Tips & Tricks to Achieve

Fast, Sensitive, and

Reproducible Separation of
Amino Acids

Anne Blackwell, Ph.D.

Biocolumns Product Support Scientist
September 12, 2017

For Research Use Only. Not for use in diagnostic procedures.

1
Some Basics

Amino Acids are the building blocks of proteins

Amino Acids require derivatization to be detected by UV or FL

- OPA/FMOC, Ninhydrin, Dansyl chloride, and PITC are common reagents used

Derivatization can be done pre-column or post column

- OPA/FMOC, Dansyl chloride, and PITC are common reagents used for pre-column
- Ninhydrin is common for post column methods
Analysis of AA can be done by several methods:
- GC, CE, HPAE-PAD
- LC/UV/FL, LC/MS

For Research Use Only. Not for use in diagnostic procedures.

2
Why is Amino acid analysis important?

• Important for protein and peptide identification and quantitation

• Part of reverse-phase characterization in biopharma

• Required by the FDA

• Important for monitoring cell culture media

• Used for the analysis of metabolic intermediates - “Bound vs. Free”

For Research Use Only. Not for use in diagnostic procedures.

3
The Agilent Amino Acid Analysis solution
0.5µm

1.7µm 2.7µm

Ready to use
AdvanceBio AAA kit
(Standards and All Agilent LC AdvanceBio AAA
Reagents) systems including Columns
Infinity II systems Fast and rugged

For Research Use Only. Not for use in diagnostic procedures.

4
Agilent AdvanceBio AAA

Previous Agilent AAA Method What’s New?

Agilent has a well established solution for Amino Acid - All reagents conveniently kitted together under a
Analysis single part number
- based on automated pre-column derivatization - Introduced an HpH chemistry on a Poroshell
capabilities of Agilent Autosamplers particle for improved column lifetime
- Uses ZORBAX Eclipse AAA column - Traditional silica columns dissolve above neutral
- Well established method using reagents and pH, but HpH chemistry stabilizes column
standards from Agilent • AA derivatization and separation are most efficient
at higher pH
- Poroshell column with 2 µm frits is less
susceptible to clogging

For Research Use Only. Not for use in diagnostic procedures.

5
Pre- vs Post-Column Derivatization
Post Column Derivatization - The historic Gold Standard of dedicated Amino Acid Analyzers

Pump Autosampler Derivatization Detector

Cation exchange
Ninhydrin or UV/Vis
Fluorescamine or FLD
OPA

Pre Column Derivatization - Offline: Pre Column Derivatization - Online:

Autosampler
Pump Autosampler Detector Pump Detector
Reversed Phase Derivatization Reversed Phase

UV or FLD UV or FLD

Derivatization Derivatization done offline, either Derivatization done online, in the autosampler – eliminates error
manually or with separate automation, associated with manual sample handling for highly consistent,
and samples are transferred to reproducible results
autosampler

For Research Use Only. Not for use in diagnostic procedures.

6
AdvanceBio AAA Reagent Kit
Part Number Material
5061-3339 100mL Borate Buffer
5061-3337 FMOC reagent - 10 ampoules, 1 mL each
5061-3335 OPA reagent, 10 mg/mL, 6 ampoules
5062-2479 Dithiodiproprionic acid (DTDPA)
Order components
5061-3330 AA, standard 1nmol 10/PK individually, or together as
5061-3331 AA standards, 250 pmol 10/PK part of a kit with a single
part number (5190-9426)
5061-3332 AA standards, 100 pmol 10/PK
5061-3333 AA standards, 25 pmol 10/PK
5061-3334 AA standards, 10 pmol 10/PK
5062-2478 AA supplements, 1g each

For Research Use Only. Not for use in diagnostic procedures.

7
 Automated Derivatization in the Autosampler
Ortho Phthalaldehyde (OPA) 1. Allows visualization by UV or FL
O SR ’ 2. Helps retain very polar compounds
H R’SH
+RNH2 NR
H
Room Temperature
O
Fluorescence: Ex 340nm, Em 450nm
Non-fluorescent DAD: 338, 10nm; Ref. 390, 20nm
Does not absorb at 338nm

Fluorenylmethoxy
chloroformate RR’NH - HCl
(FMOC) + or
RNH2 Room Temperature NRR’
or
NHR

Fluorescence: Ex 260nm, Em 325nm

Fluorescent DAD: 262, 16nm; Ref. 324,8nm
Absorbs at 262nm and
Fluorescences at 324nm Optimal pH for reaction with AA: ~10.0
For Research Use Only. Not for use in diagnostic procedures.
8
 1290 Infinity II Multisampler
Online derivatization/Injection program

• Draw 2.5 μL from borate vial (Agilent p/n 5061-3339)

• Draw 1.0 μL from sample vial
• Mix 3.5 μL in wash port 5 times
• Wait 0.2 min Method can be programmed into ANY
• Draw 0.5 μL from OPA vial (Agilent p/n 5061-3335) Agilent autosampler –
• Mix 4.0 μL in wash port 10 times default speed
- Eliminates manual labor and variability
• Draw 0.4 μL from FMOC vial (Agilent p/n 5061-3337) - Enables highly precise data
• Mix 4.4 μL in wash port 10 times default speed
• Draw 32 μL from injection diluent vial
• Mix 20 μL in wash port 8 times
• Inject
• Wait 0.1 min
• Valve bypass
1260 Infinity II Vialsampler
For Research Use Only. Not for use in diagnostic procedures.
9
 1290 Infinity II Multisampler
Online derivatization/Injection program
OpenLab ChemStation C.01

Method can be programmed into ANY

Agilent autosampler –
- Eliminates manual labor and variability
- Enables highly precise data

1260 Infinity II Vialsampler

For Research Use Only. Not for use in diagnostic procedures.
10
Robust Columns for AAA
A robust, high efficiency Fast LC column with resistance to elevated pH and
temperature offering users performance comparable to that of sub-2 µm alternatives
but with up to 50% less back pressure.

Utilizes a proprietary
technology for particle synthesis

Core P120 Particle Treated P120

• 2.7 µm particles, 110 Å pore size

• Two dimensions available: 3.0 x 100 mm, 4.6 x 100 mm
• Guard columns also available in each i.d.
• Each individual column is tested for efficiency
• Each batch is tested with amino acid standards to ensure performance

For Research Use Only. Not for use in diagnostic procedures.

11
Chromatographic Method Time (min) %B
0 2
0.35 2
13.4 57
• Flow rate – 1.5 mL/min for 4.6 mm and 0.62 mL/min for 3
mm i.d. 13.5 100
15.7 100
• Injection volume – 1µL with needle wash at the wash port
for 7s 15.8 2
18 stop
• Column temperature – 40 ºC
• Detection wavelength – 338 and 262nm
• Samples- Agilent AAA standards, media samples and
protein hydrolysate standards

For Research Use Only. Not for use in diagnostic procedures.

12
Fast and Rugged Amino Acids Separation
DAD = 338 nm DAD = 262 nm

For Research Use Only. Not for use in diagnostic procedures.

13
Order of Elution for OPA and FMOC derivatives
Peak # AA Name AA Abbreviation Derivative Type
1 Asparic Acid ASP OPA
2 Glutamic Acid GLU OPA
3 Aspraragine ASN OPA
4 Serine SER OPA
5 Glutamine GLN OPA
6 Histidine HIS OPA
7 Glycine GLY OPA
8 Threonine THR OPA
9 Arginine ARG OPA
10 Alanine ALA OPA
11 Tyrosine TYR OPA
Primary AA
12 Cysteine CYS-CYS OPA
13 Valine VAL OPA
14 Methionine MET OPA
15 Norvaline* NVA OPA
16 Tryptophan TRP OPA
17 Phenylalanine PHE OPA
18 Isoleucine ILE OPA
19 Leucine LEU OPA
20 Lysine LYS OPA

21 Hydroxyproline HYP FMOC

22 Sacrosine (IS) SAR FMOC Secondary AA

23 Proline PRO FMOC

For Research Use Only. Not for use in diagnostic procedures.

14
 Elution Profile with and without Sodium Azide
*DAD1 A, Sig=338,10 Ref=390,20 (AAA FINAL\STD WITH NAN3\1BE-0201.D)
mAU *DAD1 A, Sig=338,10 Ref=390,20, TT (AAA FINAL\STD WITHOUT NAN3\1BG-0401.D)

Cystine
Aspartic acid

Lysine
Arginine
• Historically NaN3 has been

Glutamic acid

Serine
Asparagine
400

Glycine
Glutamine

Phenylalanine
Threonine

Methionine
added to aqueous mobile

Tyrosine
Alanine

Tryptophan
Histidine

Isoleucine
Valine

Leucine
phase to reduce bacterial

Norvaline
growth. 300

• NaN3 is highly toxic.

• No effect on the 200
separation.
• Highly recommend filtering
100
mobile phases (0.45 or 0.2
µm) to reduce bacterial
growth.
0
1 2 3 4 5 6 7 8 9 min

For Research Use Only. Not for use in diagnostic procedures.

15
 Amino Acids RT RSD (%) Area RSD (%)

Reproducible Separations 1. Aspartic acid

2. Glutamic acid
1.270
0.973
1.066
1.85
mAU 3. Asparagine 0.605 1.79
1 nmol amino acid standards
400 4. Serine 0.629 1.82
4.6 x 100 mm column
DAD1 A, Sig=338,10 Ref=390,20, 5. Glutamine 0.470 1.56
350
6. Histidine 0.430 1.22
DAD = 338 nm DAD = 262 nm
300 7. Glycine 0.477 1.92
Aspartic acid

8. Threonine 0.440 1.95

250

Lysine
9. Arginine 0.251 2.15

Arginine

Cystine
Asparagine

Glutamine

200 10. Alanine 0.280 3.06

Glycine
Serine

Phenylalanine
Threonine

Methionine
11. Tyrosine 0.128 1.65

Tyrosine

Hydroxyproline
Alanine

Tryptophan
Histidine
Glutamic acid

150

Valine

Isoleucine

Leucine
12. Cystine 0.067 1.9

Norvaline

Sarcosine
100 13. Valine 0.084 2.47

Proline
14. Methionine 0.073 1.82
50 15. Norvaline 0.073 1.72
16. Tryptophan 0.054 1.57
0
17. Phenylalanine 0.051 1.66
2 4 6 8 10 12 min
18. Isoleucine 0.047 1.72
19. Leucine 0.03 1.7
• Retention time %RSD mostly under 1% 20. Lysine 0.028 1.66
• Peak area %RSD mostly under 3% 21. Hydroxyproline 0.021 4.13

For Research Use Only. Not for use in diagnostic procedures.

22. Sarcosine 0.026 1.15
23. Proline 0.021 4.36 16
System suitability as per European Pharmacopoeia (Ph.Eur)
The European Pharmacacopoeia (Ph. Eur.) defines requirements for the qualitative
and quantitative composition of amino acids and mixtures of amino acids. The
requirements for allowed impurities are also defined. Manufacturers of amino acids
are legally bound to prove that their amino acids meet these specifications before
they can distribute their products in Europe.
Leucine (Leu) is a branched-chain α-amino acid and is produced by the fermentation
process. During this process, isoleucine can be produced as a by-product. The
European Pharmacopoeia states that leucine and isoleucine should have a
resolution of not less than 1.5

≥ 1.5

Isoleucine Leucine Ref: Ph.Eur.9.0 (2.2.56) Amino Acid Analysis

For Research Use Only. Not for use in diagnostic procedures.
17
Ample Resolution of Leucine & Isoleucine
Baseline resolution of isoleucine and leucine (Rs = 4.35) Competition
meeting the regulatory requirements for these components. data Protein hydrolysate
sample
mAU
DAD1 A, Sig=338,10 Ref=390,20,
500
Aspartic acid

Rs = 4.35
400 Rs = 2.0
Arginine
Glutamic acid

Lysine
Serine

300

Isoleucine
Glycine

Leucine
Alanine

Methionine
Histidine

Tryosine

Phenylalanine
Cysteine
Threonine

Valine
200
Column Leu/Ile Rs

. Proline
( ≥ 1.5)
100 Agilent AdvanceBio AAA
4.5
4.6 × 100 mm

0
Agilent AdvanceBio AAA
4.6
2 4 6 8 10 12 min 3 × 100 mm

For Research Use Only. Not for use in diagnostic procedures.

18
Linearity and Limits of Detection & Quantitation
mAU mAU
mAU
60 70
100 Asparagine Glutamine Tryptophan
60
50
80
50
40
60 40
30
30
Asparagine 40
20
20
20 10 10
mAU 0
0 0
2.4 2.6 2.8 min 3.2 3.4 3.6 min 8.2 8.4 8.6 min
100

Tryptophan
80
Glutamine

60

40

20

0
4 6 8 min
For Research Use Only. Not for use in diagnostic procedures.
19
 Linearity and Limits of Detection & Quantitation
Linearity for Aspargine Linearity for Glutamine
2500 1200
y = 1.0107x + 0.3716
R² = 0.9997
Concentration (pmol) S/N ratio
y = 2.0654x + 2.6479
1000
2000 R² = 0.9993

800
Asparagine
1500

0.9 (LOD) 5.3

Area

Area
600

1000
400

500
1.9 (LOQ) 10.8
200

0 0
Glutamine
0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200
Concentration (pmol) Concentration (pmol)
0.9 (LOD) 3.0
Linearity for Tryptophan
3.8 (LOQ) 13.8
1800 y = 1.6446x + 6.0767

1600
R² = 0.9999 Tryptophan
1400

1200
0.9 (LOD) 4.5
1000
3.8 (LOQ) 20.5
Area

800

600

400

200 S/N >3.0 = LOD

0
0 200 400 600 800 1000 1200 S/N > 10 = LOQ
Concentration (pmol)

For Research Use Only. Not for use in diagnostic procedures.

20
AAA of Cell Culture Media – MEM
L-Arginine, L-Cystine, L-Glutamine, L-Histidine, L-Isoleucine, L- Leucine, L-Lysine, L-Methionine, L- Phenylalanine, L-
Threonine, L-Tryptophan, L- Tyrosine and L-Valine, L-Glutamic acid

mAU
MEM Eagle , Sig=338,10 Ref=390,20,

Lysine
150

Threonine
100

Isoleucine

Leucine
Valine

Phenylalanine
Tyrosine
Glutamic acid

Histidine

Methionine
50

Cystine
Glutamine

Arginine

Alanine
0
Glutamine

50
Glutamic acid

Phenylalanine
Isoleucine
Methionine

Leucine
Histidine

Valine
Tyrosine
Threonine

Alanine
Glycine
Serine

Arginine
Asparagine

Cystine
100

Norvaline

Lysine
250pmol AA std , Sig=338,10 Ref=390,20,

2 3 4 5 6 7 8 9 min

For Research Use Only. Not for use in diagnostic procedures.

21
AAA of Cell Culture Media – NEAA cell culture supplement

L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic acid, Glycine, L-Proline and L-Serine

Aspartic acid
mAU NEAA cell culture supplement , Sig=338,10 Ref=390,20,

Asparagine
Glutamic acid

Glycine

Alanine
60

Serine

Proline
40

20

Glutamine

Proline
20
Glutamic acid

Hydroxyproline
40

Phenylalanine
Isoleucine

Leucine
Histidine

Valine
Methionine
Tyrosine
Threonine

Alanine
Glycine
Serine
Aspartic acid

Asparagine

Arginine

60

Sarcosine
Cystine

Norvaline

Lysine
80 250pmol AA std , Sig=338,10 Ref=390,20,

2 4 6 8 10 12 min

For Research Use Only. Not for use in diagnostic procedures.

22
AAA of Cell Culture Media – RPMI 1640
L-Arginine, L-Glutamic acid, L-Asparagine, L-Cystine, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine,
L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine and L-Valine

mAU
RPMI 1640 Media , Sig=338,10 Ref=390,20,
200

150
Aspartic acid

100 Glutamic acid

Serine

Hydroxyproline
Isoleucine

Lysine
Leucine
Cystine
Asparagine
50

Glycine

Phenylalanine
Threonine

Methionine
Tyrosine

Tryptophan
Valine
Histidine

Proline
Arginine
0
Glutamine

Proline
Hydroxyproline
50

Isoleucine
Tryptophan
Phenylalanine
Glutamic acid

Histidine

Leucine
Methionine
Valine
Tyrosine
Threonine

Alanine
Glycine

Sarcosine
Serine

Arginine
Asparagine

100

Cystine

Lysine
Norvaline
Aspartic acid

250pmol AA std , Sig=338,10 Ref=390,20,

150
2 4 6 8 10 12 min

For Research Use Only. Not for use in diagnostic procedures.

23
Poroshell HPH C18 last longer in phosphate buffer

110
100
90
Efficiency

80
% Initial

Non-HPH silica column

70
Poroshell HPH-C18
60
50
0 1000 2000 3000
mL Stress Buffer

Lifetime of SPP columns in phosphate buffer, pH 8, at elevated temperature. Mobile phase:

Premixed 60% 30 mM sodium phosphate buffer at pH 8 and 40% acetonitrile; Flow rate 0.4
mL/min; UV absorbance 254 nm; 65 °C; Columns: 2.1 x 50 mm, 2.7 µm; Analyte: Naphthalene.
For Research Use Only. Not for use in diagnostic procedures.
24
Traditional Lifetime Testing
Using conventional column testing Eclipse Plus C18 column test looks great and will perform
well for most customers. However it is not a realistic test for most lab workflow.

For Research Use Only. Not for use in diagnostic procedures.

25
Improvements in Lifetime Testing for AA Analysis

Past lifetime testing consisted of 500 injections without stopping

• This test, while it could differentiate long life columns, was not indicative of a customer workflow

Customers will typically run ~20 to 50 or 100 samples, then stop, and resume a day
or more later
• This is much more stressful on the column, and will cause columns to fail sooner versus running 500
injections straight through.
The lifetime testing for the Poroshell HPH-C18 was adapted to more customer
focused workflow, with breaks between series of injections to give a true indicator of
the lifetime ni the customers’ hands

For Research Use Only. Not for use in diagnostic procedures.

26
Test like a customer runs: 100 injections (3 days), Store (4 days)
Most labs run a batch of samples (25-100) and then shut off for a few days so we did too
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-05 10-21-37LFS-RDWS022\WJL00010000025.D)
Injection 25

3.442

6.452

8.051

9.671
4.107
4.366

5.123

5.486

7.884
mAU

10.104
9.166
9.008
4.532
1.637
80

1.076

7.603
60

7.502

11.153
Stop @

4.793
40
20 100 and 200
0
0 2 4 6 8 10 12 min
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-15 16-13-54LFS-RDWS022\WJL00010000225.D)

8.011

9.622
5.105

5.448

7.842
mAU

10.070
9.116
4.342

6.417
Injection 225

3.421

8.965
4.091
1.656

4.503
80

7.568
1.081

60

11.120
7.472
40 Stops @

4.763
20
0
300 and 400
0 2 4 6 8 10 12 min
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-18 14-02-17LFS-RDWS022\WJL00010000400.D)
Injection 400

5.079

5.415

7.976

9.586
mAU

4.319

10.041
7.807

9.084
6.388
3.403

4.073

8.932
4.481
80
1.589

7.539
1.061

60

11.083
7.441
40

4.742
20
0
0 2 4 6 8 10 12 min
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-25 12-56-57LFS-RDWS022\WJL00010000500.D)

Injection 500
4.290

5.060

5.385

6.364

7.771
7.942

9.039
3.381

9.533

9.971
mAU

8.887
1.546

4.451
1.070

4.049

7.509
80
60

11.023
7.420
Stop @
4.715

40
20 525
0
0 2 4 6 8 10 12 min

Looks great after 500 injections run 100 injections shut down 4 days repeat
For Research Use Only. Not for use in diagnostic procedures.
27
Amino Acid Analysis in Fermentation Applications

DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000008.D)

mAU
175 Soy Sauce diluted 1/100

3.400
150

1.123

6.065
125

5.846
2.225

4.792
3.192
100

2.775

5.538
2.863

5.662
75

0.705

2.579
50

4.978

5.224
4.062
0.484

2.993
2.920

5.759
1.893
1.275

6.665
25
0
1 2 3 4 5 6 7 min
DAD1 B, Sig=262,16 Ref=324,8, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000008.D)
0.486
0.531
0.673
0.693

mAU
0.598

30
Many other foods (such as soy
0.368
0.561
0.547
0.433

1.123
1.186

20 sauce) and pharmaceuticals that

10 are produced using fermentation
0
processes are monitored by AAA
1 2 3 4 5 6 7 min

For Research Use Only. Not for use in diagnostic procedures.

28
Amino Acids Analysis for Batch Comparison
DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000002.D)
mAU
200 Kronenburg Blanc

3.516
150

3.399
2.768

3.185

4.874
100

0.688

5.198
4.051

5.744
2.577

5.847
5.656
4.793
1.104

2.228

5.335
5.474
2.170
50
0
-50
0 1 2 3 4 5 6 7 min
DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000004.D)
mAU

3.386
200 Old Dominion Cherry Blossom Lager

3.524
2.084
150

4.791
2.775

3.192

4.054
100

5.660
5.548

5.860
2.582
1.126

4.884
2.907
0.692

5.218
50

5.743
2.237
0
-50
0 1 2 3 4 5 6 7 min
DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000006.D)
mAU Blue Moon Belgian Style Wheat

3.379
3.515

4.782
2.766
200

3.187

5.648
4.047

5.846
5.535
150
2.085
1.124

6.071
2.578
0.694

4.881
2.233

100

5.218

5.747
4.978
50
0
-50
0 1 2 3 4 5 6 7 min

Quantity and diversity of amino acids is evident, can be used to monitor reactions and compare batches

For Research Use Only. Not for use in diagnostic procedures.

29
Tips & Tricks - Maintenance

• Replace derivatization reagent, borate buffer, amino acid standard daily

• Recalibrate for retention times and response factors daily
• Check column and guard column performance by following specs (Rs for 2 pairs
of AA)
• Replace mobile phase A and B with fresh ones every other day
• Exchange guard column if high back pressure develops
• Avoid using MAX mixing speed during sample derivatization
• The max speed on newer LCs is much faster than older LCs (1100s, 1200s), and can cause
excessive wear on the autosampler.

For Research Use Only. Not for use in diagnostic procedures.

30
Tips & Tricks - Troubleshooting
Poor chromatographic resolution?
• Cell culture media does not require any sample preparation, however appropriate dilutions have to be made to suit
detector response
• In all cases, use the low-volume heat exchanger with short red tubing to minimize extra column volume
• Ensure proper connections
• Damaged guard or analytical column

Low intensity chromatogram?

• OPA/FMOC reagent deteriorated
• Air bubble in vial insert

Column storage?
• Never leave the column in mobile phase A even if it’s just overnight
• For short term always store the column in mobile phase B
• For long term, store column in 50/50 acetonitrile/H2O
For Research Use Only. Not for use in diagnostic procedures.
31
Damaged Column
DAD1 A, Sig=338,10 Ref=390,20, TT (AAA FINAL\AAA 46 X 100 COLUMN DAMAGE 2 (2)\1DF-0101.D)
mAU

350

After three days in mobile phase A

300

250

200

150

100

50

2 4 6 8 10 12 14 min

For Research Use Only. Not for use in diagnostic procedures.

32
 Tips & Tricks – Saving Time
• After Injection 1, during the sample derivatization for Injection 2, the initial mobile phase condition is
flowing through the LC and the column
• Save time by shortening the length of the re-equilibration time at the end of the method by the
amount of time consumed by sample derivatization
23 minutes
High Organic Wash Time (min) %B
0 2
0.35 2
13.4 57

Separation 13.5 100

15.7 100
Initial mobile phase Sample 15.8 2
conditions are Derivatization Re-equilibration
running through the 18 stop
column!
For Research Use Only. Not for use in diagnostic procedures.
33
Tips & Tricks – Saving Time
• After Injection 1, during the sample derivatization for Injection 2, the initial mobile phase condition is
flowing through the LC and the column
• Save time by shortening the length of the re-equilibration time at the end of the method by the
amount of time consumed by sample derivatization

23 + 23 = 46 minutes for 2 samples Time (min) %B

0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stop

For Research Use Only. Not for use in diagnostic procedures.

34
Tips & Tricks – Saving Time
• After Injection 1, during the sample derivatization for Injection 2, the initial mobile phase condition is
flowing through the LC and the column
• Save time by shortening the length of the re-equilibration time at the end of the method by the
amount of time consumed by sample derivatization

20.8 + 20.8 = 41.6 minutes for 2 samples Time (min) %B

0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stop

For Research Use Only. Not for use in diagnostic procedures.

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How-To Guide – Step by Step Instructions & Method Details

All the information you need

Document number 5991-7694EN

For Research Use Only. Not for use in diagnostic procedures.

36
Summary

• We used the Agilent AdvanceBio AAA solution for the automated online
derivatization and separation of amino acids.
• Area and RT precision of the method were excellent, and Leu/Ile resolution met
the system suitability requirement.
• Linearity curves with ten standard concentrations of three amino acids, ranging
from 0.9pmol to 1nmol, had excellent coefficient of linearity values, indicating that
the method was quantitative and accurate.
• The LOD and LOQ for the amino acids were 0.9pmol and 3.8 pmol respectively,
indicating that the method was sensitive.
• This method was able to separate and detect, amino acids from a variety of
samples, including cell culture media, protein hydrolysate, and fermentation
reactions.
For Research Use Only. Not for use in diagnostic procedures.
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