Agilent Biocolumns

Amino Acid Analysis
Application Compendium

Contents

1

Background2

Getting Started3

How to Guide - Amino Acid Analysis Accurate Results with
AdvanceBio End-to-End Solution - 5991-7694EN

4

Featured Application Notes

23

Determination of Amino Acid Composition of Cell Culture Mediaand Protein Hydrosylate Standard - 5991-7922EN

23

Automated Amino Acid Analysis Using an Agilent Poroshell
HPH-C18 Column - 5991-5571EN

33

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Amino Acid
Analysis
Background
Determining the amino acid composition of a protein is a well-established technique that isused with other techniques to confirm the correct structure. Acid hydrolysis is typically used tohydrolyze the protein into its constituent amino acids before analysis. Amino acids are also key
ingredients in the cell culture medium used to prepare recombinant proteins. It is often desirable
to monitor the consumption of individual amino acids during the fermentation reaction and so
the same chromatographic approach may be used.
Amino acids are inherently zwitterionic and possess a variety of side chains, including neutral,hydrophobic, hydrophilic, acid, and basic groups. They also lack a suitable UV chromophore,
making the separation and detection of the twenty or so naturally occurring amino acids
challenging. Agilent introduced a unique method of amino acid analysis combining precolumn
derivatization, employing the liquid handling capabilities of the multisampler, together withreversed-phase separation to baseline resolution of all the common amino acids.
To perform precolumn derivatization the sample is first neutralized in borate buffer at high pH
to ensure that the amino terminus of each amino acid is neutralized. Primary amines are thenreacted with ortho-phthaldehyde (OPA) and secondary amines (proline, hydroxyproline etc.) arereacted with 9-fluorenylmethylchloroformate (FMOC-Cl). This enables the subsequent separation
by reversed-phase to be performed, but the high pH conditions required to obtain baseline
resolution means that the latest pH stabilized columns provide the longest column lifetime.

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2

R

H
N
H

C
H

O
C
O

H

Getting Started

Amino Acidand Cell Culture
Analysis

Small molecule chromatography (<150 Å)
Delivers robust, high-resolution separations

AdvanceBio Amino Acid Analysis (AAA)LC/UV or LC/FLD with sample derivatization
Attribute

Advantage

Exceptional resolution

More reliable results

High pH–resistant C18
stationary phase

Longer column lifetimes

HPLC and UHPLC
compatible

Increased flexibility

The AdvanceBio Amino Acid Analysis kit combines all
the reagents and calibration standards necessary forthe analysis into a single part number, 5190-9426. Eachcomponent can also be ordered separately if needed. The'How-To" Guide on the following pages contains detailed
instructions for mobile phase preparation, setting up
the automated, online amino acid derivatization in theautosampler, and the LC method.
The AdvanceBio Amino Acid Analysis column is packed
with C18 silica particles that have been chemicallymodified for high pH stability. Amino acid separations
are most efficient at high pH, and this improvement
over previous amino acid analysis solutions significantly
extends the lifetime of the column under these mobile
phase conditions. To maximize the benefit of thisadvancement, take care to never store the column in
mobile phase A. Short term storage should be in mobile
phase B, while long-term storage should be in 50 %acetonitrile. The column is meant to withstand short-term
exposure to basic pH during a gradient, but prolonged
exposure to high pH will still lead to shortened column
lifetimes.

AdvanceBio MS Spent Media

LC/MS without sample derivatization

3

Attribute

Advantage

HILIC LC separation/
MS detection

One method for multiple
metabolite classes

No sample
derivatization needed

Use any
LC/MS system

PEEK-lined stainlesssteel column hardware

Excellent peak shapeand recovery

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Amino Acid Analysis:“How-To” Guide
Accurate results with AdvanceBio end-to-end solution
The Agilent AdvanceBio Amino Acid Analysis (AAA) end-to-end solution optimizes workflow
efficiency by combining the advantages of the Agilent InfinityLab LC Series instrumentation andcolumn technology with proven precolumn derivatization chemistry. It is part of the AdvanceBio
family that delivers consistent, exceptional performance for the complete characterization ofproteins, antibodies, conjugates, new biological entities, and biopharmaceuticals.
This complete, single vendor solution (including
chemicals/standards, columns, and application support)
provides fast, sensitive, and automated amino acidanalysis. It is based on the latest InfinityLab LC Series
instrument and column technology. The automatedonline derivatization in the Agilent 1290/1260 Infinity II
vialsampler eliminates tedious manual procedures anddelivers reproducible reaction results. AdvanceBio AAA
columns provide the speed and resolution of sub-2 μm
columns, but with 50 % less backpressure and reduced
risk of column clogging.

The AdvanceBio AAA solution has evolved from proven
Agilent ortho-phthalaldehyde/ 9-fluorenyl-methyl
chloroformate (OPA/FMOC) reagents for amino acidderivatization. Together with AdvanceBio AAA columns
and standards, these reagents provide an ideal,
quantitative and qualitative amino acid analysis thatcombines speed and sensitivity. When used according to
the protocol described in this document, the AdvanceBio
AAA solution enables the user to separate the amino acids
commonly found in protein/peptide hydrolysates.
.

Separation of AA standard
DAD1 A, Sig=338,10 Ref=390,20, 1nm AAA standard

Sarcosine

ProlineLysineLeucineHydroxyproline

Tryptophan
Phenylalanine
Isoleucine

CystineArginine

ValineMethionine
Norvaline

100

Glutamic acid

200
150

Tyrosine

Asparagine
Serine250

Alanine300

Glutamine
Histidine
GlycineThreonine350

Aspartic acid

mAU

50
0
2

Expert applications
and technicalsupportAdvanceBio AAA
reagents andstandards
Ready to use

Agilent analytical
LC systems
Efficient LC
solutions

AdvanceBio AAA columns
Fast, rugged amino acidseparation

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4

6

8

10

min

Fast, reliable data

4

AdvanceBio AAA columns:
Superficially porous particle (SPP) technology
AdvanceBio AAA columns are based on Agilent’s
innovative 2.7 μm superficially porous particle (SPP)
Poroshell technology—particles consist of a 1.7 μm solidcore with a 0.5 μm porous shell.
The 2.7 μm SPPs provide 40-50 % lower backpressure
with 80-90 % of the efficiency of sub-2 μm totally
porous particles. The SPPs have a narrower particle size
distribution than totally porous particles, which results in amore homogeneous column bed, and reduces dispersion
in the column. At the same time, the thin porous shellprovides lower resistance to mass transfer. What’s more,since the columns incorporate a 2 μm frit, they are as
resistant to clogging as 3.5 and 5 μm columns.
Until recently, all silica-based SPP materials possessed
limited lifetime in higher pH buffers, including phosphatebuffers. To achieve longer lifetimes, it is necessary toprotect the base particle by either surface modification or
special bonding modification. The surface is chemicallymodified using a proprietary process to form an organic
layer that is resistant to silica dissolution at high pH
conditions.

5

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AdvanceBio AAA columns:
Superficially porous particle (SPP) technology

Agilent detectors – flexibility in detection

The AdvanceBio AAA columns ensure excellent selectivity
for amino acid analysis.

Uncompromised sensitivity for simultaneous
multiwavelength detection.

Fast and rugged amino acid separation

Diode Array Detector with spectral data:–– The speed and resolution of a sub-2 μm column withup to 50 % less backpressure

Identification and peak purity analysis with more selectivity
and fewer matrix effects.

Multiple Wavelength Detector:–– More forgiving for dirty samples, due to 2 μm frits

Fluorescence Detector:–– Unique chemical modification for high pH stability andcolumn lifetime

Superior sensitivity in the multi-signal mode in the
femto-mole range.

–– Guard column options reduce your operating costs byextending the life of the columns

Everyday efficiency with confidence–– Higher speed and higher resolution thanks to theoperating power up to 600 bar and 5 mL/min–– Injector programming for automated online
derivatization–– High-sensitivity UV detection based on diode array
technology for uncompromised sensitivity for
simultaneous multiwavelength detection–– Optional full spectral detection for identification andpeak purity analysis–– Wide flexibility for other LC or UHPLC applications with
100 % HPLC compatibility

2

4

6

10

ProlineSarcosine

Hydroxyproline

LysineLeucineTryptophan

8

Phenylalanine
Isoleucine

Norvaline

CystineValineMethionine

Tyrosine

AlanineArginine

GlycineThreonine

Glutamine
Histidine

Asparagine
SerineGlutamic acid

mAU
80
60
40
20
0

Aspartic acid4.6 mm id

12min

3.0 mm id
mAU
120
100
80
60
40
20

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6

Advance your confidence:
Agilent AdvanceBio Amino Acid Analysis (AAA)
Achieve fast, sensitive, and reproducible separation ofamino acids in biological samples

Steps for AAA analysis
1. Prepare HPLC mobile phases
2. Prepare amino acid standards
3. Prepare Internal Standard (ISTD) stock solution
4. Perform online derivatization
5. Set parameters for detection
6. Run high throughput routine analysis
7. Ensure system suitability per European
Pharmacacopoeia (Ph. Eur.)
8. Optimize cell culture media and protein hydrolysate
standard
Learn more about analyzing amino acids with utmost
confidence, visit

www.agilent.com/chem/advancebioaaa

7

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Step 1:

Injection diluent
The injection diluent is 100 mL of mobile phase A and
0.4 mL concentrated H3PO4. This solution is prepared in a
100 mL bottle that should be stored at 4 °C.

Prepare HPLC mobile phases

0.1 N HCI

Mobile phase A:
10 mM Na2HPO4, and 10 mM Na2B4O7 pH 8.2
To prepare 1 L, weigh out 1.4 g anhydrous Na2HPO4 and3.8 g Na2B4O7•10H2O in 1 L water. Adjust to approximately
pH 8.4 with 1.2 mL concentrated HCl, then add small
drops of acid and adjust to a final pH of 8.2. Allow stirring
time for complete dissolution of borate crystals before
adjusting pH. Filter through 0.45 μm regenerated cellulose
membranes (p/n3150-0576).

Mobile phase B:
Acetonitrile:methanol:water (45:45:10, v:v:v)
All mobile-phase solvents are HPLC grade.
Mobile phase A is consumed at a faster rate than mobile
phase B. Therefore, we recommend preparing 2 L ofmobile phase A for every 1 L of mobile phase B.

Extended amino acid and internal standard stock solutions
are prepared in 0.1 N HCl solution. To prepare 0.1 N
HCl, add 4.2 mL concentrated HCl (36%) to a 500 mL
volumetric flask that is partially filled with water. Mix, andfill to the mark with water. Store at 4 °C.

Derivatization reagents
Derivatization reagents (borate buffers, OPA, and FMOC)
are ready-made solutions supplied by Agilent. Simply
transfer these reagents from their container into an
autosampler vial. Recommended precautions include:–– OPA is shipped in ampoules under inert gas to preventoxidation. Once opened, the OPA is potent for about
7 to 10 days. We recommend transferring 100 μL
aliquots of OPA to microvial inserts and storing in arefrigerator. Replace the OPA autosampler microvial
daily.
–– FMOC is stable in dry air but deteriorates in moisture.
It should also be transferred to microvial inserts in
100 μL aliquots, and stored in a refrigerator. Like theOPA, an open FMOC ampoule transferred to
10 microvial inserts is potent for about 7 to 10 days.
–– Borate buffer can be transferred to a 1.5 mL
autosampler vial without a vial insert. Replace every
three days.

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8

Step 2:

Step 3:

Prepare amino acid standards

Prepare Internal Standard (ISTD) stock solution

Solutions of 17 amino acids (AA) in five concentrations
are available from Agilent (10 pmol/μL to 1 nmol/μL) for
calibration curves. Store solutions at 4 °C.

For primary amino acid ISTD stock solutions, weigh58.58 mg norvaline into a 50 mL volumetric flask. For
secondary amino acids, weigh 44.54 mg sarcosine into
the same 50 mL flask. Fill halfway with 0.1 N HCl andshake or sonicate until dissolved. Finally fill to mark withwater for a final concentration of 10 nmol for each aminoacid/μL (standard sensitivity). For high-sensitivity ISTD
stock solution, take 5 mL of standard-sensitivity solution
and dilute with 45 mL of water. The final concentration of
the high-sensitivity ISTD is 1 nmol for each amino acid/µL.
Store at 4 °C.

To make the extended amino acid (EAA) stock solution,weigh:–– 59.45 mg asparagine–– 59.00 mg hydroxyproline–– 65.77 mg glutamine–– 91.95 mg tryptophan
Add the weighed out amino acids to a 25 mL volumetricflask, fill halfway with 0.1 N HCL and shake or sonicate
until dissolved. Then fill to mark with water for a total
concentration of 18 nmol/μL of each amino acid.
For the high-sensitivity EAA stock solution, take 5 mLof this standard-sensitivity solution and dilute with
45 mL water (1.8 nmol/μL). Solutions containing extended
standards are unstable at room temperature. Keep themfrozen and discard at first signs of reduced intensity.

Calibration curves may be made using two to fivestandards depending on experimental need. Typically
100 pmol/μL, 250 pmol/μL, and 1 nmol/μL are used ina three-point calibration curve for "standard sensitivity"
analysis.
The following tables should be followed if an internal
standard or other amino acid (for example, the extended
amino acids) is added. Table 1 describes "standard
sensitivity" concentrations typically used in UV analysis.
Table 2 is typically used for "high sensitivity" fluorescenceanalysis.
Table 1. Standard sensitivity calibration standards
Concentration of Final AA Solution(pmol/µL)
900

90

Take 5 mL of 18 nmol EAA
Dilute with water

5 mL
-

5 mL
15 mL

5 mL
45 mL

Diluted EAA mix

5 mL

20 mL

50 mL

Take 5 mL of diluted EAA mix

5 mL

5 mL

5 mL

Add 10 nmol ISTD solution

5 mL

5 mL

5 mL

EAA-ISTD mix

10 mL

10 mL

10 mL

Take 100 µL of EAA-ISTD mix

100 µL100 µL100 µLFor 1 nmol AA, add

900 µL--For 250 pmol AA, add

9225

900 µL-For 100 pmol AA, add--

900 µL

Final AA solution with EAA
and 500 pmol/µL ISTD

1 mL

1 mL

1 mL

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Table 2. High sensitivity calibration standards
Concentration of Final AA Solution(pmol/µL)

Step 4:

90

22.5

9

Take 5 mL of 1.8 nmol EAA
Dilute with water

5 mL
-

5 mL
15 mL

5 mL
45 mL

Diluted EAA mix

5 mL

20 mL

50 mL

Take 5 mL of diluted EAA mix

5 mL

5 mL

5 mL

Add 1 nmol ISTD solution

5 mL

5 mL

5 mL

High-sensitivity EAA-ISTD mix

10 mL

10 mL

10 mL

Take 100 µL of EAA-ISTD mix

100 µL100 µL100 µLFor 100 nmol AA, add

900 µL--

900 µL-1. Draw 2.5 μL from borate vial (p/n5061-3339)

For 25 pmol AA, add

Perform online derivatization
Depending on the autosampler model, the automatedonline derivatization program differs slightly. For the
Agilent G7129A well plate automatic liquid sampler with
100 μL capillary* (WPALS), with injection program is asfollows:

For 10 pmol AA, add--

900 µL2. Draw 1.0 μL from sample vial

Final AA solution with EAA
and 50 pmol/µL ISTD

1 mL

1 mL

1 mL

3. Mix 3.5 μL in wash port five times
4. Wait 0.2 minutes
5. Draw 0.5 μL from OPA vial (p/n 5061-3335)
6. Mix 4.0 μL in wash port 10 times default speed
7. Draw 0.4 μL from FMOC vial (p/n 5061-3337)
8. Mix 4.4 μL in wash port 10 times default speed
9. Draw 32 μL from injection diluent vial
10. Mix 20 μL in wash port eight times
11. Inject
12. Wait 0.1 minutes
13. Valve bypass* Note: other autosampler models may have a different volume capillary
installed, which will require adjustment of volumes

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10

The location of the derivatization reagents and samples is
up to the analyst and the ALS tray configuration. Using theG7129A with a 2 × 56 well plate tray (p/n G2258-44502),
the locations are:A
B–– Vial 1: Borate buffer–– Vial 2: OPA–– Vial 3: FMOC–– Vial 4: Injection diluent
C–– P1-A-1: Sample
Note: Use the correct vials, closures, and pumps parameters

Conical vial inserts with polymer feet (Figure 1A) are
required to hold the OPA and FMOC reagents because of
the limited volumes involved. The inserts are compatible
with wideopening screw-top (Figures 1B and 1C) or crimptop vials. For this procedure, an airtight seal is needed forboth FMOC, which is highly volatile, and OPA, as it slowly
degrades in the presence of oxygen. Snap-cap vials should
therefore not be used in this procedure. Be careful not
to use vials or caps designed for other instruments, toprevent damage to the auto injector.

Figure 1. Insert, vial, and cap for amino acid analysis using the Agilent
4226A autosampler: A) Conical insert (Agilent p/n 5181-1270),
B) amber wide opening vial (Agilent p/n 5182-0716), and C) screw cap(Agilent p/n 5182-0721).

Pump parameters for all methods include compressibility(×10-6 bar) A: 40, B: 80, with minimal stroke A, B of 20 μL.

Increase precision with Autosampler automation
Ortho-phthaladehyde (OPA) for primary AA
O
H
H+RNH2+R’SH
Room temperature

SR’
NR

Automated reagent additionO

9-fluorenylmethyl chloroformate (FMOC) for secondary AA
RR’NH
+ or
RNH2
O
O

Increase precision
Eliminates manual processes–HCI
Room temperature
O

CI

O

NRR’
or
NHR

Figure 2. Online derivatization of OPA and FMOC: Separation of polaramino acids on RP-phase and detection by UV and Fluorescence

11

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Step 5:Set parameters for detection
Thermostatted column compartment (TCC)
Left and right temperatures should be set to 40 °C. Enable
analysis when the temperature is within ± 0.8 °C.

Diode array detector (DAD)
Signal A: 338 nm

10 nm
bandwidth

Reference
wavelength
390 nm20 nm
bandwidth

Signal B: 262 nm

16 nm bandwidth

Reference
wavelength
324 nm8 nm
bandwidth

Signal C*: 338 nmix

10 nm bandwidth

Reference
wavelength
390 nm20 nm
bandwidth*Signal C is not required if the instructions below are followed.

To detect both OPA and FMOC derivitized amino acids ina single chromatogram it is necessary to switch detector
wavelength between the last eluting OPA derivitized aminoacid, lysine (peak 20 in the standard), and before the firsteluting FMOC derivitized amino acid, hydroxyproline (peak21 in the standard).
Determining the appropriate transition point using the
DAD is possible by initially collecting two channels (Signal
A 338 nm, to detect OPA derivitized amino acids andSignal B 262 nm, to detect FMOC derivitized amino acids).
This will determine the ideal point at which to switch thewavelength during the run. Subsequent runs can be madeusing a single channel with the detector timetable functionused to program a wavelength switch from 338 to 262 nm
at the appropriate time. Between the elution of OPA-lysine
and FMOC-hydroxyproline, allow time for both OPA andFMOC derivitized amino acids to be detected in a single
chromatogram.
Peak width settings of > 0.01 minutes are used for allcolumns.

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12

Fluorescence detection
FLD should always be the last detector module in the flow
stream to avoid damage to the pressure sensitive flow cell(max 20 bar).
Peak width 0.01 min, stop time 18 min (adjust as needed)
Excitation 340 nm; Emission 450 nm; Filter 390 nm(Default filter)

Timetable Signal:

Gradient program

0.00 min Excitation 340 nm, Emission 450 nm; Gain(as needed)

Time (min)

%B

0

2

5.53 min Excitation 260 nm, Emission 325 nm;

0.35

2

PMT Gain 10 (as needed; transition between lysine andhydroxyproline)

13.4

57

13.5

100

15.7

100

15.8

2

18

end

To determine the transition point needed withfluorescence detection (FLD), it is necessary to perform
two separate runs: the first using Excitation 340 nm,
Emission 450 nm to detect the OPA derivitized aminoacids and the second using Excitation 260 nm, Emission
325 nm to detect the FMOC derivitized amino acids. BothOPA and FMOC derivitized amino acids can be detected ina single chromatogram, using the detector timetablefunction. This function programs a wavelength switch at
the appropriate point after the last eluting OPA derivitizedamino acid, lysine (peak 20 in the standard) and before thefirst eluting FMOC derivitized amino acid, hydroxyproline(peak 21 in the standard).

Flow rate: 1
 .5 mL/min for 4.6 mm id columns and0.62 mL/min for 3 mm id columns.
Injection
volume:	1 µL with needle wash at the port for 7 s.

Note: If the concentration of amino acids is below 100 pmol, we
recommend the use of fluorescence detection.

13

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Typical Separations
A separation of 20 amino acids using an AdvanceBio AAA
column is shown in Figure 3.
The following parameters are noted:–– No change in elution profile of amino acids with andwithout NaN3 in mobile phase.
–– NaN3 is used only as a preservative to containbacterial/fungal growth.
–– Filtering the mobile phase using 0.45 μm filter is highly
recommended. Note: If the concentration of aminoacids is below 100 pmol, we recommend the use offluorescence detection.

*DAD1 A, Sig=338,10 Ref=390,20 (AAA FINAL\STD WITH NAN3\1B E-0201.D)*DAD1 A, Sig=338,10 Ref=390,20, TT (AAA FINAL\STD WITHOUT\1B G-0401.D)

300

LysineLeucinePhenylalanine
Isoleucine

Tryptophan

Norvaline

CystineValineMethionine

Tyrosine

Arginine
AlanineGlutamine
Histidine
GlycineThreonine

Asparagine
SerineGlutamic acid

400

Aspartic acid

mAU

200

100

0
1

2

3

4

5

6

7

8

9

min

Figure 3. Separation of 20 amino acid standards using an Agilent AdvanceBio AAA 4.6 x 100 mm column with and without 5 mM sodium azide inmobile phase.
Note: Addition of 5 mM sodium azide (NaN3) to mobile phase A is optional to prevent microbiological growth and extend shelf life of buffers.

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14

Step 6:
Run high-throughput amino acid analysis

The first 20 amino acids in Figure 4, the primaryamino acids, are derivatized with OPA. The last three,hydroxyproline, sarcosine, and proline, are derivatized with
FMOC. A programmable wavelength switch from 338
to 265 nm takes place after lysine (peak 20) elutes andbefore hydroxyproline (peak 21) elutes.

2

4

6

–– The method can easily be scaled to different column
dimensions.
–– In this case, the only changes to the method weremade by altering the flow rate, changed geometricallywith the diameter of the column.
–– The low-volume heat exchanger was used with short
red tubing to minimize extra column volume.

810

4.6 mm id
Sarcosine
ProlineCystineValineMethionine
Norvaline
Tryptophan
Phenylalanine
Isoleucine
LeucineLysineHydroxyproline

Tyrosine

Asparagine
SerineGlutamine
Histidine
GlycineThreonine
Arginine
AlanineGlutamic acid

mAU
80
60
40
20
0

Aspartic acid

The chromatogram in Figure 4 illustrates typical routine
standard sensitivity in high-throughput applications thatcan be obtained using Agilent AdvanceBio AAA columns.
These separations were produced using the Agilent 1260
Infinity II HPLC binary system with AdvanceBio AAA, 100mm, 2.7 μm columns of different internal diameters,
and DAD detection. A single run can be completed
in under 20 minutes (including re-equilibration) withadequate resolution. The primary amino acids (1-20,
OPA-derivatized), were monitored at 338 nm, while thesecondary amino acids (21-23, FMOC-derivatized), weremonitored at 262 nm.

12

min

3.0 mm id
mAU
120
100
80
60
40
20
0
2

4

6

8

10

12

min

Figure 4. Separation of AA standards using Agilent AdvanceBio AAA columns of different internal
diameters using the amino acid method.

15

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Retention time and area precision for 100 pmoland 1000 pmol analysis (n=6)
Table 3. Retention time and area RSD precision for amino acids (100 pmol) separated using an AdvanceBio AAA, 4.6 x 100 mm column (six replicates.)
Amino Acids

Average RT

RT RSD (%)

Area RSD (%)

Amino Acids

Average RT

RT RSD (%)

Area RSD (%)

1. Aspartic acid

0.851

1.270

1.066

13. Valine

7.201

0.084

2.47

2. Glutamic acid

1.428

0.973

1.850

14. Methionine

7.363

0.073

1.82

3. Asparagine

2.639

0.605

1.790

15. Norvaline

7.602

0.073

1.72

4. Serine

2.835

0.629

1.820

16. Tryptophan

8.055

0.054

1.57

5. Glutamine

3.285

0.470

1.560

17. Phenylalanine

8.341

0.051

1.66

6. Histidine

3.465

0.430

1.220

18. Isoleucine

8.503

0.047

1.72

7. Glycine

3.681

0.477

1.920

19. Leucine

9.000

0.030

1.70

8. Threonine

3.837

0.440

1.950

20. Lysine

9.428

0.028

1.66

9. Arginine

4.458

0.251

2.150

21. Hydroxyproline

9.747

0.021

4.13

10. Alanine

4.764

0.280

3.060

22. Sarcosine

10.980

0.026

1.15

11. Tyrosine

5.762

0.128

1.650

23. Proline

11.620

0.021

4.36

12. Cystine

6.870

0.067

1.900

Table 4. Retention time and area RSD precision for amino acids (1000 pmol) separated using an AdvanceBio AAA, 4.6 x 100 mm column (six replicates).
Amino Acids

Average RT

RT RSD (%)

Area RSD (%)

Amino Acids

Average RT

RT RSD (%)

Area RSD (%)

1. Aspartic acid

0.837

0.151

2.60

13. Valine

7.118

0.025

2.40

2. Glutamic acid

1.400

0.512

2.19

14. Methionine

7.281

0.025

1.78

3. Asparagine

2.583

0.124

2.13

15. Norvaline

7.573

0.019

1.77

4. Serine

2.772

0.114

1.74

16. Tryptophan

7.970

0.024

2.03

5. Glutamine

3.220

0.092

1.80

17. Phenylalanine

8.238

0.027

1.98

6. Histidine

3.405

0.077

1.39

18. Isoleucine

8.413

0.025

2.17

7. Glycine

3.598

0.068

1.48

19. Leucine

8.925

0.020

1.81

8. Threonine

3.766

0.059

2.26

20. Lysine

9.357

0.022

2.00

9. Arginine

4.422

0.027

1.66

21. Hydroxyproline

9.718

0.014

3.14

10. Alanine

4.685

0.031

1.87

22. Sarcosine

10.961

0.015

5.91

11. Tyrosine

5.695

0.034

2.04

23. Proline

11.911

0.011

2.58

12. Cystine

6.794

0.030

2.22

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16

Step 7:
Ensure system suitability as per European
Pharmacopoeia
The 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 provethat their amino acids meet these specifications before
they can distribute their products in Europe.
Leucine (Leu) is a branched-chain α-amino acid, producedby the fermentation process. During this process,isoleucine can be produced as a by-product. The European
Pharmacopoeia states that leucine and isoleucine shouldhave a resolution of not less than 1.5 [1]

O

CH3
H3C

O

OH

≥ 1.5

CH3

HO

NH2

NH2

Isoleucine

CH3

LeucineTable 5. System suitability testing using Agilent AdvanceBio AAA
columns and AA standards

SystemSuitability

AdvanceBio
AAA, C18,
4.6 × 100 mm,
2.7 μmAdvanceBio AAA,
C18, 3.0 × 100 mm,
2.7 μmResolution
between Leucine
and Isoleucine(≥1.5)

4.5

4.6

Reference:
1. European pharmacopoeia 9.0 (2.2.56)
Amino Acid Analysis

17

Return to Contents Section

Step 8:Optimize cell culture media and proteinhydrolysate standard
Cell cultures are widely used to produce
biopharmaceuticals and other biologically active
compounds. The composition of the cell culture media
affects the yield and structure of the desired products
and must be carefully optimized. Cell culture media
is typically composed of mixtures of amino acids,
vitamins, carbohydrates, inorganic salts, as well as
different peptides, proteins, and other compounds. As
the cells grow, they consume nutrients and release target
biopharmaceuticals as well as waste products. Aminoacids serve as the building blocks of proteins, as well asintermediates in many metabolic pathways. Therefore,amino acids are typically added to cell culture media toprovide nutritional requirements for the cells.

Amino acid compositional analyses of commonly usedcell culture media and protein hydrolysate are shown in
Figures 5-8. This analysis confirms that the amino acidcomposition of cell culture media accurately matches with
their theoretical composition. Such applications are usefulfor monitoring and adjusting amino acid composition.
This analysis is an essential part of optimizing the
manufacturing process to ensure high quality andoptimum yield of the final biopharmaceutical product.
The following cell culture media are used forcompositional analysis using amino acid method with an
AdvanceBio AAA 4.6 x 100 mm column (Figures 5-8).
1. Minimum Essential Medium Eagle (MEM) M4655: 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.
2. Non-Essential Amino Acid (NEAA) Cell Culture Supplement M7145: L-Alanine,
L-Asparagine, L-Aspartic acid, L-Glutamic acid, Glycine, L-Proline, and L-Serine.
3. RPMI 1640 R0083: L-arginine, 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.

Determination of amino acid flux in cultured cells is an
important indicator of the metabolic rate and health of
those cells. It can also be used as an indicator of theremaining carbon and nitrogenous fuel available. Thisis especially true in hepatocyte and hepatoma cell lines,
where extensive gluconeogenesis, urea production, andprotein synthesis may consume larger quantities of aminoacids than other cell types.
HPLC with precolumn derivatization is a standard
technique in the analysis of amino acids. Precolumn
derivatization of free amino acids in solution for HPLC
separations with UV or fluorescence detection is at times
done manually offline. Some immediate drawbacks tooffline derivatization are sources of error due to operator
skill, competence, and laboratory technique; extra sample
manipulation; extra time required; and increased risk ofcontamination. Automated online derivatization minimizes
these error sources, immediately improves precision,and saves time. A rugged high-resolution HPLC method
including online derivatization, therefore, can increase
productivity compared to offline methods.

Return to Contents Section

+

-

+

+

18

60

80

19

2

4

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

6

8

Return to Contents Section
Proline8

10
ProlineHydroxyproline

7

Sarcosine

Lysine6

Glutamine

LysineLeucinePhenylalanine
Isoleucine

Tryptophan

Norvaline

ValineMethionine

CystineTyrosine

AlanineArginine

GlycineThreonine

Histidine

Asparagine
Serine250pmol AA std, Sig=338,10 Ref=390,20,

LeucineNorvaline
Tryptophan
Phenylalanine
Isoleucine

5

ValineMethionine

Alanine4

Tyrosine

AlanineGlycineSerine3

Arginine

Histidine
GlycineThreonine

20
Glutamine

Asparagine

Threonine

Tyrosine

LeucinePhenylalanine
Isoleucine

Tryptophan

ValineMethionine

Cystine

AlanineArginine

Histidine

Glutamine

Glutamic acid100

Cystine40
Glutamic acid2

Serine60

Asparagine

mAU

Glutamic acid100
Glutamic acid50

Aspartic acid50

Aspartic acid150
LysinemAU
MEM Eagle, Sig=338,10 Ref=390,20,

0

9
min

Figure 5. Amino acid analysis of Eagles MEM media (blue trace) and comparison with amino acid standards using the Agilent
AdvanceBio AAA solution.

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

40

20

0

12

min

Figure 6. Amino acid analysis of Non-Essential Amino Acid (NEAA) media (blue trace) and comparison with amino acid standard using theAgilent AdvanceBio AAA solution.

mAU

2

4

6

Sarcosine

ProlineProlineHydroxyproline

Lysine8

Hydroxyproline

LeucineLeucineLysineTryptophan
Phenylalanine
Isoleucine

ValineMethionine
Norvaline

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

150

CystineTyrosine

AlanineAsparagine
Serine100

Aspartic acid50

Glutamic acid0

Tryptophan
Phenylalanine
Isoleucine

CystineArginine
Arginine

ValineMethionine

Histidine
GlycineThreonine
Glutamine
Histidine
GlycineThreonine

Asparagine
Serine50

Glutamic acid

100

Aspartic acid150

Tyrosine

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

200

10

12

min

Figure 7. Amino acid analysis of RPMI 1650 media (blue trace) and comparison with amino acid standard using the Agilent AdvanceBio AAA solution.

DAD1 A, Sig=338,10 Ref=390,20,

LysineProlineLeucinePhenylalanine
Isoleucine

CystineValineMethionine

Rs=4.35

Tyrosine

AlanineArginine

200

Histidine
GlycineThreonine

300

Serine400

Glutamic acid500

Aspartic acid

mAU

100

0
2

4

6

8

10

12

min

Figure 8. Amino acid analysis of protein hydrolysate. The resolution between leucine and Isoleucine with the AdvanceBio AAA, 4.6 x 100 mm,
2.7 μm column is much higher than the reported value for system suitability requirement.

Return to Contents Section

20

Maintenance and troubleshooting

Troubleshooting:

The Agilent AdvanceBio AAA solution includes technicaland application support. The following maintenanceand troubleshooting tips are recommended to keep
your InfinityLab LC Series instrument systems runningsmoothly.

Poor chromatographic resolution

Daily Maintenance:–– Always use short red tubing with the low-volume heatexchanger be to minimize extracolumn volume–– Replace derivatization reagent, borate buffer, aminoacid standards, and wash water, which are placed in
autosampler tray.
–– Recalibration of retention times and response factors.
–– Check column and guard column performance using
system suitability report.

–– Exhausted guard column–– Damaged analytical column–– Post column band broadening due to too longconnections.

Low Intensity Chromatogram–– OPA reagent has deteriorated–– FMOC reagent has deteriorated–– Glycine contamination–– Every two days replace mobile phase A and B withfreshly made solvents

21

Return to Contents SectionOrdering Information
Columns, supplies and chemicals

SizePart No.

AdvanceBio AAA LC column

4.6 x 100 mm, 2.7 µm

655950-802

AdvanceBio AAA guard columns

4.6 x 5 mm, 2.7 µm, 3/pk

820750-931

AdvanceBio AAA LC column

3.0 x 100 mm, 2.7 µm

695975-322

AdvanceBio AAA guard columns

3.0 x 5 mm, 2.7 µm, 3/pk

823750-946

Borate Buffer

0.4 M in water, pH 10.2, 100 mL

5061-3339

FMOC Reagent

2.5 mg/mL in ACN, 10 x 1 mL ampoules

5061-3337

OPA Reagent

10 mg/mL in 0.4 M borate buffer and 3-mercaptoproprionic acid, 6 x 1 mL ampoules

5061-3335

Dithiodipropionic Acid (DTDPA) reagent

5g5062-2479

Inserts, with polymer feet

250 µL, 100/pk

5181-1270

Vial, screw top, amber with write-on spot

2 mL, certified, 100/pk

5182-0716

Cap, screw, green, PTFE/white silicone septum

100/pk

5182-0721

Vial, screw top, clear, flat bottom

for LC, 6 mL, certified, 100/pk

9301-1377

Cap, screwfor 6 mL vials, 100/pk

9301-1379

Septumfor 6 mL vials, 100/pk

9301-1378

AA standard

1 nmol/µL, 10 x 1 mL

5061-3330

AA standard

250 pmol, 10/pk

5061-3331

AA standard

100 pmol/µL, 10 x 1 mL

5061-3332

AA standard

25 pmol/µL, 10 x 1 mL

5061-3333

AA standard

10 pmol/µL, 10 x 1 mL

5061-3334
5062-2478

Amino acids supplement kit

Learn more about the Agilent AdvanceBio family of
innovations, designed specifically for biomoleculecharacterization, visit
www.agilent.com/chem/advancebio

Return to Contents Section

22

Application Note
Amino Acid Analysis

Determination of Amino Acid
Composition of Cell Culture Mediaand Protein Hydrosylate Standard
The Agilent AdvanceBio Amino Acid Solution

Authors

Abstract

M. Sundaram Palaniswam
Agilent Technologies, Ltd

This study presents a method for analyzing primary amino acids in cell culture
media using the Agilent AdvanceBio Amino Acid Analysis (AAA) solution withabsorbance detection. Derivitization using an online injector program with OPA
and FMOC decreases sample preparation time, and increases reproducibility overtraditional offline methods. The effectiveness of this solution for routine analysis
was confirmed using a system suitability test and retention time and area precision
studies. The AdvanceBio AAA solution provides sensitive and high‑resolution
separation of all amino acids in cell culture media. The limit of detection (LOD), limitof quantification (LOQ), and linearity for selected amino acids for qualitative assays
are also reported.

23

Return to Contents Section

Introduction

Reagents, samples, and materials

Amino acids are the basic building blocks of proteins. They
constitute all proteinaceous material of the cell including thecytoskeleton and the protein component of enzymes, receptors,
and signaling molecules. In addition, amino acids are used forthe growth and maintenance of cells. Cell culture media plays
a key role in the biopharma industry. A large proportion of theamino acids supplied from cell culture media are diverted topathways that could influence the fate of the cells in a culture.
The identification of the optimal concentration of amino acids
is important in fed batch and perfusion culture. Therefore, thedesign of an amino acid supplementation strategy might bestreamlined by identifying the amino acid demands of a cell
culture due to host cell growth and product production.

Cell culture media for compositional analysis, Minimum Essential
Medium Eagle (M4655), Non-Essential Amino Acid (M7145),
RPMI 1640 (R0083), Na2HPO4, and Na2B4O7•10H2O, werebought from Sigma-Aldrich. Protein hydrolysate was obtained
from Fisher Scientific. Acetonitrile and methanol used werebought from Lab-Scan (Bangkok, Thailand). HPLC grade andhighly purified water from a Milli-Q water purification system(Millipore Elix 10 model, USA) was used.

HPLC with precolumn derivatization is commonly used for the
analysis of amino acids. Precolumn derivatization of free aminoacids in solution for HPLC separations with UV or fluorescence
detection is sometimes done manually, offline. Some immediatedrawbacks to offline derivatization are sources of error due tooperator skill, competence, and laboratory technique. Other
drawbacks include extra sample manipulation, extra timerequired, and increased risk of contamination. Automated online
derivatization minimizes these error sources, immediately
improves precision, and saves time. Thus, a rugged highresolution HPLC method including online derivatization, canincrease productivity compared to offline methods. Consistentautomated OPA derivatization, using the injector programmingof the HPLC’s autosampler and highly efficient Agilent
AdvanceBio AAA columns, generate a rapid-reproducible
amino acid method ideal for cell culture media. This method
is convenient because the cell media samples are simply
transferred to autosampler vials and analyzed. The selectivityof the AdvanceBio AAA column and the mobile phase gradientprovides high resolution of 23 amino acids.

Mobile phase A contained 10 mM Na2HPO4, and 10 mM Na2B4O7,
pH 8.2. Mobile phase B contained acetonitrile, methanol, andwater (45:45:10, v:v:v). Since mobile phase A is consumed at afaster rate than B, it is convenient to make 2 L of A for every 1 L of
B produced.

ColumnAgilent AdvanceBio AAA, C18, 4.6 × 100 mm, 2.7 μm(p/n 655950-802)

Preparation of HPLC mobile phase

Injection diluent
The injection diluent was 100 mL mobile phase A, plus 0.4 mL
concentrated H3PO4 in a 100 mL bottle, stored at 4 °C. To prepare
0.1 N HCl, 4.2 mL concentrated HCl (36%) was added to a 500
mL volumetric flask that was partially filled with water, mixed,
then filled to the mark with water. This solution was then used
for making extended amino acid and internal standard stock
solutions. It was stored at 4 °C.

Agilent AdvanceBio AAA standards and reagents kit,p/n 5190-9426, includes:
Part number Component
5061–3339

Borate buffer: 0.4 M in water, pH 10.2, 100 mL

Materials and Methods

5061–3337

FMOC reagent, 2.5 mg/mL in ACN, 10 × 1 mL ampules

Instrumentation

5061–3335

OPA reagent, 10 mg/mL in 0.4 M borate buffer and3-mercaptoproprionic acid, 6 × 1 mL ampules5062–2479

Dithiodipropionic acid (DTDPA) reagent, 5 g

5061–3330

AA standard, 1 nmol/µL, 10 × 1 mL

5061–3331

AA standard 250 pmol, 10/pk

5061–3332

AA standard, 100 pmol/µL, 10 × 1 mL

5061–3333

AA standard, 25 pmol/µL, 10 × 1 mL

5061–3334

AA standard, 10 pmol/µL, 10 × 1 mL

5062–2478

Amino acids supplement kit, 1 g eachAnalyses were performed using an Agilent 1290 Infinity LC, which
was equipped with an Agilent 1290 Infinity binary pump delivery
system (G4220A), Agilent 1290 Infinity autosampler (G4226A),
Agilent 1290 Infinity thermostatted column compartment(G1316C), and Agilent 1290 Infinity DAD (G4212A).

Return to Contents Section24Derivatization reagentsInternal Standard (ISTD) stock solution

Derivatization reagents (borate buffers, OPA, and FMOC) are
ready-made solutions supplied by Agilent, and were transferredfrom their containers to autosampler vials. Precautions included:

For primary amino acid ISTD stock solutions, 58.58 mg norvaline
was weighed into a 50 mL volumetric flask. For secondary aminoacids, 44.54 mg sarcosine was weighed into the same 50 mL
flask. This flask was filled halfway with 0.1 N HCl, and shaken
or sonicated until dissolved, then filled to mark with water fora final concentration of 10 nmol each amino acid/μL (standard
sensitivity). For high‑sensitivity ISTD stock solution, 5 mL ofstandard‑sensitivity solution was diluted with 45 mL of water, andstored at 4 °C.

–– OPA is shipped in ampules under inert gas to preventoxidation. Once opened, the OPA is potent for approximately 7
to 10 days. Therefore, 100 μL aliquots of OPA were transferredin microvial inserts and refrigerated. The OPA autosampler
microvial was then replaced daily. Each ampule lasted 10days (one vial/day).
–– FMOC is stable in dry air, but deteriorates in moisture.
Therefore, FMOC was transferred in 100 μL aliquots tomicrovial inserts and refrigerated. An open FMOC ampule
transferred to 10 microvial inserts should last 10 days.
–– Borate buffer was transferred to a 1.5 mL autosampler vial
without a vial insert, and replaced every 3 days.

Preparation of amino acid standards–– Solutions of 17 amino acids in five concentrations areavailable from Agilent (10 pmol/μL to 1 nmol/μL) for
calibration curves. Each 1 mL ampule of standards was
divided into 100 μL portions in conical vial inserts, and stored
at 4 °C.
–– The extended amino acid (EAA) stock solution was produced
by weighing 59.45 mg asparagine, 59.00 mg hydroxyproline,
65.77 mg glutamine, and 91.95 mg tryptophan into a 25 mL
volumetric flask. This flask was filled halfway with 0.1 N HCL,
and shaken or sonicated until the amino acids were dissolved.
It was then filled to mark with water to produce a total
concentration of 18 nmol/μL of each amino acid.
–– For the high-sensitivity EAA stock solution, 5 mL of thisstandard-sensitivity solution was diluted with 45 mL water(1.8 nmol/μL). Solutions containing extended standards were
unstable at room temperature, and were kept frozen, anddiscarded at the first signs of reduced intensity.

25

Calibration curves may be made using two to five standards,
depending on experimental need. Typically, 100 pmol/μL, 250
pmol/μL, and 1 nmol/μL are used in a three-point calibration
curve for standard analytical sensitivity analysis. The following
tables should be followed if an internal standard or other aminoacids (for example, the extended amino acids) are added. Table 1
describes standard analytical sensitivity concentrations typicallyused in UV analysis.

Table 1. Chromatographic parameters used for intact andreduced analysis.
Concentration of Final AA solution(pmol/µL)

Concentration of Final AA solution(pmol/μL)
900

225

90

Take 5 mL of 18 nmol EAA

5 mL

5 mL

5 mL

Dilute with water

–

15 mL

45 mL

Diluted EAA mix

5 mL

20 mL

50 mL

Take 5 mL of diluted EAA mix

5 mL

5 mL

5 mL

Add 10 nmol ISTD solution

5 mL

5 mL

5 mL

EAA–ISTD mix

10 mL

10 mL

10 mL

Take 100 µL of EAA–ISTD mix

100 µL100 µL100 µLFor 1 nmol AA, add:

900 µL––For 250 pmol AA, add:

900 µL–For 100 pmol AA, add:––

900 µL

Final AA solution with EAA
and 500 pmol/µL ISTD

1 mL

1 mL

1 mL

Return to Contents Section

Online derivatization
Depending on the autosampler model, the automated online
derivatization program differs slightly. For the Agilent G4226A
well plate automatic liquid sampler (WPALS), the injection
program was:
1. Draw 2.5 μL from borate vial (p/n 5061-3339).

To detect both OPA and FMOC derivatized amino acids in a single
chromatogram, it was necessary to switch detector wavelengths.
This switch took place between the last eluting OPA derivatizedamino acid, lysine (peak 20 in the standard), and the first eluting
FMOC derivatized amino acid, hydroxyproline (peak 21 in thestandard).
With the DAD, determining the appropriate transition point waspossible by initially collecting two channels. Signal A, 338 nm,
detected OPA derivatized amino acids, and signal B, 262 nm,
detected FMOC derivatized amino acids. From this analysis, theideal point at which to switch wavelength during the run wasdetermined. Subsequent runs were then made using a single
channel, with the detector timetable function used to program awavelength switch from 338 to 262 nm at the appropriate time
between the elution of OPA-lysine and FMOC-hydroxyproline. This
switch allowed both OPA and FMOC-derivatized amino acids tobe detected in a single chromatogram. Peak width settings of>0.01 minutes were used for all columns.:

2. Draw 1.0 μL from sample vial.
3. Mix 3.5 μL in wash port five times.
4. Wait 0.2 minutes.
5. Draw 0.5 μL from OPA vial (p/n 5061-3335).
6. Mix 4.0 μL in wash port 10 times default speed.
7. Draw 0.4 μL from FMOC vial (p/n 5061-3337).
8. Mix 4.4 μL in wash port 10 times default speed.
9. Draw 32 μL from injection diluent vial.
10. Mix 20 μL in wash port eight times.
11. Inject.

Linearity, limit of detection (LOD), and limit ofquantification (LOQ) determination

12. Wait 0.1 minutes.
13. Valve bypass.
The location of the derivatization reagents and samples is up to
the analyst and the ALS tray configuration. Using the G1367C
with a 2 × 56-well plate tray (p/n G2258-44502), the locations
were:–– Vial 1: Borate buffer–– Vial 2: OPA

As an example extended amino acid (EAA) stock solution,
asparagine (59.45 mg), glutamine (65.77 mg), and tryptophan(91.95 mg) were used for linearity, LOD, and LOQ determination.
These standards were weighed into a 25 mL volumetric flask,
which was filled halfway with 0.1 N HCl, and mixed or sonicateduntil they dissolved. The flask was then filled to mark with water
for a total concentration of 18 nmol/μL of each amino acid.
Linearity was studied in the range of 0.9–1,000 pmol/μL of thesestandard amino acids. Appropriate AA standard solutions wereprepared in triplicate and injected into the chromatograph. The
LOD and LOQ were estimated from the calibration function.
LOD and LOQ were calculated as 3 (SD(a)/b) and 10 (SD(a)/b),
respectively, where SD(a) is the standard deviation of theintercept, and b is the slope of the calibration function.

–– Vial 3: FMOC–– Vial 4: Injection diluent–– P1-A-1: Sample

Thermostatted column compartment (TCC)
Left and right temperatures were set to 40 °C. Analysis wasenabled when the temperature was within ±0.8 °C.

Gradient program

Diode array detector (DAD)

Time (min)%B

Signal A: 338 nm, 10 nm bandwidth, reference wavelength
390 nm, 20 nm bandwidth.

0

2

0.35

2

Signal B: 262 nm, 16 nm bandwidth, reference wavelength
324 nm, 8 nm bandwidth.

13.4

57

13.5

100

15.7

100

15.8

2

18

end

Signal C: 338 nm, 10 nm bandwidth, reference wavelength
390 nm, 20 nm bandwidth.

Flow rate: 1.5 mL/min for 4.6 mm id

Return to Contents Section

26

Table 3. Retention time and area RSD precision for amino acids (1,000 pmol) separated on an Agilent AdvanceBio AAA, 4.6 × 100 mm, column (n = 6).
Amino acid

RT RSD (%)

Area RSD (%)

Amino acid

RT RSD (%)

Area RSD (%)

1. Aspartic acid

0.151

2.60

13. Valine

0.025

2.4

2. Glutamic acid

0.512

2.19

14. Methionine

0.025

1.78

3. Asparagine

0.124

2.13

15. Norvaline

0.019

1.77

4. Serine

0.114

1.74

16. Tryptophan

0.024

2.03

5. Glutamine

0.092

1.8

17. Phenylalanine

0.027

1.98

6. Histidine

0.077

1.39

18. Isoleucine

0.025

2.17

7. Glycine

0.068

1.48

19. Leucine

0.020

1.81

8. Threonine

0.059

2.26

20. Lysine

0.022

2

9. Arginine

0.027

1.66

21. Hydroxyproline

0.014

3.14

10. Alanine

0.031

1.87

22. Sarcosine

0.015

5.01

11. Tyrosine

0.034

2.04

23. Proline

0.011

2.58

12. Cysteine

0.030

2.22

System suitability as per the European
Pharmacopoeia (Ph. Eur.)

H3C

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 that is produced
by a fermentation process (Figure 2). During this process,isoleucine can be produced as a by‑product. The Ph. Eur. statesthat leucine and isoleucine should have a resolution of not lessthan 1.5 [1].
Ten concentration points for three amino acids were assayedin triplicate. The three standard amino acids showed good
linearity in the tested range. The area response obeyed theequation y = mx + C, where the intercept C was zero within 95 %confidence limits, and the square correlation coefficient (R2) was
always greater than 0.99. Figure 3 shows the linearity curve forasparagine, glutamine, and tryptophan in the concentration range
evaluated.
The LOD and LOQ were approximately 0.9 pmol and 3.8 pmol,
respectively, using UV detection, indicating that the method wassensitive. Table 5 shows the observed LOD and LOQ values of
asparagine, glutamine, and tryptophan.

27

CH3 O
OH

O

≥1.5

CH3

HO

NH2 CH3NH2Isoleucine

LeucineFigure 2. Isoleucine and leucine chemical relationship.

Table 4. System suitability testing using AdvanceBio AAA columns
and AA standards.
Concentration of Final AA solution (pmol/µL)

System suitability
Resolution between
leucine and isoleucine(≥1.5)

Agilent AdvanceBio
AAA, C18,
4.6 × 100 mm,
2.7 μmAgilent AdvanceBio
AAA, C18,
3 × 100 mm,
2.7 μm4.5

4.6

Table 5. LODs and LOQs for three amino acids.

Asparagine

Glutamine

Tryptophan

Asparagine

Glutamine

Concentration S/N(pmol)
ratio

Concentration(pmol)

S/N
ratio

Concentration(pmol)

S/N
ratio

0.9 (LOD)

5.3

0.9 (LOD)

3.0

0.9 (LOD)

4.5

1.9 (LOQ)

10.8

3.8 (LOQ)

13.8

3.8 (LOQ)

20.5

Return to Contents Section

TryptophanPrecision of retention time and area (n = 6)

150
100

Sarcosine

200

Proline250

CystineValineMethionine
Norvaline
Tryptophan
Phenylalanine
Isoleucine
LeucineLysineHydroxyproline

300

Glutamic acid

The chromatogram in Figure 1 illustrates the standard analyticalsensitivity achieved in high-throughput separations of aminoacids. This chromatogram was obtained using an Agilent
1290 Infinity LC with an Agilent AdvanceBio AAA, 4.6 × 100 mm,
2.7 μm column using the amino acid method with DAD detection.
A single run was completed in 18 minutes (including reequilibration) with adequate resolution. The primary amino acids(1-20, OPA-derivatized), shown in Figure 1, were monitored at 338
nm, while the secondary amino acids (21-23, FMOC-derivatized)
were monitored at 262 nm.

Tyrosine

350

Arginine
AlanineHigh-throughput routine analysis

DAD1 A, Sig=338,10 Ref=390,20,400

Asparagine
SerineGlutamine
Histidine
GlycineThreoninemAU

Aspartic acid

Results and Discussion

50
0
2

4

6

8

10

12 minFigure 1. Separation of amino acid standard (1 nmol) on an Agilent
AdvanceBio AAA 4.6 × 100 mm, 2.7 μm column, using the amino acid
method.

Tables 2 and 3 summarize the average retention times and area
RSDs for all the amino acids for the 100 and 1,000 pmol from six
replicates of an amino acid method. The retention time RSDs for
all amino acid peaks, including the early eluting peak 1 were lessthan 1.2%, demonstrating excellent gradient reproducibility. Peak
area RSDs were less than 5 %, indicating precise sample injection.
The RSD values demonstrate the robustness and precision of theamino acid method.

Table 2. Retention time and area RSD precision for amino acids (100 pmol) separated on an Agilent AdvanceBio AAA, 4.6 × 100 mm, column (n = 6).
Amino acid

RT RSD (%)

Area RSD (%)

Amino acid

RT RSD (%)

Area RSD (%)

1. Aspartic acid

1.270

1.066

13. Valine

0.084

2.47

2. Glutamic acid

0.973

1.85

14. Methionine

0.073

1.82

3. Asparagine

0.605

1.79

15. Norvaline

0.073

1.72

4. Serine

0.629

1.82

16. Tryptophan

0.054

1.57

5. Glutamine

0.470

1.56

17. Phenylalanine

0.051

1.66

6. Histidine

0.430

1.22

18. Isoleucine

0.047

1.72

7. Glycine

0.477

1.92

19. Leucine

0.03

1.7

8. Threonine

0.440

1.95

20. Lysine

0.028

1.66

9. Arginine

0.251

2.15

21. Hydroxyproline

0.021

4.13

10. Alanine

0.280

3.06

22. Sarcosine

0.026

1.15

11. Tyrosine

0.128

1.65

23. Proline

0.021

4.36

12. Cysteine

0.067

1.9

Return to Contents Section

28

Amino acid analysis of cell culture media and proteinhydrolysate standard

It is evident that the amino acid composition of cell culture
supplements, as determined by this method, matches accurately
with their theoretical composition. In addition, baseline resolutionof isoleucine and leucine was observed with a resolution factor of
4.35 for the protein hydrolysate standard, meeting the regulatory
requirements for these components significantly better thancompetitive columns. Such applications are useful in monitoring
and adjusting amino acid composition, which is an essential partof optimizing the manufacturing process to ensure high quality
and optimum yield of the final biopharmaceutical product.

A
Linearity for aspargine

Area

2,000
1,500
1,000

y = 2.0654x + 2.6479
R2 = 0.9993

500
0

0

200

400

600

800

1,000

1,200

Concentration (pmol)
1,200BLinearity for glutamine

1,000
800

Area

We analyzed the amino acid composition of commonly usedcell culture supplements. These standards included: Minimum
Essential Medium Eagle (MEM), Non-Essential Amino Acid
(NEAA), RPMI 1640 R0083, and protein hydrolysate standard.
The results were then compared with the amino acid standards.
Figures 4 to 7 show the overlays of amino acid composition of
the media and the amino acid standards.

2,500

y = 1.0107x + 0.3716
R2 = 0.9997

600
400
200
0

0

200

400

600

800

1,000

1,200

Concentration (pmol)
1,800

C

1,600

Linearity for tryptophan

1,400

Area

1,200

y = 1.6446x + 6.0767
R2 = 0.9999

1,000
800
600
400
200
0

0

200

400

600

800

1,000

1,200

Concentration (pmol)

Figure 3. Linearity curve with 10 standard concentrations ofasparagine, glutamine, and tryptophan ranging from 0.9 to 1,000 pmol,
showing excellent coefficient values.

29

Return to Contents SectionmAU

MEM Eagle, Sig = 338.10 Ref = 390.20
4

150

19

100

50

7

12
10

5

1

11
8

6

2 3

9

18

17
16

14
13

15

0

50

100

Peak ID
1. Glutamic acid
2. Asparagine
3. Serine
4. Glutamine
5. Histidine
6. Glycine
7. Threonine
8. Arginine
9. Alanine
10. Tyrosine
11. Cystine
12. Valine
13. Methionine
14. Norvaline
15. Tryptophan
16. Phenylalanine
17. Isoleucine
18. Leucine
19. Lysine

250 pmol AA std, Sig = 338.10 Ref = 390.20
2

3

4

5

6

7

8

9

min

Figure 4. Amino acid analysis of MEM media (blue trace) mirrored with AA standards (red trace).

NEAA cell culture supplement, Sig = 338.10 Ref = 390.20

mAU
60
40
20
0

5
20
40

1

21
34

60
80

23

2
6

78

9

10

11

17

250 pmol AA std, Sig = 338.10 Ref = 390.20
2

4

16 18 19

1314

6

12

22

15

20

8

10

12

min

Peak ID
1. Aspartic acid
2. Glutamic acid
3. Asparagine
4. Serine
5. Glutamine
6. Histidine
7. Glycine
8. Threonine
9. Arginine
10. Alanine
11. Tyrosine
12. Cystine
13. Valine
14. Methionine
15. Norvaline
16. Tryptophan
17. Phenylalanine
18. Isoleucine
19. Leucine
20. Lysine
21. Hydroxyproline
22. Sarcosine
23. Proline

Figure 5. Amino acid analysis of NEAA media (blue trace) and comparison with AA standards (red trace).

Return to Contents Section

30

mAU
200

RPMI 1640 Media, Sig = 338.10 Ref = 390.20

5

150
100

1
4

50

2

7

3
6

0

8

9

10

11

12 14
13

18 19

20
21

17
16

23

50
22
100

15
250 pmol AA std, Sig = 338.10 Ref = 390.20

150

2

4

6

8

10

12

min

Peak ID
1. Aspartic acid
2. Glutamic acid
3. Asparagine
4. Serine
5. Glutamine
6. Histidine
7. Glycine
8. Threonine
9. Arginine
10. Alanine
11. Tyrosine
12. Cystine
13. Valine
14. Methionine
15. Norvaline
16. Tryptophan
17. Phenylalanine
18. Isoleucine
19. Leucine
20. Lysine
21. Hydroxyproline
22. Sarcosine
23. Proline

Figure 6. Amino acid analysis of RPMI 1650 media (blue trace) and comparison with AA standards (red trace).

mAU
500

DAD1 A, Sig = 338.10 Ref = 390.20
1

400
7
300

16

2
3

5
4

200

Rs = 4.35
86

9

10 1112

15
13
14
17

100

0

2

4

6

8

10

12

Peak ID
1. Aspartic acid
2. Glutamic acid
3. Serine
4. Histidine
5. Glycine
6. Threonine
7. Arginine
8. Alanine
9. Tyrosine
10. Cysteine
11. Valine
12. Methionine
13. Phenylalanine
14. Isoleucine
15. Leucine
16. Lysine
17. Prolinemin

Figure 7. Amino acid analysis of protein hydrolysate standard. The resolution between leucine and isoleucine with the Agilent AdvanceBio AAA,
4.6 × 100 mm, 2.7 μm column is much higher than the reported value for system suitability requirement for this pair.

31

Return to Contents Section

Conclusion
Amino acid analysis is an important approach for thecharacterization of protein and peptide-based products. Studying
the roles of amino acids during bioprocesses leads to better
understanding the feeding strategy, and to improving the yieldand quality of the product. In addition, the determined amino acidcomposition can confirm sample identity, and give a measureof sample purity. This Application Note demonstrates several
Agilent tools for the analysis of amino acids. We first used theAgilent 1290 Infinity LC and the Agilent AdvanceBio AAA kit
for the automated online derivatization of amino acids using
OPA/FMOC chemistries. The derivitized amino acids were thenseparated on an AdvanceBio AAA LC column to achieve a fast,sensitive, and reproducible separation of amino acids. Area
and RT precision of the method were excellent, and met thesystem suitability requirement. Linearity curves with 10 standard
concentrations of three amino acids, ranging from 0.9 pmol to 1
nmol, had excellent coefficient of linearity values, indicating thatthe method was quantitative and accurate. The LOD and LOQ
for the amino acids were 0.9 pmol and 3.8 pmol, respectively,
indicating that the method was sensitive. In addition, thismethod was able to separate and detect amino acids from cell
culture media and protein hydrolysate standard. The amino acidcomposition determined using this method correlated well with
their theoretical compositions..

Reference
1. European Pharmacopoeia 9.0 (2.2.56) Amino Acid Analysis

For More Information
These data represent typical results. For more information on our
products and services, visit www.agilent.com/chem.

Return to Contents Section

32

Application Note
Amino Acid Analysis

Automated Amino Acid Analysis
Using an Agilent Poroshell HPH-C18
ColumnAuthors

Abstract

William Long
Agilent Technologies, Inc

In this application note, an automated precolumn OPA/FMOC amino acid method,previously developed on 3.5 and 1.8 μm Agilent ZORBAX Eclipse Plus C18 columns,
is expanded to include 2.7 μm Agilent Poroshell HPH-C18 superficially porous
columns. This column exhibits good lifetime and transferability to different column
dimensions, both of which are shown in this work. Applications of the column tofermentation products are also shown.

33

Return to Contents Section

Introduction
Superficially porous particle (SPP) technology is based onparticles with a solid core and a superficially porous shell.
These particles consist of a 1.7 μm solid core with a 0.5 μm
porous shell. In total, the particle size is about 2.7 μm. The
2.7 μm superficially porous particles provide 40 to 50 % lower
backpressure and 80 to 90 % of the efficiency of sub-2 μm
totally porous particles. The superficially porous particles have anarrower particle size distribution than totally porous particles.
This results in a more homogeneous column bed, and reduces
dispersion in the column. At the same time, the thin porous
shell provides lower resistance to mass transfer. The result is
minimal loss of efficiency at higher flow rates [1]. Additionally,
since the columns incorporate a 2 μm frit, they are as resistant toclogging as 3.5 and 5 μm columns. Until recently, all silica-based
SPP materials possessed limited lifetime in higher pH buffers,
including phosphate buffers. To achieve these longer lifetimes,
it is necessary to protect the base particle by either surface
modification or special bonding modification. The surface ofAgilent Poroshell HPH-C18 particles are chemically modified
to form an organic layer, resistant to silica dissolution at highpH conditions, using a proprietary process. The continuous
improvement in HPLC columns and instrumentation presents
an opportunity to improve HPLC methods. A proven orthophthalaldehyde/9-fluorenylmethyl chloroformate (OPA/FMOC)derivatized amino acid method developed on HP 1090 Series
HPLC systems, and later updated for the Agilent 1100 Series, has
now evolved further taking advantage of the Agilent 1260 Infinity
Binary LC and superficially porous Agilent Poroshell HPH-C18
columns [2-8].

Experimental
Preparation of HPLC mobile phase
Mobile phase A contained 10 mM Na2HPO4, 10 mM Na2B4O7, pH
8.2, and 5 mM NaN3. For 1 L, weigh 1.4 g anhydrous Na2HPO4
plus 3.8 g Na2B4O7•10H2O in 1 L water plus 32 mg NaN3. Adjust to
about pH 8.4 with 1.2 mL concentrated HCl, then add small dropsof acid to pH 8.2. Allow stirring time for complete dissolutionof borate crystals before adjusting pH. Filter through 0.45 μm
regenerated cellulose membranes (p/n 3150-0576). Mobile
phase B contains acetonitrile:methanol:water (45:45:10, v:v:v). All
mobile-phase solvents were HPLC grade. Since mobile phase A isconsumed at a faster rate than B, it is convenient to make 2 L of
A for every 1 L of B.

Derivatization reagents (borate buffers, OPA, and FMOC) are
ready-made solutions supplied by Agilent. They simply need to betransferred from their container into an autosampler vial. Some
precautions include:–– OPA is shipped in ampoules under inert gas to preventoxidation. Once opened, the OPA is potent for about 7 to 10
days. We recommend transferring 100 μL aliquots of OPA
to microvial inserts. Label with name and date, cap, andrefrigerate. Replace the OPA autosampler microvial daily.
Each ampoule lasts 10 days.
–– FMOC is stable in dry air but deteriorates in moisture. It
should also be transferred in 100 μL aliquots to microvial
inserts. Label with name and date, cap tightly, and refrigerate.
Like the OPA, an open FMOC ampoule transferred to 10
microvial inserts should last 10 days (one vial/day).
–– Borate buffer can be transferred to a 1.5 mL autosampler vial
without a vial insert. Replace every three days.

Preparation of amino acid standards
Solutions of 17 amino acids in five concentrations are available
from Agilent (10 pmol/μL to 1 nmol/μL) for calibration curves.
Divide each 1 mL ampoule of standards (p/n 5061-3330 through5061-3334) into 100 μL portions in conical vial inserts. Cap andrefrigerate aliquots at 4 °C. To make the extended amino acid
(EAA) stock solution, weigh 59.45 mg asparagine, 59.00 mghydroxyproline, 65.77 mg glutamine, and 91.95 mg tryptophan
into a 25 mL volumetric flask. Fill halfway with 0.1 N HCL andshake or sonicate until dissolved. Fill to mark with water for a
total concentration of 18 nmol/μL of each amino acid. For thehigh-sensitivity EAA stock solution, take 5 mL of this standardsensitivity solution and dilute with 45 mL water (1.8 nmol/μL).
Solutions containing extended standards are unstable at room
temperature. Keep them frozen and discard at first signs of
reduced intensity.
For primary amino acid ISTD stock solutions, weigh 58.58 mg
norvaline into a 50 mL volumetric flask. For secondary aminoacids, weigh 44.54 mg sarcosine into the same 50 mL flask. Fillhalf way with 0.1 N HCl and shake or sonicate until dissolved,
then fill to mark with water for a final concentration of 10 nmol
each amino acid/μL (standard sensitivity). For high-sensitivity
ISTD stock solution, take 5 mL of standard-sensitivity solution
and dilute with 45 mL of water. Store at 4 °C. Calibration curves
are made using two to five standards depending on experimental
need. Typically, 100 pmol/μL, 250 pmol/μL, and 1 nmol/μL areused in a three-point calibration curve for standard-sensitivity
analysis.

The injection diluent was 100 mL mobile phase A, plus 0.4 mL
concentrated H3PO4 in a 100 mL bottle, stored at 4 °C.
To prepare 0.1 N HCl, add 4.2 mL concentrated HCl (36%) to a
500 mL volumetric flask that is partially filled with water. Mix, andfill to the mark with water. This solution is for making extended
amino acid and internal standard stock solutions. Store at 4 °C

Return to Contents Section

34

Pump parameters

Thermostatted column compartment (TCC)

Pump parameters for all methods include compressibility (×10–6bar) A: 35, B: 80, with minimal stroke A, B of 20 μL.

Left and right temperatures were set to 40 °C. Enable analysis
when the temperature is within ± 0.8 °C. See Table 5 for whichheat sink to use.

Online derivatization
Depending on the autosampler model, the automated online
derivatization program differs slightly. For the Agilent G1376C
well plate automatic liquid sampler (WPALS), with injection
program:
1. Draw 2.5 μL from borate vial (p/n 5061-3339).
2. Draw 1.0 μL from sample vial.

Signal A: 	338 nm, 10 nm bandwidth, and reference wavelength
390 nm, 20 nm bandwidth.
Signal B: 	262 nm, 16 nm bandwidth, and reference wavelength
324 nm, 8 nm bandwidth.
Signal C: 	338 nm, 10 nm bandwidth, and reference wavelength
390 nm, 20 nm bandwidth.

3. Mix 3.5 μL in wash port five times.

The DAD was programmed to switch to 262 nm, 16 nm
bandwidth, reference wavelength 324 nm, 8 nm bandwidth,
after lysine elutes, and before hydroxyproline elutes. Signal
C was determined by examining signal A and B timeframes
between peaks 20 and 21, then choosing a suitable point toswitch wavelengths. Once the switch time was established andprogrammed into the method, signals A and B were optional.

4. Wait 0.2 minutes.
5. Draw 0.5 μL from OPA vial (p/n 5061-3335).
6.

Diode array detector (DAD)

Mix 4.0 μL in wash port 10 times default speed.

7. Draw 0.4 μL from FMOC vial (p/n 5061-3337).
8. Mix 4.4 μL in wash port 10 times default speed.
9. Draw 32 μL from injection diluent vial.

Peak width settings of > 0.01 minutes were used for all columns.

10. Mix 20 μL in wash port eight times.
11. Inject.
12. Wait 0.1 minutes.
13. Valve bypass
The location of the derivatization reagents and samples is up to
the analyst and the ALS tray configuration. Using the G1367C
with a 2 × 56 well plate tray (p/n G2258-44502), the locations
were:• Vial 1: Borate buffer• Vial 2: OPA• Vial 3: FMOC
• Vial 4: Injection diluent• P1-A-1: Sample

35

Return to Contents Section

Results and Discussion

Conditions for Figure 1.

As can be seen in Figure 1, using the same chromatographic
conditions, the separation was very similar. The elution orderof the mixture on both columns was the same, and as shownin Figure 2, the relationship of retention times of the aminoacid samples was highly correlated between an Eclipse Plus
C18 and a Poroshell HPH-C18, with a correlation co-efficientof 0.997. As can be seen in the chromatograms, the retention
times were slightly less on the Poroshell HPH-C18 column. Somechromatographic differences are notable. Thus, separation ofleucine and lysine looks better on Poroshell HPH-C18, while theseparation between lysine and hydroxyproline and the sarcosine/
proline pair looks worse. As suggested in previous application
notes, the chromatography can be altered to enhance resolutionof desired peak pairs.

Parameter Value

mAU
60

A

50

Agilent Poroshell HPH C18, 4.6 × 100 mm, 2.7 μm(p/n695975-702) or Agilent Eclipse Plus C18, 4.6 × 100 mm,
3.5 μm (p/n959961-902)

Flow rate:

1.5 mL/min

Gradient:

Time (min)
0
0.35
13.4
13.5
15.7
15.
18

2

5

3
4

6

30

Agilent Poroshell HPH-C18,
4.6 × 100 mm, 2.7 µm

12

9 10

7
8

19

13
14

11

%B
2
2
57
100
100
82
end

20

15

1

40

Column:16 17 18
22

21

20
10

23

0
0

2

mAU
60

4

6

8

B

15
3

2

50

4

40
1

30

5
7
6

9

8

20

12

10

13

11

16

14

12 min10

19

Agilent ZORBAX Eclipse Plus C18,
4.6 × 100 mm, 3.5 µm

17 18
21

20

22
23

10
0
0

2

4

6

8

10

Peak ID
1. Aspartic acid
2. Glutamic acid
3. Asparagine
4. Serine
5. Glutamine
6. Histidine
7. Glycine
8. Threonine
9. Arginine
10. Alanine
11. Tyrosine
12. Cystine
13. Valine
14. Methionine
15. Norvaline
16. Tryptophan
17. Phenylalanine
18. Isoleucine
19. Leucine
20.Lysine
21. Hydroxyproline
22.Sarcosine
23.Proline

12 minFigure 1. Comparison of an Agilent Poroshell HPH C18 to an Agilent ZORBAX Eclipse Plus C18 column using the Amino Acid Method.

14
Retention time (min)Agilent Poroshell HPH-C18

12
y = 1.0201x – 0.5565
R2 = 0.9977

10
6
5
4
2
0

0

2

4
6
8
10
12
Retention time (min)Agilent ZORBAX Eclipse Plus C18

14

Figure 2. Correlation of retention times using Agilent Poroshell
HPH-C18 and Agilent ZORBAX Eclipse Plus C18 columns.
Return to Contents Section36Column dimensions
The method can easily be scaled to different column dimensions.
In this work, three column dimensions were studied. All columns
were 100 mm in length with 4.6, 3.0, or 2.1 mm internal diameter,
as shown in Figure 3. In this case, the only changes to themethod were made by altering the flow rate. Table 1 lists thegradient program used throughout. Flow rates are changed
geometrically with the diameter of the column. The flow rateused with the 4.6 × 100 mm column was 1.5 mL/minute. The
flow rates for the 3 and 2.1 mm columns were 0.62 and 0.21 mL/
min, respectively. In all cases, the low-volume heat exchanger
was used with short red tubing to minimize extra column volume.
Using the Agilent 1260 Infinity Binary LC with low dispersion
heating and tubing, the column pressure was approximately 175
bar. We observed that retention time of all analytes increasedslightly (without changing selectivity) as columns were changed
from larger to smaller internal diameter. This is due to theincrease in gradient delay time. As the flow rates are scaled andconsequently reduced from larger to smaller column ids, thegradient delay volume remains constant, thereby increasing thetime it takes for the gradient to reach the column. The difference
in retention between various column ids could potentially be
reduced or eliminated by scaling the gradient delay volume on the
LC system (adding or removing capillary length/diameter/volume
between the pump and column inlet).

9.486

22

9.664

Return to Contents Section

min

12

min

10

12

12.622

2.1 mm id
12.11912.30810.14810.4009.714

12.172

9.950

11.66511.8583.0 mm id

Figure 3. Agilent Poroshell HPH-C18 100 mm columns of different inside dimension using the amino acid method.

37

23
12

10

9.036
9.197

7.146
7.279
7.514
7.613
7.813
7.912
8.081
8.361
8.632
8.756

8

11.491

10

9.240

8.266

8.556
8.727

7.888

7.442
7.604

21
9.762

9.062

8.082

8.384
8.555

7.256
7.415

7.119

7.355

6.684
6.817
7.050

4.6 mm id

8
6.484

5.523

5.156

6

6

19

8

6.011

5.066

4.713

15

1718

7.152

6.473
6.612
6.852

4

16

6

4.379

2

3.999
4.184
4.434
4.589

3.286
3.508

4

14
13
7.699

6.925

11

5.801

4.500

3.389
3.551
3.801
3.958

10
4.861

2.751
2.963

1.701

C

B14139

1.092

2
mAU
120
100
80
60
40
20
0

4

1.755

0.459
0.599

1.602

B

B14139

1.029

2
mAU
120
100
80
60
40
20
0

8

3.578
3.750
3.995
4.153

20
0

56

2.926
3.139

40

4

20

12

9

7

1.666

60

0.979

80

0.440
0.590

mAU

3

2
1.503

A

B141391min

Lot-to-lot variability
Batch-to-batch or lot-to lot reproducibility is also an importfactor in method development. It is recommended that, before
a method is adopted, one of the earliest validation steps is to
examine the method performance on at least three columns
made from different lots.
Following good validation practice, three columns loaded withparticles from different production batches were examinedfor 4.6, 3.0, and 2.1 × 100 mm columns. The overlays of thesethree sets are shown in Figures 4A-C. As can be seen in Figure
4A, the amino acid separation on the 4.6 × 100 mm column
achieved good peak as well as baseline separation shape for allcompounds. No change in elution order was noted, and lot-to-lot
reproducibility looked good. A slight change in retention time canbe seen in Figure 4A though the k’ remained constant. However,a slight change in the wavelength switch time is required as it
is tied to the elution times of lucine and hydroxyproline. Similar
reproducibility is evident in Figures 4B and 4C for the smaller idcolumns.

9.486

12.07912min

11.336

9.624
9.327

10

11.861

11.491

10

8.959

8.283
8.453

7.987

9.422

8

23

11.491

7.152

9.762

21

7.033

12

min

8

10

11.786

11.2779.568

11.442

8.868

7.909

ALS injector programming
8.197
8.365

7.080
7.238
7.518

11.689

22

9.062

8.082

8.384
8.555

6.925

19

1718

8

6.793

5.657

6

7.157
7.317
7.599

6.876

6.473
6.612
6.852
5.721

4.418

4.771

6.362
6.514
6.794

4

7.256
7.415
7.699

5.801

4.500

4.861

3.389
3.551
3.801
3.958

2.751
2.963

2

16

14
13

10.557

0

6

20

15

6.308
6.443
6.721

20

B14141

4

4.022

60
40

C

0.925

80

2

0.437
0.572

mAU

4.377

0

2.633
2.828

20

6

1.603

0.440
0.589

40

11

4
2.684
2.885

B14138

60

1.575

80

2

B
0.956

mAU

10

4.702

0

8

3.316
3.478
3.714
3.871

20

56

3.259
3.426
3.651
3.808

40

4

12

9

7

1.666

0.440
0.590

60

1

1.503

B14139

0.979

A

1.459

80

3

2

1.414

mAU

12

min

Figure 4A. Separation of amino acid and internal standards on three lots of Agilent Poroshell HPH-C18, 4.6 × 100 mm, 2.7 μm (p/n 695975-702).

Return to Contents Section

38

mAU
120
100
80
60
40
20
0

39

B141382

4

6

8

Return to Contents Section

10
12

10

10
12

12
12.62221

12.5101011.796
11.97911.45210.708

11.57810

12.5028
11.93
12.11912.3088

12.116

11.49811.60811.792

10.891

10.222

9.890

9.605

10

12.01012.20019 20

10.14810.400

9.753

9.170

7.442
7.604

11.66511.85812.172

9.240

8.556
8.727

8.266

7.888

9.950

7.355

6.684
6.817
7.050

5.066

6.011

21

12.01012.1629.714

810.30316 17 18

9.881

9.555

8

10.041

15

9.080

8.479
8.650

8.189

7.793

7.330
7.500

7.042

8

9.795

14
13

9.611

9.303

8.398
8.568

7.237

6.531
6.675
6.963

5.861

4.379

4.713

7.119

9.664

12

10.10710.310

6

19
16 17
18

9.644

6
12

9.459

11

9.036
9.197

10

9.379

6
8.115

7.023

6

9.389

4

15

8.720
8.985
9.138

4
7.267
7.434

5.827

6

7.721

6.194
6.460
6.617
6.704
6.934
7.144

4.485
4.809

4

14
13

8.664
8.941
9.102

4

11

7.146
7.279
7.514
7.613
7.813
7.912
8.081
8.361
8.632
8.756

5.156

4.157

9

7.066
7.195
7.440
7.533
7.736
7.828
7.993
8.274

5.523

8

6.484

7

6.403

6

10

6.462

2
5

5.440

4

8

5.147

3
4.802

4

4.475

3.578
3.750
3.995
4.153

2.926
3.139

1.602

7

5.089

4.854

2
4.170

2

3.999
4.184
4.434
4.589

3.309
3.487
3.680
3.847

2.663
2.841

1.755

1.029

6

5.493

4.765

2
3.328
3.502
3.699
3.862

1.508

1.396

0.921

0.459
0.599

4

3.923
4.107
4.344
4.506

3.286
3.508

2
2.683
2.862

2

3.221
3.433

1.517

0.576

3

7.103
7.226
7.503
7.617
7.866
8.043
8.313

4.843

C
2

3.986
4.168
4.406
4.553

B14140

3.272
3.479

B
1.701

1
1.404

0.943

mAU C
120 B14138
100
80
60
40
20
0

1.651

0.454
0.582

mAU B
120 B14140
100
80
60
40
20
0

1.680

B14139
1.092

A

1.073

0.625

mAU A
120 B14139
100
80
60
40
20
0

1.110

mAU
120
100
80
60
40
20
0
0.620

mAU
120
100
80
60
40
20
0

0.648

1
20

22 23

12
min

12
min

12
min

Figure 4B. Separation of amino acid and internal standards on three lots of Agilent Poroshell HPH-C18, 3 × 100 mm, 2.7 μm (p/n 695975-502).

9

22
23

min

min

min

Figure 4C. Separation of amino acid and internal standards on three lots of Agilent Poroshell HPH-C18, 2.1 × 100 mm, 2.7 μm (p/n 695775-702).

2

4

6

8

11.1539.671

10.10411.12010.070min

Injection 225

101210.041

Injection 400
11.083

min

10

12

min

Injection 500
11.023

9.622
9.586
9.533

8.887
9.039

12

9.971

8.965
9.116
8.932
9.084

7.441
7.539
7.807
7.976
7.420
7.509
7.771
7.942

8
6.364

5.385

5.060
4.715

4.049
4.290
4.451

6

Injection 25

10

8
6.388

5.415

5.079

6

7.472
7.568
7.842
8.011

6.417

5.448

8

4.742

4.073
4.319
4.481

4
3.381

2

9.008
9.166

6.452

5.123

5.486

4.793
5.105

6

4.763

4.091
4.342
4.503

3.421

4
3.403

1.589

1.061

4.107
4.366
4.532

3.442

1.637
1.656

1.081

4

2

1.546

mAU
D
80
60
40
20
0
0

2

1.070

mAU
A
80
60
40
20
0
0
mAU
B
80
60
40
20
0
0
mAU
C
80
60
40
20
0
0

1.076

There are two approaches to achieving high pH stability in silica
HPLC columns. One way is to employ special bonding chemistry,
as in the Agilent ZORBAX Extend C18 column. This column
uses bidentate bonding to protect the silica from dissolution athigh pH. Another way to achieve high pH stability is to modify
the silica itself, making it less soluble. The surface of Poroshell
HPH particles are chemically modified to form an organic layer,
resistant to silica dissolution at high pH conditions, using aproprietary process [11].

7.884
8.051

Column lifetime is an important consideration for
chromatographers analyzing amino acid samples. Most silica
columns lose efficiency after prolonged exposure to these
conditions. Kirkland et al. [9] and Tindall and Perry [10] discussed
possible reasons for the reduced lifetime of silica columns in
phosphate buffer, but both agree that columns do not last as
long.

Figure 5 is an overlay of four chromatograms. Single 4 L bottles
of mobile phase A and B were prepared. A single 2.1 × 100
mm column was used for lifetime testing from a series of 500
analyses over a period of four weeks. In this series, approximately
102 injections were made each week using freshly opened aminoacid standard mix and reagents. At the end of the sequence,the column was flushed with 100 % B mobile phase for 40
minutes and the instrument was shut down. In this manner, themethod was run for 3.5 days and the column was stored withno analysis for 3.5 days. This simulated typical practice in a lab
where samples are run for an extended time, and then a column
is washed and stored. Storing a column in 100 % mobile phase
B was recommended in the original amino quant methods,
and is common practice in many successful laboratories thatfrequently run amino acids. A realistic lifetime study was carriedout, showing excellent lifetime of the column over one month ofuse, with over 500 standard injections, shutting down and storing
after each sequence. As can be seen in Figure 5, the 17 aminoacid sample lost no resolution and only a slight retention time
shift was seen.

7.502
7.603

Lifetime

10

12

min

Figure 5. Column lifetime test using an Agilent Poroshell HPH-C18, 2.1 × 100 mm column running an amino acid method.

Return to Contents Section

40

Conclusions

References

Agilent Poroshell HPH-C18 has selectivity similar to totally porous
Agilent ZORBAX Eclipse Plus C18. This allows easy transfer ofexisting methods such as the amino acid method. In this work, nochanges to the chromatographic conditions were made althoughchanges in the gradient could be done to improve resolution on
selected amino acids. In most cases, Poroshell HPH-C18 wasslightly less retentive than totally porous Eclipse Plus C18. The
method was investigated with 4.6, 3.0, and 2.1 mm × 100 mm
columns. Use of the low volume column heater is recommended.
In total, four particle lots were investigated, requiring only slightchanges to the wavelength switch time. A realistic lifetime study
was carried out, showing excellent lifetime of the column overone month of use, with over 500 standard injections, shutting
down and storing after each sequence.

1. Wang, X.; Barber, W. E.; Long, W. J. Applications of superficiallyporous particles: High Speed, high efficiency or both? J.
Chromatogr. A. 2012, 1228, 72-88.
2. Schuster, R.; Apfel, A. A new technique for the analysis
of primary and secondary amino acids; Application note,
Hewlett-Packard Publication number 5954-6257, 1986.
3. Schuster, R. Determination of amino acids in biological,
pharmaceutical, plant and food samples by automatedprecolumn derivatization and high-performance liquid
chromatography. J. Chromatogr. B. 1988, 431, 271-284.
4. Henderson, Jr., J. W.; Ricker, R. D.; Bidlingmeyer, B. A.;
Woodward, C. Rapid, Accurate, Sensitive and Reproducible
HPLC Analysis of Amino Acids; Application note, Agilent
Technologies, Inc. Publication number 5980-1193E, 2000.
5. Woodward, C.; Henderson, Jr., J. W.; Todd Wielgos, T. HighSpeed Amino Acid Analysis (AAA) on Sub-Two Micron
Reversed-phase (RP) Columns; Application note, Agilent
Technologies, Inc. Publication number 5989-6297EN, 2007.
6. Gratzfeld-Huesgen, A. Sensitive and Reliable Amino Acid
Analysis in Protein Hydrolysates using the Agilent 1100 Series
HPLC; Application note, Agilent Technologies, Inc. Publication
number 5968-5658EN, 1999.
7. Greene, J.; Henderson, Jr., J. W.; Wikswo, J. P. Rapid andPrecise Determination of Cellular Amino Acid Flux Rates
Using HPLC with Automated Derivatization with Absorbance
Detection; Application note, Agilent Technologies, Inc.
Publication number 5990-3283EN, 2009.
8. Henderson, Jr., J. W.; Brooks, A. Improved Amino Acid
Methods using Agilent ZORBAX Eclipse Plus C18 Columns
for a Variety of Agilent LC Instrumentation and Separation
Goals; Application note, Agilent Technologies, Inc. Publication
number 5990-4547EN, 2010.
9. Kirkland, J. J.; van Straten, M. A.; Claessens, H. A. Reversedphase high-performance liquid chromatography of basic
compounds at pH 11 with silica-based column packings. J.
Chromatogr. A. 1998, 797, 111-120.
10. Tindall, G. W.; Perry, R. L. Explanation for the enhanced
dissolution of silica column packing in high pH phosphate andcarbonate buffers. J. Chromatogr. A. 2003, 988, 309-312.
11. Anon. Extending Column Lifetime in Pharmaceutical Methods
with High pH-Stable Poroshell HPH Chemistries; Technicaloverview, Agilent Technologies, Inc. Publication number 59915022EN, 2014.

41

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Published in the USA, April 30, 2021

42