High Performance Liquid

Chromatography
You’ve Got a Problem to Solve

I need a quantitative
separation of I’ll get
carbohydrates in some on it!
of our products
as soon as possible.

I’ll need a separation

technique.
Separation Techniques

I have two separation techniques in my lab,

High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
Invention of Chromatography by M.
Tswett Ether Chromatography

Chlorophyll
Colors

CaCO3
Comparing Chromatography to the
Flow of a River...
Light leaf
Heavy stone Water flow

Base
Chromatography

 Chromatography is a separation technique which used to separate a

mixture of compounds into its individual components based in certain
physical and chemical properties
 Mobile phase  the solvent system which carries the mixture to be
separated
 Stationary phase  immobile surface which is particulate in nature. This is
the region over the compound gets separated
Mobile Phase / Stationary Phase

 A site in which a moving

phase (mobile phase)
and a non-moving
Mobile phase (stationary phase)
phase make contact via an
Strong Weak interface that is set up.
 The affinity with the
mobile phase and
Stationary stationary phase varies
phase with the solute. 
Separation occurs due
to differences in the
speed of motion.
Three States of Matter and
Chromatography Types

Mobile phase

Gas Liquid Solid

Gas

Stationary
phase Liquid
Gas Liquid
chromatography chromatography
Solid
HPLC

 Although GC is widely used, it is limited to samples that are thermally stable

and easily volatilized.
 For non volatile samples, such as peptides and carbohydrates, can be
analyzed by GC, but only after they have been made more volatile by
suitable chemical derivatization
 Comparison of HPLC and GC

Sample Volatility Sample Polarity

HPLC HPLC
• No volatility requirement • Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase

GC GC
• Samples are nonpolar
• Sample must be volatile
and polar
 Comparison of HPLC and GC
11

Sample Thermal Lability Sample Molecular Weight

HPLC HPLC
• Analysis can take place
• No theoretical upper limit
at or below room
temperature
• In practicality, solubility is
limit.

GC GC
• Sample must be able
to survive high • Typically < 500 amu
temperature injection
port and column
 Comparison of HPLC and GC

Sample Preparation Sample Size

HPLC HPLC
• Sample must be filtered
• Sample size based upon
column i.d.
• Sample should be in
same solvent as mobile
phase

GC GC

• Solvent must be volatile • Typically 1 - 5 L

and generally lower
boiling than analytes
 Comparison of HPLC and GC
Separation Mechanism Detectors

HPLC HPLC
• Both stationary phase • Most common UV-Vis
and mobile phase take • Wide range of non-
part destructive detectors

GC GC
• Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds
Comparison of HPLC and GC
Separation in HPLC

 In HPLC, a liquid or a solid sample dissolved in a suitable solvent, is carried

through a chromatographic column by a liquid mobile phase.
 Separation is determined by
 Solute/stationary phase interaction
 Ion exchange
 Size exclusion
 Solute/mobile-phase interaction
 In each case, however, the basic instrumentation is essentially the same
Flow Channel Diagram for High Performance Liquid
Chromatograph

Detector

Column

Pump Column oven

(thermostatic
column chamber)
Eluent Sample injection unit Drain
(mobile phase) (injector)
Data processor
Degasser
HPLC Columns

 Analytical column
 the most commonly used column for HPLC are constructed from stainless steel
with internal diameter between 2.1-4.6 mm and length ranging from 30-300 mm.
These column are packed with 3-10 µm porous silica particles that may have an
irregular or spherical shape.
 microcolumns (int diameter 44-200 µm and lengths of up to several meters)
 open tubular column (int diameter 1-50 µm and length of 1 m)
HPLC Column

 Guard column
 A guard column is placed before the analytical column
 It contain the same particulate packing material and stationary phase as the
analytical column
 It significantly shorter and less expensive, length 7.5 mm
 Because they are intended to be sacrificial, guard column are replaced regularly

 Two problems that are caused to shorten the lifetime of an analytical column:
 Solutes binding irreversibly to the stationary phase, degrade the column’s
performance by decreasing the available stationary phase
 Particulate material injected with the sample may clog the analytical column
Stationary Phases

 In LC, the stationary phase is a liquid film coated on an packing material

consisting 3-10 µm porous particles
 It may be partially soluble in the mobile phase, causing it to bleed from the
column over time.
 Bonded stationary phases are attached by reacting the silica particles with
an organochlorosilane of the general form Si(CH3)2RCl

 Such columns are designated as end-capped

Normal Phase / Reversed Phase

Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (hydrophilic)
The properties of a stationary phase
Normal-phase chromatography
Liquid chromatography using a polar stationary
phase and a nonpolar mobile phase
If R is a polar functional group, then the
stationary phase will be polar
Example: those fro which R contains a cyano (-
C2H4CN), diol (-C3H6OCH2CHOHCH2OH), or
amino (-C3H6NH2) functional group
Since the stationary phase is polar, the mobile
phase is a nonpolar or moderately polar solvent
The properties of a stationary phase

 Stationary Phase
 Silica gel: -Si-OH
 Cyano type: -Si-CH2CH2CH2CN
 Amino type: -Si-CH2CH2CH2NH2
 Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH

 Mobile Phase
 Basic solvents: Aliphatic hydrocarbons, aromatic
hydrocarbons, etc.
 Additional solvents: Alcohols, ethers, etc.
Relationship between Hydrogen Bonding and
Retention Time in Normal Phase Mode

SiOH HO
Strong
SiOH
Weak
Very weak
OH

Steric hindrance
The properties of a stationary phase

 Reverse-phase chromatography
 Liquid chromatography using a non polar stationary phase and a polar mobile
phase
 It the more commonly encountered form of HPLC
 The most common nonpolar stationary phases use an organochlorosilane for
which the R group such as n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chain
The properties of a stationary phase

 Stationary phase: Low polarity

 Octadecyl group-bonded silical gel (ODS)

 Mobile phase: High polarity

 Water, methanol, acetonitrile
 Salt is sometimes added.
Relationship Between Retention Time
and Polarity

C18 (ODS) OH

Weak
Strong
CH3
Comparison of Normal Phase and
Reversed Phase

 Normal Phase  Reversed Phase

 Effective for separation  Wide range of
of structural isomers applications
 Offers separation  Effective for separation
selectivity not available
of homologs
with reversed phase
 Stationary phase has
 Stabilizes slowly and is
prone to fluctuations in long service life
retention time  Stabilizes quickly
 Eluents are expensive  Eluents are inexpensive
and easy to use
Mobile phases

 The elution order of solutes in HPLC is governed by polarity.

 Normal-phase
 The least polar solute spends less time  first to elute from the colum
 Retention times are controlled by selecting the mobile phase, with a less polar
mobile phase leading to longer retention times
 Reverse-phase
 The order of elution is reversed
 The most polar solute being the first to elute
 Increasing the polarity of the mobile phase leads to longer retention times,
whereas shorter retention times required a mobile phase of lower polarity
Choosing a mobile phase
Degasser

Problems caused by dissolved air in the eluent

Unstable delivery by pump
More noise and large baseline drift in detector cell

In order to avoid these problems, the

eluent must be degassed.
Online Degasser

Regulator Vacuum chamber

Helium Polymeric film tube
cylinder

To pump
To pump
To draft

Drain valve

Eluent container Eluent container

Helium purge method Gas-liquid separation membrane method

Isocratic vs gradient elution

 Isocratic elution
 When a separation uses a single mobile phase of fixed composition
 It is often difficult, however to fins a single mobile-phase composition that is
suitable for all solutes
 Gradient elution
 Changing the composition of the mobile phase with time
 Example: for a RPLC, the intial mobile phase composition is relatively polar, as the
separation progress, the mobile phase’s composition is made less polar
Representative HPLC Detectors

UV-VIS absorbance detector

Photodiode array-type UV-VIS absorbance detector
Fluorescence detector
Refractive index detector
Evaporative light scattering detector
Electrical conductivity detector
Electrochemical detector
Mass spectrometer
UV-VIS Absorbance Detector

C: Concentration
Detection cell

Ein Eout

A
l

A = e·C·l = –log (Eout / Ein) C

(A: absorbance, E: absorption coefficient)
Advantages of High Performance
Liquid Chromatography

 High separation capacity, enabling the batch

analysis of multiple components
 Superior quantitative capability and
reproducibility
 Moderate analytical conditions
 Unlike GC, the sample does not need to be vaporized.
 Generally high sensitivity
 Low sample consumption
 Easy preparative separation and purification of
samples
Separation in HPLC
Separation Process and Chromatogram for
Column Chromatography

concentration

Chromatogram
Output

Time
 How can We Analyze the Sample?

Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose 5

2
3
Zorbax NH2 (4.6 x 250 mm) mAU
4
70/30 Acetonitrile/Water 1
6
1 mL/min Detect=Refractive Index

time
 Injector

Separation in based upon differential

Mixer migration between the stationary and
mobile phases.
Stationary Phase - the phase which
Pumps remains fixed in the column, e.g. C18,
Silica

Mobile Phase - carries the sample

Column
through the stationary phase as it
moves through the column.

Detector

Waste
Solvents

High Performance Liquid Chromatograph

 Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
 The Chromatogram

to - elution time of unretained peak

tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time
 Modes of High Performance Liquid
Chromatography
Types of Compounds Mode Stationary Mobile Phase
Phase

Neutrals Reversed C18, C8, C4 Water/Organic

Weak Acids Phase cyano, amino Modifiers
Weak Bases
Ionics, Bases, Acids Ion C-18, C-8 Water/Organic
Pair Ion-Pair Reagent

Compounds not Normal Silica, Amino, Organics

soluble in water Phase Cyano, Diol

Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer

Exchange Exchange Counter Ion
Resin

High Molecular Weight Size Polystyrene Gel Filtration-

Compounds Exclusion Silica Aqueous
Polymers Gel Permeation-
Organic
Qualitative Analysis

 Identification based on retention time

 Acquisition of spectra with detector
 UV spectra
 MS spectra
 Transfer to other analytical instruments after preparative separation
Quantitative Analysis

 Quantitation performed with peak area or height.

 Calibration curve created beforehand using a standard.
 Absolute calibration curve method
 Internal standard method
 Standard addition method
Calibration Curve for Absolute
Calibration Curve Method
Area
Concentration
A1 Calibration curve
C1
A4

A2

Peak area
A3
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
Concentration
Substances That Must Not Be Injected
into the Column
Insoluble substances (e.g., microscopic
particles and precipitation)
Substances that are precipitated in the
eluent
Substances that irreversibly adsorb to the
packing material
Substances that dissolve, or chemically react,
with the packing material
Filtration and Centrifugal Separation

 In general, filter every

sample before injection!
 It is convenient to use a
disposable filter with a
pore diameter of approx.
0.45 µm.
 Centrifugal separation is
applicable for samples
that are difficult to filter. Filter Syringe
HPLC Applications

Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates

tetracyclines
Pharmaceuticals corticosteroids
antidepressants
Consumer Products
barbiturates
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine