC
system. Consumables must be ordered separately.

ICS-2100 Ion Chromatography System with Full EG, Chromeleon,

and Windows XP Workstation, without Degas..................................... 069656

ICS-2100 Ion Chromatography System with Full EG, Chromeleon,

Windows XP Workstation and Degas................................................... 069657

ICS-2100 Ion Chromatography System with Full EG and

Chromeleon, without Degas or PC....................................................... 069658

ICS-2100 Ion Chromatography System with Full EG,

Chromeleon, and Degas, without PC................................................... 069659

AutoDilution License............................................................................ 069725

PEEK is a trademark of Victrex PLC.

Windows is a registered trademark of Microsoft Corporation.
MonoDisk, Reagent-Free, RFIC-EG, and RFIC-ESP are trademarks and AES, ASRS, Atlas,
AutoSuppression, Chromeleon, IonPac, and SRS are registered trademarks of Dionex Corporation.

Passion. Power. Productivity.

Dionex Corporation North America Europe Asia Pacific
1228 Titan Way U.S./Canada (847) 295-7500 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768; (32) 3 353 4294 Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735
P.O. Box 3603 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190
Sunnyvale, CA South America Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Sweden (46) 8 473 3380 Taiwan (886) 2 8751 6655
94088-3603 Brazil (55) 11 3731 5140 Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722
(408) 737-0700 www.dionex.com LPN 2198-02 PDF 06/09
©2009 Dionex Corporation
systems Reagent-Free Ion Chromatography Systems with
Eluent Generation for IC Without Manually Prepared Eluents

Since the introduction of Benefits of Eluent Generation Key benefits:

Reagent-Free™ ion chromatography RFIC-EG systems generate • Simplifies operation; no need to
(RFIC™) products in 1995, Dionex high-purity hydroxide, carbonate, or prepare eluents or regenerants
has continued to simplify IC while methanesulfonic acid (MSA) eluents
increasing the capabilities and power electrolytically using Eluent Generation • Improves analytical reproducibility,
of ion analysis. RFIC systems with Cartridges (EGC). Chemists no longer day-to-day, week-to-week,
eluent generation (RFIC-EG™ systems) need to spend time manually prepar- month-to-month
produce consistent, precisely controlled, ing eluents as traditionally done using
high-purity eluents and regenerants conventional IC systems. The eluent is
• Ensures system-to-system reproduc-
ibility and lab-to-lab consistency
electrolytically. Not only is the generated at the concentration required
eluent preparation labor decreased, for your IC application. Eluents are • Achieves sensitive results with
but reproducibility is increased. The purified on-line using Continuously pure, uncontaminated eluent
ICS-2100 and ICS-5000 systems are Regenerated Trap Columns (CR-TC),
integrated RFIC-EG systems. The and suppressed electrolytically before • Eliminates errors and variability
RFC-30 Reagent-Free Controller can detection, without the need to prepare associated with manual eluent and
be used to convert most other Dionex regenerants. The only requirement is a regenerant preparation
IC systems into RFIC‑EG systems. high-purity source of deionized water.

Passion. Power. Productivity.

• Achieves the power of hydroxide
and MSA gradient separation with No manually
prepared eluent Injector No external regenerant
an isocratic pump
Eluent
• Reduces operator exposure to Generator
CR-TC
Guard
Column
Analytical
Column
Electrolytic
Suppressor
hazardous chemicals CRD

Conductivity

•
Pump Cell
Reduces pump maintenance
because pump is exposed only to
deionized (DI) water Waste
Chromeleon
®

• Supports capillary-scale flow rates

Workstation
Conductivity
to reduce eluent and EGC Detector

consumption. 15199-03

Figure 1. RFIC-EG systems schematic diagram.

Simplify Operation with RFIC-EG Systems
RFIC-EG systems are very easy RFIC Eluent Generator (EG) Instrument Compatibility Chart*
to operate. Simply install the eluent
generator, attach a source of deionized Instrument Anions Cations
water to your pump, and begin collecting Gradient or Isocratic Isocratic Isocratic Gradient or
Hydroxide EG Carbonate EG Carbonate/ Isocratic MSA EG
data. The schematic diagram (Figure 1) Bicarbonate EG
illustrates just how easy operation is
ICS-2100 KOH, NaOH*, LiOH* K2CO3 K2CO3 + EPM MSA
with RFIC-EG systems. The compatibil-
ity chart to the right shows what eluent ICS-5000 KOH, NaOH, LiOH K2CO3 K2CO3 + EPM MSA
generator cartridges are supported on (Analytical)
each system.
ICS-5000 KOH not available not available MSA
(Capillary)
Reproducibility of Eluent Generation
Electrolytic eluent generation RFC-30 KOH not available not available MSA
produces amazingly consistent run-to-
run eluent concentrations by eliminating *Contact your local Dionex representative for software requirements.
errors associated with manual eluent
preparation. Eluent concentration
accuracy and precision translates into Column: IonPac® AS22, 4 × 250 mm Inj. Volume: 10 µL
Eluent: 4.5 mM K2CO3 /1.4 mM KHCO3 Detection: Suppressed conductivity,
highly reproducible retention times and Eluent Source: EGC II K2CO3 + Electrolytic pH Modifier (EPM) ASRS® 4 mm
peak areas. Figure 2 shows an overlay Temperature: 30 °C AutoSuppression® recycle mode

of 100 chromatograms, illustrating the Flow Rate: 1.2 mL/min

Analytes: RT RSD Area RSD
unmatched reproducibility provided by Fluoride 0.08 1.4
eluent generation. Chloride 0.76 0.2
Nitrite 0.05 0.4
Nitrate Sulfate Bromide 0.04 0.09
Fluoride
Make Your Methods Portable Nitrite
Bromide Nitrate 0.04 0.15
Phosphate 0.05 0.14
With RFIC-EG systems, the Sulfate 0.13 0.11
ability to transfer methods from one lab
to another is simplified. Whether the lab Chloride Phosphate
µS
is next door or in another country,
RFIC eluent generators ensure that your
analytical results are consistent and can
be seamlessly transferred. RFIC eluent
generators are so precise that you can
expect less than 1% deviation from lab
to lab under most analytical conditions. 0 2 4 6 8 10 12
Minutes 22900-01

Figure 2. Systems using eluent generation produce extremely reproducible retention times and
peak areas, as demonstrated with this overlay of 30 injections of an anion standard on an
ICS-2000 RFIC-EG system using an electrolytically generated carbonate/bicarbonate eluent.

2
Achieve Sensitive Results The eluent concentration is, therefore, Labor Savings
RFIC eluent generators make directly proportional to the applied Labor savings alone justify the
trace-level analysis routine. The ultra- current from the eluent generator and purchase of an EGC. Manual eluent
pure eluent produced by our full line inversely proportional to the eluent flow preparation typically consists of a long
of eluent generator cartridges, results rate. Because both of these parameters list of tasks that can include weighing or
in a stable baseline that makes peak can be precisely controlled, the resulting pipetting chemicals, diluting, filtering,
integration more accurate and reliable. eluent concentrations are more precise mixing stock solutions, transferring,
In addition to lower background, than manually prepared eluents. This dispensing, priming, degassing, filling
electrolytically generated gradients consistency is true from run-to-run, bottles, etc. This list does not take into
provide minimal baseline shifts system-to-system, and lab-to-lab. consideration potential errors associated
compared to conventional gradients. Eluent generation is fully supported with eluent makeup or time required for
by Dionex OQ/PQ validation tools system equilibration. Eluent generation
Accuracy of RFIC Eluent Generators to ensure that your laboratory meets simplifies IC by making the eluent for
The eluent concentration even the most rigorous regulatory you. Simply add water to the system
generated by an RFIC-EG system is requirements. and begin collecting data. In addition,
extremely accurate and reproducible. with an RFIC-EG system, no additional
Dionex’s patented eluent generation chemicals or reagents are needed for
technology follows Faraday’s Law. the suppressor, which saves even more
time. Table 1 illustrates the potential
labor savings of RFIC eluent generators.

Table 1. Labor Savings

Cost of Manual Preparation* Time to Start Up Labor Savings

System for Life with Eluent Generation
of Cartridge
Hourly 0.5 h 1.0 h 2.0 h 1 h 0.5 h 1.0 h 2.0 h
Labor (Hours spent per 5-day week
Rate preparing eluent. Calculations
based on 50 weeks)
$15.00 $375.00 $750.00 $1500.00 $15.00 $360.00 $735.00 $1485.00
$25.00 $625.00 $1250.00 $2500.00 $25.00 $600.00 $1225.00 $2475.00
$35.00 $875.00 $1750.00 $3500.00 $35.00 $840.00 $1715.00 $3465.00
$45.00 $1125.00 $2250.00 $4500.00 $45.00 $1080.00 $2205.00 $4455.00
*Quality Assurance Report conditions

3
RFIC EGC Lifetime
The life expectancy of an Table 2. Cartridge Life
analytical EGC is a function of a Column* Eluent Flow Conc. I Hours Days Weeks
number of user-selectable parameters. (mL/ (mM) (current (8 h/day) (5 days/
Based on eluent concentration and min) required) week)
flow rate, the number of expected
operating hours for the cartridge can AS23 (4 mm) K2CO3:KHCO3 1.0 4.5:0.8 17 2547 318 64
be determined. Table 2 shows some AS23 (2 mm) K2CO3:KHCO3 0.25 4.5:0.8 4.3 10190 1274 255
examples of calculations of EGC II
KOH, EGC II K2CO3, and EGC II MSA AS22 (4 mm) K2CO3:KHCO3 1.2 4.5:1.4 23 1906 238 48
cartridge lifetimes based on different AS22 (2 mm) K2CO3:KHCO3 0.3 4.5:1.4 5.7 7627 953 191
column applications.
Under most conditions, the AS19 (0.4 mm) KOH 10 µL/ 20 0.322 13,140 5471 781
EGC (capillary) will last 18 months

min
regardless of use, allowing Always on AS18 (4 mm) KOH 1.0 39 62.7 1282 160 32
operation without concern for acclerated
AS18 (2 mm) KOH 0.25 39 15.7 5128 641 128
consumption of the EGC.
AS18-Fast KOH 10 µL/ 39 0.627 13,140 5471 781
Unparalleled Gradient Flexibility (0.4 mm) min
In addition to saving time
AS15 (3 mm) KOH 0.5 40 32.2 2500 313 63
preparing reagents, methods
development can be speeded up by AS14 (4 mm) K2CO3:KHCO3 1.2 3.5:1.0 17 2500 313 63
adding hydroxide and MSA gradient
AS9-HC (4 mm) K2CO3 1.0 9.0 29 1500 188 38
capability to the existing isocratic
system. Electrolytically generated CS12A (4 mm) MSA 1.0 18 28.9 1389 174 35
gradient separations are as effortless CS12A (0.4 mm) MSA 10 µL/ 20 0.321 13,140 5471 781
as isocratic separations. Not only is
min
eluent preparation eliminated, but also
straightforward software control creates CS16 (4 mm) MSA 1.0 30 48.2 833 104 21
gradient profiles on demand. A change CS17 (2 mm) MSA 0.25 6 2.4 16666 2083 420
in eluent concentration is accomplished
through the software, not manual eluent *Quality Assurance Report conditions
1
Non-stop operation
preparation from a chemical stock.
Furthermore, on-line eluent generation
eliminates eluent contaminants that
cause gradient baseline shifts by RFC-30: Upgrade IC Systems to
electronically generating a high-purity, RFIC‑EG Systems
contamination-free mobile phase. This The Reagent-Free Controller
eluent generation results in higher (RFC-30) is an economical way to
sensitivity, improved resolution, upgrade an existing DX-120, DX-320,
lower baseline drift, and accurate DX-500, DX-600, or ICS-2500 ion
peak integration. chromatography system to an RFIC-EG
system. The RFC-30 is a stand-alone
eluent generation system that does not
require software. This controller and
EGC can deliver isocratic or simple
gradient eluents. The RFC-30 includes
an eluent generator, an electrolytic
EGC Capillary cartridges provide months or
suppressor controller (AES® and SRS®
even years of eluent generation, and provide 300), and control for the Continuously
the ability to run KOH or MSA gradients, and Regenerated Trap Columns (CR-TC).
isocratic carbonate/bicarbonate eluents without
the need to manually prepare eluents.

4
 RFIC-EG component Specific ation s rfc-30 Reage nt-Free
Controller Specific ati ons
EGC Specifications
Cartridge Dimensions (h × w × d): 23 × 7 × 10 cm (9 × 2.75 × 4 in.) TTL Inputs:
Two independent 0–5 V
Cartridge Weight: 1.6 kg (3.5 lb)
0V = On, 5V = Off; ac control
Concentration Range: 0.1–100 mM (0.1–80 mM EGC-LiOH)
Dimensions (h × w × d):
Flow Rate: 0.10–3.00 mL/min. 12.4 × 16.2 × 28.8 cm
Max. Operating Pressure: 21 MPa (3000 psi) (4.9 × 6.4 × 11.3 in.)
Max. Solvent Concentration: EGC II KOH–25% methanol Weight:
EGC II MSA–no solvents 2.5 kg (5.5 lb)
EGC II K2CO3–no solvents
Power Requirements:
EGC (Capillary) Specifications Consumption, 500 VA max.;
Cartridge Dimensions (h × diam.): 15 cm × 6.4 cm (6 in. × 2.5 in.) voltage, 100–240 V ac;
frequency, 50/60 Hz
Cartridge Weight: 0.375 kg (0.83 lb)
Operating Temperature Range:
Concentration Range: 0.1–200 mM
4–40 °C
Flow Rate: 1–30 µL/min (0.001–0.030 mL/min) Operating Humidity Range:
Max. Operating Pressure: 34.5 MPa (5,000 psi) 5–95% relative, noncondensing
Max. Solvent Concentration: EGC KOH (Capillary)–25% Methanol
EGC MSA (Capillary)–no solvents

RFIC CR-ATC and CR-CTC Specifications

Dimensions (h × w × l): 5.1 cm × 5.5 cm × 8.4 cm
(2.0 in × 2.15 in × 3.3 in)
Weight: 60 g (0.13 lb)
Current Output (Analytical): < 125 mA
Void Volume (Analytical): < 100 µL
Constant Voltage (Analytical): 24 V dc

Current Output (Capillary): 1 mA

Void Volume (Capillary): <2 µL
Constant Voltage (Capillary): 12 V dc

5
 RFIC-EG component Specification s ( cont.)
RFIC Suppressor Specifications
ASRS 300 and CSRS 300
Dimensions: 16.8 × 4.5 × 5.2 cm (6.6 × 1.8 × 2.1 in)
Void Volume: 4 mm: < 50 µL
2 mm: < 15 µL
Weight: 630 g (1.4 lb)
Current Range: 0–500 mA
AAES and CAES
Dimensions: 4.9 × 4.4 × 10.2 cm (1.9 × 1.8 × 4.0 in.)
Void Volume: < 35 µL
Weight: 120 g (0.3 lb)
ACES 300 and CCES 300
™ ™

Dimensions: 9.4 × 3.1 × 10.3 cm (3.7 × 1.2 × 4.1 in.)

Void Volume: < 1.5 µL
Weight: 150 g (0.33 lb)
Current Range: 0–20 mA

6
 Ordering Informati on

To order in the U.S., call 1-800-346-6390, or contact the Dionex Regional Office
nearest you. Outside the U.S., order through your local Dionex office or
distributor. Refer to the following part numbers.

EGC Cartridges Part Number

EGC III KOH Cartridge............................................................................074532
EGC III MSA Cartridge............................................................................ 074535
EGC III K2CO3 Cartridge.......................................................................... 074536
EPM III Electrolytic pH Modifier . .......................................................... 080135
(needed to generate K2CO3/KHCO3 eluent)
Carbonate mixer kit (2 mm) . ................................................................... 063443
Carbonate mixer kit (4 mm) . ...................................................................061686
EGC III NaOH Cartridge..........................................................................074533
EGC III LiOH Cartridge...........................................................................074534
Splitter/Mixer (used to operate EGC III NaOH and ................................063049
EGC III LiOH in dual-mode for Cryptand A1 column chemistry)
EGC-KOH (Capillary) Cartridge..............................................................072076
EGC-MSA (Capillary) Cartridge..............................................................072077

Electrolytic Suppressors Part Number

ASRS 300 (2 mm) Anion Self-Regenerating Suppressor.........................064555
ASRS 300 (4 mm) Anion Self-Regenerating Suppressor.........................064554
CSRS 300 (2 mm) Anion Self-Regenerating Suppressor.........................064557
CSRS 300 (4 mm) Anion Self-Regenerating Suppressor.........................064556
Anion Atlas Electrolytic Suppressor (AAES)........................................... 056116
Cation Atlas Electrolytic Suppressor (CAES).......................................... 056118
Anion Capillary Electrolytic Suppressor (ACES 300).............................072052
Cation Capillary Electrolytic Suppressor (CCES 300).............................072053

Eluent Purification Part Number

CR-ATC Continuously Regenerated Anion Trap Column........................060477
CR-CTC II Continuously Regenerated Cation Trap Column...................060478
CR-ATC (Capillary) Continuously Regenerated Anion...........................072078
Trap Column
CR-CTC (Capillary) Continuously Regenerated Cation..........................072079
Trap Column

Sample Purifications Part Number

For Hydroxide Eluents:
CRD 200 (4 mm) Carbonate Removal Device.........................................062983
CRD 200 (2 mm) Carbonate Removal Device.........................................062986
CRD 200 (Capillary) Carbonate Removal Device....................................072054
For Carbonate Eluents:
CRD 300 (4 mm) Carbonate Removal Device.........................................064637
CRD 300 (2 mm) Carbonate Removal Device.........................................064638

Reagent-Free Controller Part Number

RFC-30 Reagent-Free Controller (EGC III KOH, CR-ATC)...................060667
RFC-30 Reagent-Free Controller (EGC III MSA, CR-CTC)...................060668
RFC-30 (MSA, CR-CTC, CTS-10) for DX-120 Only.............................061414
RFC-30 (KOH, CR-ATC, CTS-10) for DX-120 Only..............................061413

7
 ACES, CCES, Reagent-Free, RFIC, and RFIC-EG are trademarks, and AES, ASRS, AutoSuppression,
Chromeleon, CSRS, IonPac, and SRS are registered trademarks of Dionex Corporation.

Passion. Power. Productivity.

Dionex Corporation
North America Europe Asia Pacific
1228 Titan Way U.S./Canada (847) 295-7500 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735
P.O. Box 3603 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190
South America Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Sweden (46) 8 473 3380 Taiwan (886) 2 8751 6655
Sunnyvale, CA
94088-3603 Brazil (55) 11 3731 5140 LPN 1624-04 PDF 4/10
Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722
(408) 737-0700 www.dionex.com ©2010 Dionex Corporation
 Thermo Scientific
Dionex UltiMate 3000
Standard Systems

Standard Systems
more UHPLC for all
Precise • Robust • Versatile
 The new benchmark in HPLC
UHPLC solutions for any laboratory

Optimum HPLC
With support for pressures up to 620 bar (9,000 psi) at flow rates of
up to 10 mL/min, detector data collection rates up to 100 Hz, and
injection cycle times as low as 15 s, the Thermo Scientific Dionex
UltiMate 3000 standard systems fully support conventional methods
while offering compatibility with UHPLC applications.

Unsurpassed Versatility
We offer the most complete choice of HPLC instruments in the
industry. No matter what your applications are, the UltiMate™ 3000
standard systems can be perfectly configured to meet your demands.

Unmatched Performance
High-quality components, innovative mechanical, optical and
electronic design, and ideally matched instruments assure highest
performance. Anytime. Anywhere.

Reliable Solvent Delivery Advanced Sample Handling Optimized

2
|
 Superior Reliability
The UltiMate 3000 standard systems with their perfect interplay
of industry-leading technology, rigorous quality assurance, and
predictive performance monitoring set a new standard of
reliability and durability in HPLC.

Unparalleled Ease of Use

Getting from samples to results is faster and easier than ever before.
Rich, intelligent functionality with Operational Simplicity™, and powerful
data analysis tools will increase the efficiency of your workflow and
boost overall lab productivity.

More than a System

With a full range of services from highly efficient installation processes
and comprehensive training programs, to qualification and validation
services, we want to provide you with more than a system.

One complete
solution for
LC and UHPLC

Fluidics High-Performance Columns Unlimited Detection Intelligent Software

|
3
 Unmatched performance
precise. robust. versatile.

Reliable Solvent Delivery

The UltiMate 3000 SD pump family offers the most complete choice in the
industry and is the right solution for a wide range of applications. All models
combine unrivaled flow and gradient performance with outstanding reliability
and durability.

• Wide operating flow rate range up to 10 mL/min at pressures of up to

620 bar

• SmartFlow™ ensures optimal performance independent of mobile

phase composition

• SpinFlow™ mixing technology perfectly balances gradient delay volume

against mobile phase mixing efficiency

2 nd Stage: 1 – Acesulfame K
Standard mixture WVL:210 nm
Longitudinal Mixing 2 – Saccharin
3 – Caffeine
4 – Aspartame
1,000 5 – Vanillin
6 – Benzoate
7 – Sorbate
8 – Benzaldehyde 50.0

mAU
3 5
6 7
1 2 26.0
7.0 % CH 3 CN 4
0.06% Formate 8
Solvent A 1
st
Stage:
Flow:
7.0

Radial Mixing 1.200 mlL /min

0 1 2 3 4 5 6
Minutes
RT Precision
3 1 – Acesulfame K 0.052% RSD
Overlay of 10 energy drink 3 – Caffeine 0.048% RSD
samples 4 – Aspartame 0.034% RSD
Solvent B
Flow direction 5 – Vanillin 0.024% RSD

1,000
Innovative mixer for exceptional high mixing performance 50.0

mAU

7.0 % CH 3 CN 26.0
0.06% Formate 1
4
5
7.0

Flow:
1.200 mlL /min

0 1 2 3 4 5 6
Minutes

Fast analysis of soft drink additives at 515 bar with excellent retention time precision of < 0.001 min SD

Pump Type Application Examples

Isocratic Routine isocratic methods
Binary Gradient Fast LC, LC-MS
Routine gradient methods, method development, small scale
Quaternary Gradient
purifications
Routine and advanced chromatographic methods, productivity
Dual-Gradient
solutions, multidimensional methods
4
|
Advanced Sample Handling
The innovative UltiMate 3000 autosamplers ensure reliable, precise, and accurate
injections over a wide range of injection volumes. The patented temperature
control protects thermally sensitive samples by eliminating temperature gradients
in the sample compartment.

• Reliable, precise, and accurate injections

• Cycle times as low as 15 s

• Flexible formats, 384 well plates to 10 mL vials

• Available also with fractionation and re-injection option

Every column
UV_VIS_1
WVL:280 nm

mAU
and every
sample
Impurity

fits
0.75 1.00 1.25 1.50
Minutes

Fractionation of a byproduct during a 1.5 min run

Autosampler Type Application Examples

Analytical Sampler Conventional LC methods
Analytical Sampler with Offline 2D-LC methods, fractionation and re-injection
Fractionation Option directly into MS, small scale purifications
Semipreparative Sampler Semipreparative purifications
The Thermo Scientific Dionex WPS-3000 autosampler series
supports an industry-leading range of sample formats

|
5
 Operational simplicity
unparalleled ease of use

Precise Thermostatting for Any Column

The Thermostatted Column Compartment uses large-area Peltier elements
and a fan-based forced-air design to provide efficient cooling and heating.
This design ensures fast equilibration upon startup, after opening the front
door, and after changing the set temperature.

• Holds up to 12 columns to facilitate automated method development

• Freely configurable switching valves for advanced chromatographic

techniques

• UHPLC+ solution kits facilitate advanced workflows such as

on-line SPE-UHPLC

From Left Pump 950 mm

waste

Dual - Gradient Pump

5 4 350 mm 1
2 6
Autosampler 250 mm Right Valve
450 mm
SPE 1 3 5
4
Detector 350 mm
350 mm 250 mm
Analytical Column

On-line SPE-LC for increased sensitivity, reproducibility, and sample throughput of methods

Advanced Solutions Benefits

Increased sensitivity, reproducibility and sample throughput of
On-line SPE-LC
methods with on-line sample preparation
Uniform response with the Thermo Scientific Dionex Corona
Inverse Gradient LC
Charged Aerosol Detector
Increased throughput by running two distinctive methods on a
Parallel Setup
system at the same time
Increased throughput by automatic alternation between two
Tandem Operation
different methods
Increased throughput by automatic alternation between two
Application Switching
different methods

Off-line 2D-LC Increased resolution with two-dimensional LC

6
|
Revolutionary Fitting System
The Thermo Scientific Dionex Viper fitting system does away with
problems experienced with conventional fitting systems. It provides
a perfect fit each time and ensures superior chromatographic
performance.

• Provides zero-dead-volume fingertight connections

• Works with virtually any valve and any column from

any manufacturer

High Performance Columns

Thermo Scientific columns meet the stringent requirements of modern HPLC and LC/MS methods,
providing exceptionally high column efficiencies, symmetrical peaks, and exceptional ruggedness.
A range of columns from general purpose to unique specialty columns are available, based on:

• Fully porous and solid core ultra-pure silica substrates

• Innovative bonding for a wide range of column selectivity

• Quality manufacturing and stringent QC to ensure consistent performance

Turnkey solutions–including
plumbing and software

|
7
 Unique detection
more data-rich results

Wide Detection Versatility

The UltiMate 3000 detector family covers a wide detection
principle range. This makes it convenient to choose the
optimum detection type for your application requirements
in terms of sensitivity, specificity, and precision.

Flexible UV-Vis Absorbance Detection

Three different detector types with a wide range of flow cells
provide full flexibility in UV-vis detection at a data collection
rate of up to 100 Hz. The Diode Array Detector is the best
choice for maximum application flexibility combined with full
wavelength spectra acquisition. The Variable Wavelength
Detector offers the best noise, drift, and linearity performance.

Detection Type Application Examples

UV-Vis Absorbance (DAD,
Standard detection for UV-vis absorbing analytes
MWD,VWD)
Fluorescence (FLD) Highly sensitive and selective detection of fluorescent analytes
Refractive Index (RI) Detection of weak UV-vis absorbing analytes in isocratic elution

Charged Aerosol (CAD) Universal detection for all non-volatile and semi-volatile analytes

Electrochemical (ECD) Oxidizable or reducible analytes

Single-Quad Mass (MS) Ionizable analytes

8
|
Charged Aerosol Detection – A Near Universal
Detection Technology
Charged aerosol detection is a unique detection technology for nonvolatile
or semivolatile analytes providing consistent response independent of
chemical structure. The Thermo Scientific Dionex Corona Charged
Aerosol Detector creates new opportunities for method development,
QA/QC, and bioanalytical chemistry.

• Wide dynamic range

• Suitable for isocratic and gradient

applications Comparison of Response Uniformity for API’s
200% 200%
180% 180%
• Inverse gradient solution for uniform 160%
Corona CAD
160%
UV at 254 nm

response and universal calibration 140% 140%

120% 120%
100% 100%
80% 80%
Peak Area

60% 60%
40% 40%
20% 20%
0% 0%
ß-Estradiol
Ciprofloxacin
Corticosterone
Digoxin
Diphenylhydantoin
Famotidine
Furosemide
Nadolol
Nalbuphine
Norflocacin
Ouabain
Primidone
Quinidine
Quinine
Ranitide
Sulpride
Trimethobenzamide
Warfarin

ß-Estradiol
Ciprofloxacin
Corticosterone
Digoxin
Diphenylhydantoin
Famotidine
Furosemide
Nadolol
Nalbuphine
Norflocacin
Ouabain
Primidone
Quinidine
Quinine
Ranitide
Sulpride
Trimethobenzamide
Warfarin
Analyte Analyte

Response directly proportional to the amount of analyte independent from optical properties

Sensitive Fluorescence Detection

The Thermo Scientific Dionex UltiMate FLD-3000 Fluorescence
Detector series sets a new industry standard in terms of innovative
technology, detection performance, and application versatility.
A unique Dual-PMT option ensures maximum sensitivity across
the entire detection range.

Enhanced Electrochemical Detection

The Thermo Scientific Dionex Coulochem III is an electrochemical
detector with unrivaled sensitivity in amperometric or coulometric
detection. It features advanced techniques such as pulsed
amperometric detection and cyclic voltammetry.

|
9
 Streamlined all chromatography workflows
delivering results instantly

Intelligent Chromatography Software

Thermo Scientific Dionex Chromeleon 7 is the next-generation
chromatography data system from Thermo Fisher Scientific.
Using Operational Simplicity™ as its guiding design principle,
Chromeleon™ 7 takes you from samples to results in the shortest
time possible.

Innovative eWorkflows enable anyone to start chromatographic

analyses and generate results with just a few selections. eWorkflows
automatically create sequences containing the correct instrument
conditions, data processing parameters, and reporting templates.
Samples are run automatically, processed immediately, and
reported instantly.

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 From sample
to results
in less clicks

Chromeleon’s automatic integration updates and

dynamic data processing reduce the analyst’s effort
and display the results immediately.

The unique and innovative integration tools,

Cobra™ peak detection wizard and SmartPeaks™
integration assistant, enhance the user experience
and make detection and integration easy and
straightforward.

Software for Mass Spectrometry

DCMSlink is a free, control-only software package, providing fully
integrated single-point control of any of our LC systems through
Thermo Scientific Xcalibur software, and mass spectrometry control
software from other leading manufacturers.

Learning new software is held to a minimum with the MS software

as the single user interface. Using DCMSlink to connect your
instrument to various MS software packages, the full benefits of
the UltiMate 3000 single and dual pump systems are ready to
support your MS applications.

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www.thermoscientific.com/dionex
©2012 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All Thermo Scientific Dionex products are
designed, developed, and manufactured
other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are under an ISO 9001 Quality System.
subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

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