Agilent 1100 User Manual

Series diode array detector (DAD)
is below 5 pmol with a signal-to­noise ratio of 2. For the Agilent
1100 Series fluorescence detector
(FLD) the limit of detection is
below 100 fmol except for Cys-SS­Cys. Due to better selectivity we
recommend the use of the FLD
below 100 pmol. The linearity cor­relation factor for well resolved
amino acids is between 0.99998
and 0.99999 in the range from
10 pmol up to 1000 pmol for DAD.

A special amino acid report is
shown and hints for maintenance
and troubleshooting are included.
Ordering information about
columns, standards, chemicals
and capillaries, and Agilent Appli­cation Services is also included.

Angelika Gratzfeld-Huesgen

Sensitive and Reliable Amino Acid
Analysis in Protein Hydrolysates using
the Agilent 1100 Series HPLC

Pharmaceutical

Technical Note

Agilent Technologies

Innovating the HP Way

Abstract

This technical note demonstrates
the performance of amino acids
analysis on the Agilent 1100 Series
modules and systems for LC.
Detailed information regarding
instrumental and chromatographic

conditions and performance are
given. The precision for retention
times over 10 runs is as low as

0.2 % RSD, and the RSD of areas is
between 0.6 and 5 %. The limit of
detection for the Agilent 1100

Introduction

Since the introduction of auto­mated amino acid analysis on the
HP 1090 Series HPLC system
using a two step precolumn
derivatization, this technique has
become a well accepted and rou­tine method for the analysis of pri­mary and secondary amino acids
in protein hydrolysates. This tech­nical note will demonstrate that
amino acid analysis can be done
on the Agilent 1100 Series mod­ules and systems for LC using the
binary pump to obtain even better
performance than was achieved
with the HP 1090 Series. The
conditions used are the same,
that were used on the HP Amino­Quant 2 based on the HP 1090
Series system.

Experimental

HPLC Instrumentation:

The following Agilent 1100 series
modules were used:

• high pressure gradient pump
order number: G1312A

• online vacuum degasser
order number: G1322A

• autosampler
order number: G1313A ( or
thermostatted autosampler
order number: G1327A)

• thermostatted column
compartment
order number: G1316A

• diode array detector for
concentrations above 100 pmol
order number: G1315A

• fluorescence detector for
concentrations below 100 pmol
order number: G1321A

• Agilent ChemStation
order number: G1319A

• Software for amino acid reports
part number G1300-10013
(user contributed software).

One modification was made for
the standard high pressure
gradient pump. The solvent mixer
(part number G1312-87330) was
replaced by a capillary (part
number 1090-87610) or Upchurch
mixer to reduce delay volume.

Amino acid Standards

Five different concentrations of
amino acid standards were used
for evaluating the precision of
retention times and areas, and the
limit of detection and linearity.
These were 10, 25, 100, 250 and
1000 pmol/µl. The standards
contained the following
compounds:

Asp Aspartic acid
Glu Glutamic acid
Ser Serine
His Histidine
Gly Glycine
Thr Threonine
Ala Alanine
Arg Arginine
Tyr Tyrosine
Cys-SS-Cys Cystine
Val Valine
Met Methionine
Phe Phenylalanine
Ile Isoleucine
Leu Leucine
Lys Lysine
Pro Proline

Derivatization Reagents

The online derivatization was
performed using ortho-phthalalde­hyde (OPA) for the primary amino
acids and 9-fluorenylmethyl
chloroformate (FMOC) for the
secondary amino acids. A 0.4 N
borate buffer was used with pH

10.4.

Preparing mobile phases,
standards and derivatization
reagents

Mobile phase A:

1. Weigh 1.36 ± 0.025 g of sodium
acetate tri-hydrate and transfer
it into a 800 ml glass beaker.

2. Add 500 ml of purified water
and stir until all crystals are
completely dissolved.

3. Add 90 µl of triethylamine
(TEA) and mix.

4. Adjust the pH to 7.20 ± 0.05 by
adding a few drops of 1–2 %
acetic acid.

5. Add 1.5 ml of tetrahydrofuran
(THF) and mix.

FMOC reagent

1. Place several microvials in the
microvial rack.

2. Open one ampule of the FMOC
reagents. This is the approxi­mate amount needed for ten
days.

3. Pipette between 50 and 100 µl
into each microvial.

4. Cap all vials. Store vials which
are not directly used in the
refrigerator.

Borate buffer

Place 1 ml of borate buffer into
a 2 ml vial.

Water

Place 1 ml purified water into a
2 ml vial.

Preparing internal standards of
5 nmol and 500 pmol

The secondary amino acids were
quantified using sarcosine as an
internal standard. The primary
amino acids can be quantitated by
either using external standard
procedures or by using norvaline
as an internal standard. If you are
working with DTDPA to convert
cystine and cysteine to the stable
Cys-MPA during hydrolysis, then
make up your ISTD stock solu­tions in borate buffer instead of
HCl to avoid solubility problems.

Mobile phase B:

1. Weigh 1.36 g ± 0.025 g of
sodium acetate trihydrate and
transfer it into a 200 ml glass
beaker.

2. Add 100 ml of purified water
and stir until all crystals are
dissolved.

3. Adjust pH to 7.20 ± 0.05 by
adding a few drops of 1–2 %
acetic acid.

4. Add this solution into a
mixture of 200 ml acetonitrile
and 200 ml methanol and mix.

Derivatization reagents:

For convenience use microvial kit
from Agilent (part number 9301-

1388)

OPA reagent

1. Place several microvials in the
microvial rack.

2. Open one ampule of the OPA
reagents. This is the approxi­mate amount needed for ten
days. Preparing more could
cause problems with oxidation.

3. Pipette between 50 and 100 µl
into each microvial.

4. Cap all vials. Store vials which
are not directly used in the
refrigerator.

1. 5 nmol ISTD standard:
Weigh 22.3 mg sarcosine
(optional 29.3 mg norvaline)
and dissolve in 50 ml

0.1 N HCl.

2. 500 pmol ISTD standard:
Take 5 ml of 5 nmol ISTD and
dilute with 50 ml 0.1N HCl.

Preparing the calibration
standards

For standard sensitivity eight
different concentration were used:

1. 900 pmol/µl amino acid stan­dards with 500 pmol/µl internal
standards.
Place 900 µl from standard
with 1000 pmol amino acids
and 100 µl from ISTD (5 nmol)
in a 2 ml vial. Mix and place
100 µl each in microvials

2. 225 pmol/µl amino acid stan­dards with 500 pmol/µl
internal standards. Use 250
pmol amino acid standard
and follow the same procedure
as under 1.

3. 90 pmol/µl amino acid
standards with 500 pmol/µl
internal standards.
Use 100 pmol amino acid
standard and follow the same
procedure as under 1 of this
section.

For high sensitivity three
different concentrations were
used:

1. 90 pmol/µl amino acid
standards with 50 pmol/µl
internal standards.
Place 900 µl from standard
with 100 pmol amino acids and
100 µl from ISTD (500 pmol) in
2 ml vial. Mix and place 100 µl
each in microvials.

2. 22.5 pmol/µl amino acid
standards with 50 pmol/µl
internal standards.
Use 25 pmol amino acid
standard and follow the same
procedure as under 1.

3. 9 pmol/µl amino acid standards
with 50pmol/µl internal stan­dards.
Use 10 pmol amino acid
standard and follow the same
procedure as under 1.

Extended Amino Acids (EAA)

In addition to the previously
analyzed 17 amino acids which
can be found in hydrolysates (see
figure 1), the amino acid supple­ment kit contains the following
amino acids as solids, which are
of interest for amino acid analysis
in food:

norvaline
sarcosine
asparagine
glutamine
tryptophan
and hydroxyproline

Table 1 shows the stock solutions
used for different purposes.

Use these stock solutions in place
of the 5 nmol ISTD or 500 pmol
ISTD stock solutions previously
described, to prepare your calibra­tion standards.

Preparing internal standards of
10 nmol and 1 nmol

The secondary amino acids were
quantitated using sarcosine as an
internal standard. The primary
amino acids can be quantitated by
either using external standard
procedures or by using norvaline
as an internal standard. If you are
working with DTDPA to convert
cystine and cysteine to the stable
Cys-MPA during hydrolysis, then
make up your ISTD stock
solutions in a borate buffer
instead of HCl to avoid solubility
problems.

1. 10 nmol ISTD standard:
Weigh 44.6 mg sarcosine
(optional 58.6 mg norvaline)
and dissolve in 50 ml

0.1 N HCl.

2. 1 nmol ISTD standard:
Take 5 ml of 10 nmol ISTD and
dilute with 50 ml 0.1 N HCl.

Standard sensitivity

9 nmol/µl extended amino acid with 5 nmol/µl internal standards

2.25 nmol/µl extended amino acid with 5 nmol/µl internal standards
900 pmol/µl extended amino acid with 5nmol/µl internal standards

High sensitivity

900 pmol/µl extended amino acid with 500 pmol/µl internal standards
225 pmol/µl extended amino acid with 500 pmol/µl internal standards
90 pmol/µl extended amino acid with 500 pmol/µl internal standards

Table 1
Stock solutions

Preparing the stock solution of
the extended amino acid
standards

For standard sensitivity an
18 nmol/µl concentration was
used:

Weigh

59.5 mg asparagine

65.7 mg glutamine

91.8 mg tryptophan

59.0 mg 4-hydroxyproline

into a 25 ml graduated flask.

For high sensitivity a 1.8 nmol
concentration was used:

5 ml of the 18 nmol EAA was
placed into a 50 ml flask and
made up to 50 ml with

0.1N HCl

Table 2 details the preparation of
the calibration standards for stan­dard sensitivity, with table 3 show­ing the calibration standards for
high sensitivity.

Concentration 90 pmol 22.5 pmol 9 pmol

of final solution

Amount of 18 nmol EAA 5 ml 5 ml 5 ml

Dilute with 0,1N HCl — 15 ml 45 ml

Amount of diluted solution from above 5 ml 5 ml 5 ml

Amount of 10 nmol ISTD 5 ml 5 ml 5 ml

Amount of mixed solution from above 100 µl 100 µl 100 µl

Amount of 1000 pmol AA standard 900 µl — —

Amount of 250 pmol AA standard — 900 µl —

Amount of 100 pmol AA standard — — 900 µl

Table 2
Preparing the calibration standards for standard sensitivity

Concentration 90 pmol 22.5 pmol 9 pmol

of final solution

Amount of 18 nmol EAA 5 ml 5 ml 5 ml

Dilute with 0,1N HCl — 15 ml 45 ml

Amount of diluted solution from above 5 ml 5 ml 5 ml

Amount of 1 nmol ISTD 5 ml 5 ml 5 ml

Amount of mixed solution from above 100 µl 100 µl 100 µl

Amount of 100 pmol AA standard 900 µl — —

Amount of 25 pmol AA standard — 900 µl —

Amount of 10 pmol AA standard — — 900 µl

Table 3
Preparing the calibration standards for high sensitivity

Results

The analysis of amino acids using
precolumn online derivatization
with OPA and FMOC can be
accomplished using UV (figure 1)
or fluorescence detection
(figure 2). We recommend the use
of fluorescence detection if the
concentration of amino acids is
below 100 pmol.

Chromatographic conditions

Column: 200 × 2.1 mm AA column

and guard column

Mobile phase: A = 20 mMol NaAc + 0.018 %

TEA adjusted to pH 7.2
with 1-2 % acetic acid,
B = 20 % of 100 mMol NaAc
adjusted to pH 7.2 with 1-2 %
acetic acid + 40 % ACN

and 40 % MeOH
Flow rate 0.45 ml/min
Compressibility A 50 x 10-6bar
Compressibility B 115 x 10

-6

bar
Stroke: A and B Auto
Gradient: start with 100%A, at 17 min

60%B, at 18 min
100%B, at 18.1 min flow 0.45,
at 18.5 min flow 0.8,
at 23.9 min flow 0.8,
at 24 min 100%B and flow 0.45,
at 25 min 0%B

DAD UV Detector

Signal A= 338/10 nm,
Ref= 390/20 nm;
Signal B= 262/16 nm,
Ref= 324/8 nmat;
15 min signal A =262/16 nm,

Ref =324/8 nm
Oven temp: 40 ºC
Post time: 5 min

Injector program

1 Draw 5.0 µl from vial 10—borate buffer
2 Draw 1.0 µl from vial 11—OPA reagent
3 Draw 0.0 µl from vial 12—water
4 Draw 1.0 µl from sample
5 Draw 0.0 µl from vial 12—water
6 Mix 8 µl in air, max speed, six times
7 Draw 1.0 µl from vial 14—FMOC
8 Draw 0.0 µl from vial 12—water
9 Mix 9 µl in air, max speed, 3 times
10 Inject

FLD Setting Agilent 1100 Series

Excitation 340 nm
Emission 450 nm
PTM gain 12
at 14.5 min

Excitation = 266 nm

Emission = 305 nm

PTM Gain 11

Time [min]

2 4 6 8 10 12 14 16

0

Absorbance
[mAU]

0

10

20

30

40

50

60

70

80

1

2

3

4

5

6

7

8

9

10

11 12

13

14 15

16

2 Glu

1 Asp

3 Ser
4 His
5 Gly
6 Thr

7 Ala
8 Arg
9 Tyr
10 Cys-SS-Cys
11 Val

15 Leu
16 Lys
17 Pro

12 Met

14 Ile

13 Phe

Figure 1
Analysis of 250 pmol/µl amino acid standard with precolumn online derivatization and using
DAD-UV detection

Time [min]

0

2

4

6

8

10

12

14

16

18

LU

0

25

50

75

100

125

175

Asp

Glu

Ser

His

Gly

Thr

Ala

Arg

Try

Cys-SS-Cys

Val

Met

Phe

Ile

Leu

Lys

Pro

150

10 pmol standard

Figure 2
Analysis of 10 pmol/µl amino acids with fluorescence detection

The limits of detection (LOD) for
both detectors is given in table 4.

Linearity for the Agilent Series
1100 DAD is excellent for well
resolved peaks in the range from
10 to 1000 pmol/µl using standards
(figure 3). In figure 3 the correla­tion coefficient for four amino
acids is between 0.99998 and

0.99999.

Compound LOD for DAD LOD for FLD

(pmol) (pmol) (fmol)

Asp 0.6 19

Glu 0.5 18

Ser 0.9 21

His 2.1 29

Gly 1.6 21

Thr 1.3 21

Ala 1.3 20

Arg 1.2 17

Tyr 1.2 19

Cys-SS-Cys 1.7 not measured

Val 1.3 17

Met 1.2 16

Phe 1.4 17

Ile 1.4 16

Leu 1.4 18

Lys 1.2 57

Pro 2.4 22

Amount [pmol]

0

500

Area

0

100

200

300

400

500

600

700

1

2

3

4

5

17, DAD1 A

Correlation: 0.99999
for Leu

Rel. Res%(1): 13.424

Area = 0.7193022*Amt -1.1877714

Amount [pmol]

0 500

Area

0

100

200

300

400

500

600

700

1

2

3

4

5

7, DAD1 A

Correlation: 0.99999
for Thr

Rel. Res%(1): 28.342

Area = 0.71286557*Amt -2.0083654

Amount [pmol]

0 500

Area

0

100

200

300

400

500

600

700

1

2

3

4

5

2, DAD1 A

Correlation: 0.99999
for Glu

Rel. Res%(1): 23.525

Area = 0.7101211*Amt -1.3335603

Amount [pmol]

0

500

Area

0

100

200

300

400

500

600

700

1

2

3

4

5

13, DAD1 A

Correlation: 0.99998
for Cys-SS-Cys

Rel. Res%(1): 24.287

Area = 0.74520727*Amt -1.3516723

Table 4
LOD’s for fluorescence and UV detection

Figure 3
Linearity over the concentration range from 10 pmol/µl to 1000 pmol/µl

Retention time and area precision
for both detectors are shown in
table 5. Retention time precision
over 6 runs is for both detectors
below 0.2 %. Area precision for the
DAD over 6 runs is below 5 % for
a 100 pmol/µl amino acid
standard. For the fluorescence
detector the precision of areas is
close to 5 %.

Software

The standard software of the
Agilent Chemstation can be used,
and no additional macros are
needed. A template for the special
amino acids report is available in
the Amino Acids Reports software
with all necessary information and
documentation.

Compound RSD RT RSD Area

DAD FLD DAD FLD

Asp 0.05 0.139 0.58 0.924

Glu 0.14 0.155 0.62 0.576

Ser 0.17 0.156 0.84 1.015

His 0.12 0.155 1.67 1.778

Gly 0.12 0.118 1.05 1.124

Thr 0.11 0.113 0.80 0.739

Ala 0.12 0.120 0.82 0.767

Arg 0.08 0.094 0.67 0.905

Tyr 0.06 0.062 3.18 1.614

Cys-SS-Cys 0.05 not measured 1.99 not measured

Val 0.05 0.058 0.84 0.919

Met 0.05 0.045 0.91 1.236

Phe 0.04 0.048 0.90 1.079

Ile 0.04 0.050 1.18 0.759

Leu 0.04 0.040 1.05 0.952

Lys 0.05 0.060 4.93 5.107

Pro 0.04 0.044 4.14 4.379

Table 5
Precision of retention times and areas of DAD and FLD over 6 runs

Amino acid reports

The following report can be
obtained using the special method
and report template of the Amino
acid software kit (G1300-10013).
The following information is
included on the supplied disk:

• Installation procedure for the

method

• Checkout example for the

report template

• Instrument optimization

procedure for amino acid
analysis

• Customizing supplied operation

• Calculation specifications and

template and method details

• Common errors

A detailed description on how to
obtain and interpret this report is
included on the disk as a Word for
Windows document which can be
printed out. An example of the
printout is shown in figure 4.

Figure 4
An example of the report printout

Amino Acid Analysis Report

Acquisition method name: DATA:AAS.M Seq. Line: 6
Aquisition operator name: RG Vial#: 35
Sequence name: TOXIC.S Injection#: 1
Sample name: LYSOZYME
Data file name: C:\HPCHEM\1\DATA\1411F35A.D
Data analysis method name: C:\HPCHEM\1\METHODS\DEFAA.M
Injected on: 15 Nov 91 9:28 am

# Compound Ret time Area Height Width Symmetry

[min] [mAU*s] [mAU] [min]

1 ASP 1.285 226.5 80.78 0.043 0.666
2 GLU 1.477 65.8 38.79 0.025 0.468
3 SER 4.401 102.1 23.48 0.065 0.859
4 HIS 5.328 5.8 1.17 0.074 0.947
5 GLY 5.524 113.5 21.59 0.081 0.760
6 THR 5.902 74.5 14.91 0.077 0.811
7 CYS 6.094 73.4 14.86 0.075 0.847
8 ALA 7.199 139.0 28.94 0.074 0.739

9 ARG 7.459 122.1 25.03 0.075 0.786
10 TYR 8.833 27.9 5.51 0.077 0.705
11 VAL 10.730 59.1 11.67 0.077 0.770
12 MET 10.955 14.4 2.70 0.081 0.811
13 NVA 11.333 281.2 52.08 0.083 0.650
14 PHE 12.380 30.9 6.02 0.079 0.803
15 ILE 12.595 60.2 10.69 0.086 0.734

16 LEU 13.270 88.6 15.94 0.085 0.716
17 LYS 13.875 31.1 5.51 0.086 0.700
18 SAR 16.957 256.7 39.76 0.100 0.849
19 PRO 17.579 21.8 1.80 0.171 1.073

# Compound Ret time Amount Amount Residues Relative to

[min] [pmol] [ng] mole % / mole ALA
________________________________________________________________________
1 ASP 1.285 511.34 58.8 18.4 24.49 21.72
2 GLU 1.477 141.22 18.2 5.1 6.76 6.00
3 SER 4.401 243.88 21.2 8.8 11.68 10.36
4 HIS 5.328 15.56 2.1 0.6 0.75 0.66
5 GLY 5.524 261.22 14.9 9.4 12.51 11.10
6 THR 5.902 169.82 17.2 6.1 8.13 7.21
7 CYS 6.094 231.97 28.1 8.3 11.11 9.85
8 ALA 7.199 282.51 20.1 10.2 13.53 12.00
9 ARG 7.459 247.77 38.7 8.9 11.87 10.52

10 TYR 8.833 65.62 10.7 2.4 3.14 2.79
11 VAL 10.730 116.89 11.6 4.2 5.60 4.97
12 MET 10.955 25.44 3.3 0.9 1.22 1.08
13 NVA 11.333 500.00 - - - -
14 PHE 12.380 62.91 9.2 2.3 3.01 2.67
15 ILE 12.595 119.58 13.5 4.3 5.73 5.08
16 LEU 13.270 188.84 21.3 6.8 9.05 8.02
17 LYS 13.875 74.50 9.5 2.7 3.57 3.16
18 SAR 16.957 500.00 - - - -
19 PRO 17.579 22.71 2.2 0.8 1.09 0.96

Expected molecular weight (AA_ProteinMW) : 14400
Calculated minimum molecular weight : 12770
Expected MW / Calculated MW : 1.1277

min

2 4 6 8 1012141618

Norm.

0

100

200

300

400

500

DAD1 A, Sig=338,10 Ref=390,20 of 1411F35A.D

ASP

GLU

SER

HIS

GLY

THR CYS

ALA

ARG

TYR

VAL

MET

NVA

PHE

ILE

LEU

LYS

DAD1 B, Sig=262,16 Ref=324,8 of 1411F35A.D

SAR

PRO

Maintenance

In order to keep the system run­ning smoothly we recommend to
do the following:

Daily

1. Replace derivatization
reagents, borate buffer, amino
acid standards and wash water,
which are placed in the
autosampler tray.

2. Recalibration of retention
times and response factors
daily.

3. Check column and guard
column performance using sys­tem suitability report.

Every 2 days

1. Replace mobile phases A and B
with freshly-made solvents.

High column pressure occurs

1. Exchange guard column.

2. If this does not help, exchange
analytical column.

Troubleshooting

Changes in retention times—
amino acids elute earlier

• Poor resolution of His and Gly
is caused by absence of triethy­lamine.

• AA’s between Ser and Tyr elute
earlier, concentration of THF
too high. 0.4 % is already influ­encing the RT’s.

• Low concentration of sodium
acetate is influencing the later
peaks more than the earlier
ones.

• pH too high e.g. 7.6, specially
the peaks between 10 and 14
min are eluting too early.

• Oven temperature too high.
Nearly all peaks are eluting
earlier.

• High concentration of sodium
acetate decreases the resolu­tion between Ala/Arg and
Val/Met.

• High concentration of acetoni­trile lets peaks above 10 min
elute earlier.

• High concentration of
methanol decreases resolution
between Val/Met.

• If concentration of triethy­lamine is too low resolution
between Cys-Cys/Val is getting
poor.

Amino acids elute later

• THF absence is strongly
marked throughout the chro­matogram. Note especially the
splitting of the Glu peak . As
the concentration of THF is
increased, the peak splitting
decreases and the retention
time improve.

• If the pH is too low, e.g. 6.8,
loss in resolution for Val/Met
and Cys-Cys/Val/Met ( for
DAD) occurs.

• If the oven temperature is too
low e.g. 38ºC all peaks are elut­ing later.

Poor chromatographic
resolution

• Exhausted guard column.

• Damaged analytical column.

• Post column band broadening
due to too long connections.

Low Intensity chromatogram.

• OPA reagent has deteriorated.

• FMOC reagent has
deteriorated.

• Glycine contamination.

Ordering

Table 6 contains details for
ordering instrumentation and
supplies.

Amini acid standards

Each amino acid standard con­tains the following amino acids:

Asp Aspartic acid
Glu Glutamic acid
Ser Serine
His Histidine
Gly Glycine
Thr Threonine
Ala Alanine
Arg Arginine
Tyr Tyrosine
Cys-SS-Cys Cystine
Val Valine
Met Methionine
Phe Phenylalanine
Ile Isoleucine
Leu Leucine
Lys Lysine
Pro Proline

Order Number Description

Instrumentation

G1312A Agilent 1100 Series binary pump
G1312-67301 0.12 mm id capillary
or
A-330 Upchurch semi prep filter 5022-2165

G1313A Agilent 1100 Series autosampler
#011 Vial kit
or
G1327A Agilent 1100 Series thermostatted autosampler
#011 Vial kit

Optimum performance of the thermostatted autosampler requires the following capillary connections:

G1312-67305 (Pump to autosampler) Outlet capillary
5021-1823 (Autosampler to column compartment) Flexible tubing 400 mm, 0.12 mm id (no fittings)
G1316-87303 (Column compartment to inlet column) Capillary, 70 mm, 0.12 mm id
5021-1823 (Outlet column to DAD) Flexible tubing 400 mm, 0.12 mm id (no fittings)
G1322A Agilent 1100 Series vacuum degasser
G1316A Agilent 1100 Series column compartment
G1315A Agilent 1100 Series diode-array detector
#018 Standard flow cell (10 mm, 13 µl)
G1321A Agilent 1100 Series fluorescence detector
G1319A Agilent ChemStation

Supplies
5063-6588 Amino Acid Separation Kit contains:

79916AA-572 Column for amino acid analysis, 200 × 2.1 mm
79916KT-110 Guard columns, Hypersil ODS, 20 × 2.1 (3 per pack)
79900CH-010 Guard cartridge holder
79841-87609 Column connector, 35 mm, 0.12 mm id.
9301-1388 Crimpcap microvials (100 per pack)
5181-1210 Crimpcaps, butyl rubber (100 per pack)

Amino Acid Standards

5061-3330 1 nmol/µl AA standard 10 × 1 ml ampules
5061-3331 250 pmol/µl AA standard 10 × 1 ml ampules
5061-3332 100 pmol/µl AA standard 10 × 1 ml ampules
5061-3333 25 pmol/µl AA standard 10 × 1 ml ampules
5061-3334 10 pmol/µl AA standard 10 × 1 ml ampules
5062-2478 Supplemental Amino Acid Kit includes 1 g each norva

line, sarcosine, asparagine, glutamine, tryptophan
and 4-hydroxyproline

Reagents for amino acid analysis

5061-3335 OPA reagent, 10 mg/ml each in 0.4 M borate buffer

o-phthalaldehyde (OPA) and 3-mercaptoproprionic
acid, 6 ampules

5061-3337 FMOC reagent, 2.5 mg/ml in acetonitrile, 9-fluorenyl

methylchloroformate, 1 ml
5061-3339 Borate buffer, 100 ml
5062-2479 DTDPA (dithiodipropionic) reagent for analysis of

cysteine, 5 g

Software

G1300-10013 Software for special method set up and reporting—

software description included as document file

(User contributed software)
5968-5796E Sensitive and reliable amino acid analysis in protein

hydrolysates using the Agilent 1100 Series HPLC

Support

(not included in kit

)

H1170A Application Optimization Service—onsite, including

travel from 1–10 continuous days

Table 6
Ordering information

Agilent chemical analysis
professional services for
chromatography

If you do not have the technical
recources available to set up new
applications and methods, you
may choose to use Agilent’s Appli­cation Optimization Service
(AOS).

Our application experts are
specialists in implementing and
optimizing applications,
integrating new analytical
technologies into the laboratory,
and complying with global quality
and regulatory standards.

Each application expert has a
minimum of five years practical
laboratory experience and is
factory trained—at an R&D level.
Most have advanced degrees in
science or technology.

With the Agilent Technologies
AOS application experts at your
side, you can:

• accelerate integration of new
analytical technologies into
your laboratory operations to
remain competitive

• decrease research and develop­ment cycle time to reduce the
time it takes to get your
products to market

• establish a benchmark for
instrument performance to
ensure confidence in analytical
results.

Additionally, all of our application
experts adhere to ISO-registered
quality processes.

For the latest information and services visit
our world wide web site:
http://www.agilent.com/chem

Agilent Technologies

Innovating the HP Way

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All Rights Reserved. Reproduction, adaptation
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copyright laws.

Publication Number 5968-5658E