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:
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Innovating the HP Way
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Publication Number 5968-5658E
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