Rapid, Accurate, Sensitive, and ReproducibleHPLC Analysis of Amino Acids

Amino Acid Analysis Using Zorbax Eclipse-AAA Columnsand the Agilent 1100 HPLC

John W. Henderson, Robert D. Ricker, Brian A. Bidlingmeyer,and Cliff Woodward

An ideal, quantitative amino acidanalysis combines speed andsensitivity with reliability of boththe derivatization reaction andthe analytical technique. Thesegoals are achieved with auto-mated, online derivatization usingo-phthalaldehyde (OPA) forprimary amino acids and 9-fluorenylmethyl chloroformate(FMOC) for secondary aminoacids; the automatedderivatization is then integratedwith rugged HPLC analysis. Thecomplete procedure is rapid,accurate, sensitive, and reproduc-ible using the Agilent 1100 HPLC.

Combining OPA and FMOC chemis-tries enables fast pre-columnderivatization of amino acids (AA)for chromatographic analysis. Thereaction mixture is buffered at apH of 10.2, which allows directderivatization of acid hydrolyzedprotein/peptide samples. Theprimary AA�s are reacted first withOPA using 3-mercaptopropionicacid (3-MPA). The secondary AA�sdo not react with the OPA; but arethen derivatized using FMOC. Theincorporation of 3-MPA into theindoles decreases their hydropho-bicity, and as a result, the OPA-derivatives elute chromatographi-cally before the FMOC derivatives.Excess FMOC and its degradationproducts elute after the last of thesecondary AA�s and do not inter-fere with the analysis.The derivatization process is fast

and is easily automated using theAgilent 1313A autosampler. Be-cause of the advantageous reactionspeeds, both derivatizations arecomplete at room temperature.The automated procedure providesa high degree of reproducibility.Total analysis from injection toinjection can be achieved in aslittle as 14 min (10-min analysistime) on the 75-mm column. On the150-cm column total run time is 26min (16-min analysis time). Bothanalyses provide high samplethroughput.

SEPARATION OPTIONS

The Zorbax Eclipse-AAA columncontains batch-qualified reversed-phase material. When used accord-ing to the protocol described inthis Technical Note, the columnenables the user to separate the

amino acids commonly found inprotein/peptide hydrolysates.

The A and B mobile-phase compo-nents are easy to prepare, and thegradient consists of linear seg-ments (refer to ExperimentalConditions, Mobile Phase section,for details). This combinationcontributes to a rugged protocolthat can be accomplished on theAgilent 1100 HPLC using eitherbinary or quaternary solvent-delivery systems.The chromatogram in Figure 1illustrates typical routine sensitiv-ity in high-throughput applicationsthat can be obtained on the Agilent1100 HPLC binary system using the1100 Diode Array Detector (DAD).A single run can be completed in14 minutes (including re-equilibra-tion) with adequate resolution.Separation conditions are listed in

mAU

0

5

10

min0 4 6 8 10 12

mAU

0

5

10

15

2

1 2

3

4

5

6

78

9

10 11 1221

2019

1817

16

1514

13

22

23

2421

A

B

338 nm

262 nm

Figure 1: Routine Analysis, High-Throughput Separation of 24 AminoAcids Using the Eclipse-AAA Protocol. The column dimensions are 4.6 x75 mm, 3.5 µm. See Table 1 for peak identification. Detection: A. 338 nm(OPA amino acids), B. 262 nm (FMOC-amino acids).

the Experimental Conditionssection. The primary amino acids(OPA-derivatized) shown in Figure1A, are monitored at 338 nm whilethe secondary amino acids (FMOC-derivatized) shown in Figure 1B,are monitored at 262 nm. Theamount injected was 125 pmoles ofeach amino acid, in 0.5 µL.

Table 1. Amino Acid Elution OrderUsing Eclipse-AAA Protocol

Peak Amino Acid 3-LetterNo. Code1 Aspartate ASP2 Glutamate GLU3 Asparagine ASN4 Serine SER5 Glutamine GLN6 Histidine HIS7 Glycine GLY8 Threonine THR9 Citrulline CIT10 Arginine ARG11 Alanine ALA12 Tyrosine TYR13 Cystine CY214 Valine VAL15 Methionine MET16 Norvaline NVA17 Tryptophan TRP18 Phenylalanine PHE19 Isoleucine ILE20 Leucine LEU21 Lysine LYS22 Hydroxyproline HYP23 Sarcosine SAR24 Proline PRO

When more resolution is desiredthan is available from the high-throughput separation on a 75-mmcolumn (Fig. 1), a 150-mm columnlength should be used. Figure 2shows a typical routine sensitivity,high-resolution separation ob-tained from two different ZorbaxEclipse-AAA 150-mm lengthcolumns � one with 3.5µm particlesand the other with 5µm particles.The chromatograms show the 338nm UV signal that detects the OPA-derivatized primary amino acids.These separations are quite similarin appearance, but the chromato-gram for the 3.5-µm column demon-

mAU

0

4

8

min0 5 10 15 20 25

mAU

02468

1

2

3

4

5678

9

10 11 12 131415

1716

1819

20

3

21

12 4

5

67 8

9

1110 12 1314

15

1617

1819

20

21

5 µm, Zorbax Eclipse-AAA

3.5 µm Zorbax Eclipse-AAA

mAU

05

mAU

05

10

min0 10 15 20 25

LU

0500

1000

5

12

3

4

5678

9

10 11 12 131415

16 171819

2021

21

2223

24

12

3

4

5

678

9

10 11 12 141516 17

18

24

2322

21

2019

A

B

C

338 nm

262 nm

FLD

mAU

02468

min0 10 15 20 25

mAU

0

4

8

12

5

12

3

4

5

678

9

10 11 12 1314

15

1617

1819

20

21

23

24

22

21

338 nm

262 nm

A

B

Figure 2: High-Resolution Analysis of 21 Amino Acids: on the 5µm and3.5µm Zorbax Eclipse-AAA Column. Column dimensions are 4.6 x 150mm. See Table 1 for peak identification. Detection: 338 nm (OPA aminoacids).

Figure 3: High Sensitivity, High-Resolution Analysis of Amino Acids UsingDifferent Detection Modes and the Zorbax Eclipse-AAA Protocol. Thecolumn dimensions are 4.6 x 150 mm, 3.5 µm. See Table 1 for peak identi-fication. Detection: A. UV 338 nm (OPA amino acids), B. UV 262 nm(FMOC-amino acids), C. fluorescence (see Experimental Conditions).

Figure 4: Routine Analysis, High Resolution of 24 Amino Acids Using theEclipse-AAA Protocol. The column dimensions are 4.6 x 150 mm, 5 µm.See Table 1 for peak identification. Detection: A. UV 338 nm (OPA aminoacids), B. UV 262 nm (FMOC-amino acids).

strates higher resolution resultingfrom higher efficiency. Back pres-sure on the column containing thesmaller, 3.5 µm, particles is 240-300bar (3530-4410 psi), while the backpressure of the column having 5µm particles is 160-210 bar (2350-3090 psi). Thus, if only primaryamino acids are of interest and the15-cm column length is used, thelarger 5-µm packing is a betterchoice because of less systembackpressure.

The value of the 15-cm columncontaining 3.5-µm particles, overits 5-µm counterpart, is clearlyillustrated by comparing Figures 3and 4. Figure 3A shows separationof the primary amino acids,monitored at 338 nm; Figure 3Bshows the separation monitored at262 nm; and Figure 3C shows theanalysis using fluorescence detec-tion. Note the resolution betweenpeaks #21 (lysine) and #22 (hy-droxyproline) in Figures 3 and 4.The increase in resolution betweenthese peaks, when using the longercolumn with smaller particles(150mm, 3.5 µm), provides a longertime-window that facilitateswavelength switching of the DAD orFLD between peaks #21 and #22.

When monitoring at 262 nm (Fig.3B), a small baseline �hump� elutesbetween 7 and 10 minutes due toderivatization byproducts. Sinceonly the primary AA�s are moni-tored (338 nm) during this time,the �hump� has no impact on theirdetection or resolution. It is best tomonitor at two wavelengths fordetection of secondary amino acidssuch as hydroxyproline. If this isnot desirable, wavelength switchingcan be used.

The specific time to switch fluores-cence (or UV) wavelengths maydiffer due to minor variations intemperature, mobile phase, etc. InFigure 3C the FLD signal moni-tored at 450 nm (Ex = 340 nm), isprogrammed to change to 305 nm

(Ex = 266 nm) after peak #21(lysine) elutes but before peak #22(hydroxyproline) elutes. In thiscase, the switch was programmedto occur at exactly 15 minutes. Forspecific details, see the Experimen-tal Conditions Section, DetectionSettings. After Peak #24 (proline)elutes, the gradient increases to100% channel B to elute reactionbyproducts from the column. Afterthe step gradient up to 100% B for3.7 minutes, a programmed returnto the starting conditions equili-brates the column for the nextinjection. Also note in the fluores-cence chromatogram, that Peak #13(cystine) does not fluoresce underthese conditions and is not de-tected.

Lysine�Hydroxyproline Separa-tion and Wavelength Switching

Analysis of lysine and hydroxypro-line has a major impact on thechoice of detection parameters andcolumn configuration, as well asthe resulting runtime. Amino acidseluting prior to hydroxyproline(up to and including lysine) arederivatized with OPA and aredetected at 338 nm. Hydroxypro-line elutes immediately after lysineand is the first FMOC-derivatizedamino acid to elute; detection mustbe at 262nm. The simplest solutionis continuous collection of 338 nmand 262 nm data in two separatesignals using the Agilent 1100 DAD

or MWD (multiwavelength detec-tor).

If the DAD and MWD are notavailable, the wavelength collectedin a single channel can be switchedunder carefully chosen conditions,for detection of both OPA andFMOC-derivatized amino acids.Collection of data in a singlechannel may be necessary, forinstance, when using the Agilent1100 VWD (variable wavelengthdetector). Increased resolutionbetween lysine and hydroxyprolineis possible when using more-complex gradient-profiles. Checkon the web for additional informa-tion at www.agilent.com/chem inthe �Technical Support� / �UserContributed Software�.

When hydroxyproline is not ofinterest in the sample (e.g., inanalysis of protein hydrolysates),it is possible to use any of thecolumn configurations and switchwavelength at a time betweenlysine elution and elution of thefirst FMOC-amino acid (sarcosineor proline). In this scenario, the 4.6x 75 mm column size is adequateand has the advantage of half theanalysis time.

COMPARISON WITHAMINOQUANT METHODON THE HP1090 HPLC

Figure 5A shows a chromatogram

mAU

010203040

min0 5 10 15 20 25

mAU

02468

A

BAsp/Glu

Asp/Glu

His/Gly

Ala/Arg

Val/Met Phe/Ile

His/Gly

Val/Met

Phe/IleArg/Ala

Figure 5: Comparison of Amino-Acid Analyses. A) AminoQuant Methodon the HP 1090 HPLC with a Hypersil AA Column. B) Zorbax Eclipse-AAA Column (4.6 x 150 mm, 5 µm) on the Agilent 1100 HPLC.

0

1000

2000

3000

4000

5000

0 100 200 300 400 500 600

pmoles

Pea

k A

rea

Asp

His

Thr

Cys

Lys

B

0

20

40

60

0 100 200 300 400 500 600

pmoles

Pea

k A

rea

Asp

His

Thr

Cys

Lys

A

the Agilent 1100 HPLC. Thisseparation approach offers im-proved retention of five criticalpairs compared to the originalAminoQuant method on theHP1090 HPLC system. The repro-ducibility of the analysis of aminoacids on the Eclipse-AAA columnis comparable to that of theoriginal AminoQuant method, andthe mobile phase is more straight-forward to prepare with only a pHadjustment of the buffer (solventA) needed.

from the original AminoQuantmethod (Application Notes, HPPub. No. 5954-6257) on the HP1090HPLC using the specified mobilephase, column and flow rate. Thereare five critical pairs (asp/glu, his/gly, ala/arg, val/met and phe/ile) inthis separation. The asp/glu pairelutes very closely to the voidvolume.

Figure 5B shows a chromatogramobtained using the Zorbax Eclipse-AAA column. The resolution of allof the critical pairs is improved,especially the first pair, asp/glu,which now has increased retentionand is moved significantly awayfrom the column�s void volume.Note also, that arginine elutesbefore alanine when using theEclipse-AAA column, compared tothe original AminoQuant methodon the same HP 1090 instrument.

REPRODUCIBILITY

Table 2 shows the results fromreplicate injections (n=6) of theamino acid reaction mixtureseparated using a Zorbax Eclipse-AAA column (4.6 x 150 mm, 3.5µm). Each run represents anindividual derivatization and itschromatographic separation.Retention time reproducibility isquite good with an average % rela-tive standard deviation (%rsd) of0.18%. The reproducibility of thederivatization, as represented bythe peak area has an average %rsdof 2.0. These data are comparableto those published for the originalAminoQuant method on the HP1090 instrument (0.23 % and 2.3 %respectively (LC/GC International,Volume 5, Number 2, Feb 1992, pp.44-49).

LINEARITY AND SENSITIVITY

Linearity for the Eclipse-AAAprotocol is demonstrated for therange of 4.5 pmoles to 450 pmoles,using amino acid standards (0.5µlsample). Figure 6 shows calibrationcurves for several amino acids

using DAD or FLD detection. Thecorrelation coefficient for all 24amino acids is between 0.99900and 1.00000 using either the DADor FLD detector for calibration.

Detection of derivatized aminoacids at two low levels, 5 pmolesand 50 pmoles, is shown in Figures7 and 8 for the DAD and FLD,respectively. Using the DAD (Fig. 7),each amino acid in the standardmix can be resolved at a level ofapproximately 10 pmoles. The FLD(Fig. 8) shows higher sensitivitythan the DAD.

Amino Retention Peak AreaAcid % rsd % rsdASP 0.58 0.8GLU 0.33 3.0ASN 0.16 2.2SER 0.12 2.8GLN 0.12 2.4HIS 0.11 2.7GLY 0.15 2.5THR 0.12 1.1CIT 0.10 3.5ARG 0.36 2.3ALA 0.11 0.9TYR 0.12 0.7CY2 0.17 0.6VAL 0.16 0.5MET 0.17 1.1NVA 0.15 0.7TRP 0.18 0.8PHE 0.14 0.8ILE 0.14 1.1LEU 0.18 1.0LYS 0.19 3.2HYP 0.13 4.2SAR 0.14 6.8PRO 0.12 2.7Mean 0.18 2.0

Figure 6: Calibration curves foranalysis of amino acid derivativesby UV (A) or FLD (B) detection.Linearity is demonstrated for theconcentration range of 4.5 to 450pmoles in 0.5 µl of sample.

Table 2: Reproducibility of theZorbax Eclipse-AAA Protocol forthe Analysis of Amino Acids. AnAgilent 1100 System with quater-nary pump was used. Valuesrepresent six replicate analyses.

CONCLUSIONUsing OPA and FMOC chemistries,amino acid analysis of protein andpeptide hydrolysates can beperformed in ten minutes using theZorbax Eclipse-AAA column and

The choice of column dependsupon the analysis speed andresolution desired:

ZORBAX Eclipse-AAA4.6 x 75 mm (3.5 µm) for routinesensitivity, high-throughput workusing the DAD.

ZORBAX Eclipse-AAA4.6 x 150 mm (5 µm), for routinesensitivity, high-resolution work atlower back pressures, using theDAD

ZORBAX Eclipse-AAA4.6 x 150 mm (3.5 µm), for highsensitivity, high-resolution workusing the FLD.

ZORBAX Eclipse-AAA3.0 x 150 mm, (3.5 µm), available inFall of 2000, is for high sensitivity,high-resolution work with lesssolvent and sample consumption.

Using the Agilent 1313A auto-sampler to automate the pre-column derivatization procedureresults in a speedy and reproduc-ible reaction with minimal opera-tor intervention. This protocol canbe used for routine analysis of bothprimary and secondary aminoacids using the DAD and collectingtwo wavelengths, or with pro-grammed wavelength switching.

For high-sensitivity work, thefluorescence detector is required.

EXPERIMENTAL CONDITIONS

Chromatograms shown in Figures1 � 8 were obtained using thefollowing experimental conditions:

InstrumentThe recommended chromato-graphic system is the Agilent 1100HPLC: G1312A Binary pump withG1315A Diode Array Detector(DAD), 6-mm or 10-mm flow cell,and/or G1315A FluorescenceDetector (FLD). While the resultsshown here were obtained thebinary pump, this procedure hasalso been used with the Agilent1100 quaternary pump (G1311A).

mAU

-1

0

1

2

3

min0 2 4 6 8 10

mAU

-1

0

1

2

3

21

3

4

5 67 8 9

10

1112 13 14 15

16

1 2

34 5 6 7 8 9 10

11

12

13 14

15 16

5 pmol

50 pmol

LU

0

50

100

150

200

min0 2 4 6 8 10

LU

0

50

100

150

200

21

3

4

5 6

7

89

10

1112 1314

15

17

1 2

3

45

6 7 8 910

11 12

13 14 15 17

5 pmol

50 pmol

HPLC ColumnsZORBAX Eclipse-AAA4.6 x 75 mm, 3.5 µmPN 966400-902

ZORBAX Eclipse-AAA4.6 x 150 mm, 3.5 µmPN 963400-902

ZORBAX Eclipse-AAA4.6 x 150 mm, 5 µmPN 993400-902Optional guard column*ZORBAX Eclipse�AAA4.6 x 12.5 mm, 5 µm, 4/PKPN 820950-931

ZORBAX Eclipse-AAA3.0 x 150 mm, 3.5 µmAvailable in Fall of 2000.* The optional guard columnshould be installed directly preced-ing the analytical column, using alow dead-volume connector.

Mobile Phase

A: 40 mM Na2HPO

4 pH 7.8 [5.5 g

NaH2PO

4, monohydrate + 1 liter

water, adjust to pH 7.8 with NaOHsolution (10 N)]B: ACN: MeOH: water (45:45:10, v/v/v)It is convenient to make MobilePhase A as a 10X stock solutionwith no pH adjustment. Thesolution can be kept for severalweeks and can be diluted andtitrated to pH 7.8, as needed.All mobile-phase solvents shouldbe HPLC grade.

Pump Settings

Flow: 2 mL/minStoptime: 14 min (75-mm column)or 26 min (150-mm column)Post time: off

Auxiliary Pump Settings:Max. flow ramp: 100 mL/min2

Compressibility A: 50 x 10-6

Minimal Stroke A: 20 µLCompressibility B: 115 x 10-6

Minimal Stroke B: Auto

Figure 8: Fluorescence-Detector Response Using Different ConcentrationsAmino Acids. Gain for the Photomultiplier Tube (PMT) = 10.

Figure 7: UV-Detector Response 338 nm (OPA-derivatives) Using DifferentConcentrations of Amino Acids.

46

7 8 10 12 1311

14

15

16

18 19

20 21

DAD

FLD

21

1 2

4

67 8

10 11 12

13

14 15

16

1819 2021

1 2 64 7

8 10 11 1213

14 15

1819 20 24

24

2019

181514

13

1211

10

87

6

2

4

1

CBA

Gradients:

For 75 mm column lengthTime (min) % B0 01 09.8 5710 10012 10012.5 014 0

For 150 mm column lengthTime (min) % B0 01.9 018.1 5718.6 10022.3 10023.2 026 0Note: To extend column life, flushcolumn with 10 column volumes of100% B when column will not beused for periods of overnight orlonger.

Detector SettingsDAD:Required Lamps:UV lamp: yesVis. lamp: noUV: 338 nm, 10 nm bandwidth(bw), reference: 390 nm, 20 nm bw(for OPA-amino acids)262 nm, 16 nm bw, reference: 324nm, 8 nm bw (for FMOC-aminoacids)Peakwidth: >0.03 min (0.5 s)Slit: 4 nm

FLD:For 75 mm columnTime Ex/Em PMT(min) (nm) Gain0 340/450 108.5* 266/305 9

For 150 mm columnTime Ex/Em PMT(min) (nm) Gain0 340/450 1015* 266/305 9

*The specific time to switch fluo-

rescence wavelengths may differdue to variations in temperature,mobile phase, etc.

Peakwidth: >0.5 min

Autosampler:See vial positioning (Fig. 9)

Injector program:Draw 2.5 µL from vial 1 (boratebuffer)Draw 0.5 µL from sample (e.g.,choose vial position #11 for aminoacid sample)Mix 3 µL �in air�, max speed, 2xWait 0.5 minDraw 0 µL from vial 2 (needle washusing water in uncapped vial)Draw 0.5 µL from vial 3 (OPA)Mix 3.5 µL �in air�, max speed, 6xDraw 0 µL from vial 2 (needle washusing water in uncapped vial)Draw 0.5 µL from vial 4 (FMOC)Mix 4 µL �in air�, max speed, 6x[Optional needle rinse for highsensitivity use: Draw 0.0 µL fromvial 6 (ACN, acetonitrile)]Draw 32 µL from vial 5 (water)Mix 18 µL �in air�, max speed, 2xInject

Auxiliary:Drawspeed: 200 µL/minEjectspeed: 600 µL/minDraw position: 0.0 mm

because of the limited volumesinvolved. The inserts are compat-ible with wide-opening screw-top(Fig. 10B-C) or crimp-top vials. Forthis procedure snap-cap vialsshould not be used because anairtight seal is needed for bothFMOC, because it is highly volatile,and OPA, because it slowly de-grades in the presence of oxygen.Be careful not to use vials or capsdesigned for other instruments, asthese may damage the AgilentG1313A autosampler.

Column Compartment:Temperature: 40°C (left and rightside)Enable analysis: When temperatureis within setpoint +/- 0.8°C

Derivatization ReagentsBorate Buffer:Agilent PN 5061-3339Solution is 0.4 N in water, pH 10.2.Keep refrigerated (4°C). Dispenseas necessary.

FMOC Reagent:Agilent PN 5061-3337Pipette 100-µL aliquots of the 1-mLFMOC reagent into conical inserts,cap immediately and refrigerate(4°C); solution is useable for 7 - 10days, maximum, after dispensing.

WATER

FMOC

OPA

WATER

AA SAMPLE

BORATE

Figure 9: Position of reagent vialsin the Agilent 1313A autosampler.This positioning of vials is de-signed for the listed injectorprogram.

Figure 10: Insert, Vial, and Cap.Photo of conical insert A (AgilentPN 5181-1270), amber wide-openingvial B (Agilent PN 5182-0716), andscrew cap C (Agilent PN 5182-0721), for amino acid analysisusing the Agilent 1100autosampler.

Vials:Conical vial inserts with polymerfeet (Fig. 10A) are required to holdthe OPA and FMOC reagents

OPA Reagent:Agilent PN 5061-3335Pipette 100-µL aliquots of the 1-mLOPA reagent into conical inserts,cap immediately and refrigerate(4°C); solution is useable for 7 - 10days, maximum, after dispensing.

Water: Deionized, HPLC grade

See Ordering Information fordescriptions and part numbers.

SAMPLE PREPARATIONNote: Each reagent vial should bereplaced every day. Each 1 mLampoule of reagent containssufficient solution to last about tendays (1000 µL/100 µL = 10 days).

Amino Acid Mix for Chromato-graphic ComparisonsFor chromatographic analyses, 17amino acids from the 250 pmol/µLstandard mix (PN: 5061-3331), pluscitrulline and the 6 supplementalamino acids, were combined at aconcentration of approximately250 pmol/µL. The mixture wasprepared by combining the twostock solutions described below.Add 1 µL of the supplementalamino acid stock solution to afresh aliquot of the 250-pmolestandard (100µL) in the conicalvial insert. Mix using a vortexmixer to complete the 24-compo-nent standard ready for injection(250 pmol/L).

250 pmole standard:Divide 1 mL ampoule of 250pmol/µL amino acids (PN 5061-3331)into 100 µL portions in conicalvial inserts, cap and refrigeratealiquots at 4°C.

Supplemental amino acid stocksolution:Weigh about 0.25 mmoles of eachauxiliary amino acid (gln, asn, trp,nva, hyp, sar) from kit (PN 5062-2478) into a 20-mL vial. Add 5 mLdeionized water and sonicate in a

hot water bath until dissolved.Add another 5 mL water tocomplete dilution. Store in refrig-erator (4°C). Citrulline (Sigma-Aldrich Co., St. Louis, MO) wasadded in this mix at the sameconcentration.

For storage, do not combinesupplemental amino acids withamino acid standards. Some ofthese supplemental amino acidsdegrade in HCl (especiallyglutamine, and to a lesser extent,asparagine).

Amino Acid Mix for CalibrationCurvesFor the construction of calibrationcurves, 17 amino acids, plus the 4extended amino acids, are com-bined at various concentrationswith fixed amounts of internalstandards. The internal standards(ISTD) (norvaline and sarcosine)are part of the supplemental aminoacid kit (PN: 5062-2478). Theremaining amino acids in this kit(gln, asn, trp, hyp) form theextended amino acids (EAA). Tomake the appropriate solutions,refer to Tables 3 and 4 for low andhigh sensitivity standards, respec-tively.

Amino acid standards (10 pmol/µLto 1nmol/µL):Divide each 1 mL ampoule ofstandards PN 5061-3330 through5061-3334) into 100 µL portions inconical vial inserts, cap andrefrigerate aliquots at 4°C. Calibra-tion curves may be made usingfrom 2 to 5 standards, dependingon experimental need.

Extended amino acid (EAA) stocksolution:This solution is made using four ofthe six amino acids in the supple-mental amino acid kit (PN: 5062-2478). For use with low-sensitivitystandards (Table 3), make a 25-mL

solution containing 18 nmol/µL ofglutamine, asparagine, tryptophan,and 4-hydroxy-proline in deionizedwater. Sonicate the solution untildissolved. Store the solutionrefrigerated at 4°C. For use withhigh-sensitivity standards (Table4), make a 1.8 nmol/µL solution bydiluting 5 mL of the 18 nmol/µLstandard with 45 mL deionizedH

2O.

Internal standards (ISTD) stocksolution:These solutions are made usingtwo of the six amino acids in thesupplemental amino acid kit (PN:5062-2478). For use with low-sensitivity standards (Table 3),make a 25-mL solution containing10 nmol/µL of norvaline andsarcosine in deionized water.Sonicate the solution until dis-solved. Store in refrigerator (4°C).For use with high-sensitivitystandards (Table 4), make a1 nmol/µL solution by diluting5 mL of the 10 nmol/µL standardwith 45 mL deionized H

2O. Store in

refrigerator (4°C).

ADDITIONAL SUPPORT

User-contributed ChemstationMethod files for each column type,written documentation, as well asan amino acid report and macro,are available by download via theAgilent web site atwww.agilent.com/chem, under�Technical Support� / �User Con-tributed Software�.

Table 3: Preparation of Low-Sensitivity Amino Acid Standard Solutions.Prepare the three low-sensitivity standards by mixing together stocksolutions in the volumes shown.

Concentration of Final AA Solutions (pmol/µL)900 225 90

Take 5 mL 18 nmol EAA 5mL 5mL 5mLDilute with 0.1NHCl � 15mL 45mLDiluted EAA mix 5mL 20mL 50mL

Take 5 mL diluted EAA mix 5mL 5mL 5mLAdd 10 nmol ISTD solution 5mL 5mL 5mLEAA-ISTD mix 10mL 10mL 10mL

Take 100 µL EAA-ISTD mix 100µL 100µL 100µLAdd 1000 pmol AA standard 900µL � �Add 250 pmol AA standard � 900µL �Add 100 pmol AA standard � � 900µLFinal AA Solution withEAA and 500 pmol/µL ISTD 1 mL 1 mL 1 mL

Table 4: Preparation of High-Sensitivity Amino Acid Standard Solutions.Prepare the three high-sensitivity standards by mixing together stocksolutions in the volumes shown.

Concentration of Final AA Solutions (pmol/µL)90 22.5 9

Take 5 mL 1.8 nmol EAA 5mL 5mL 5mLDilute with 0.1NHCl � 15mL 45mLDiluted EAA mix 5mL 20mL 50mL

Take 5 mL diluted EAA mix 5mL 5mL 5mLAdd 1 nmol ISTD solution 5mL 5mL 5mLEAA-ISTD mix 10mL 10 mL 10 mL

Take 100µL EAA-ISTD mix 100µL 100µL 100µLAdd 100 pmol AA standard 900µL � �Add 25 pmol AA standard � 900µL �Add 10 pmol AA standard � � 900µLFinal AA Solution withEAA and 50 pmol/µL ISTD 1mL 1mL 1mL

ORDERING INFORMATION

Eclipse-AAA HPLC ColumnsDescription Size Particle Agilent

(mm) Size (µm) Part No.Analytical routine sensitivity,high-resolution 4.6 x 150 5µm 993400-902Analytical high sensitivity, highresolution work using the FLD 4.6 x 150 3.5µm 963400-902Analytical, routine sensitivity,high-throughput 4.6 x 75 3.5µm 966400-902Analytical high sensitivity, highresolution work�DAD or FLD 3.0 x 150 3.5µm 961400-302Guard (4/pk) 4.6 x 12.5 5µm 820950-931Guard Hardware Kit � � 820777-901

Derivatization ReagentsDescription Agilent

Part No.Borate Buffer: 0.4 M in water, pH 10.2, 100mL 5061-3339FMOC Reagent, 2.5 mg/mL in ACN, 10 x 1 mL ampoules 5061-3337OPA Reagent, 10mg/mL in 0.4M borate buffer and3-mercaptoproprionic acid, 6 x 1mL ampoules 5061-3335DTDPA Reagent for analysis of cysteine, 5g 5062-2479

VialsDescription Agilent

Part No.100 µL Conical insert with polymer feet, 100/pk 5181-1270Amber, wide-opening, write-on, screw-topvial, 2mL,100/pk 5182-0716Green Screw Cap, PTFE/silicone septum, 100/pk 5182-0721

StandardsDescription Agilent

Part No.Amino Acid Standard in 0.1 M HCl, 10 x 1mL ampoules

1 nmol /ml 5061-3330250 pmol /ml 5061-3331100 pmol /ml 5061-333225 pmol /ml 5061-333310 pmol /ml 5061-3334

Supplemental Amino Acids:Nva, Sar, Asn, Gln, Trp, Hyp, 1g each 5062-2478

Additional SupportDescriptionUser-contributed Chemstation method files, custom amino-acid reportand macro, and any additional documentation are available through theweb at www.agilent.com/chem under �Technical Support� / �User Con-tributed Software�.

Printed in USAPart No. 5980-1193EFor more information on our products, visit the Agilent Technologies home page at: www.agilent.com/chem/supplies

Copyright 2000 Agilent Technolo-gies All Rights Reserved. Reproduc-tion, adaptation or translationwithout prior written permissionis prohibited, except as allowedunder the copyright laws.