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Group/Presentation Title

Agilent RestrictedMonth ##, 200X

High-Speed Amino Acid Analysis (AAA) on 1.8µmReversed-phase (RP) Columns

Squeezing More into Less

Cliff Woodward1, John Henderson1 and Todd Wielgos2

1Agilent Technologies and 2Baxter Healthcare





Introduction: Can we Squeeze More into Less?

Current State:

1. Derivatization and analysis are totally automated2. Cycle time approximately 35 minutes3. Peak shapes of early eluters Asp and Glu relatively

broad4. Reproducibility of secondary AAs less than other AAs5. Linearity by linear regression is almost 1.06. Average area reproducibility is below 3%7. Scalability is limited (3.0 or 4.6mm id)

8. Number of usable columns is limited (4); number ofphases is 1

?





o- Phthalaldehyde (OPA) and 9-Fluorenylmethyl

chloroformate (FMOC) Reactions with Amines

RR’NH

+ orRNH2

Fluorescence: Ex 266nm, Em 305nmDAD: 262, 16nm; Ref. 324,8nm

+RNH2

- HCl

Room Temperature

+ R’SH

Room Temperature

Fluorescence: Ex 230nm, Em 450nmDAD: 338, 10nm; Ref. 390, 20nm

Absorbs at 230nm and 338nm

H

H

O

O

NR

SR

Non-fluorescent, absorbs at 230nm

Does not absorb at 338nm

FluorescentAbsorbs at 266nm andFluoresces at 305nm

OPA

FMOC

’

NRR’

orNHR





Amino Acid Analysis (AAA) on RRHT Eclipse Plus C18, 4.6x 50mm, 1.8µ Column: DAD 125pMole on column

min1 2 3 4 5 6

7

*Degradation peaks from reagents

Primary AAs (OPA) Secondary AAs (FMOC)

FMOC and by-products→

1

2

3

4

5

6

8 9 1011

12

1314

15

16

17 1819

22

23

20

21

*

*

*





Names and Order of Elution for OPA and FMOCDerivatives of Amino Acids

FMOCPROProline23

FMOCSARSarcosine*22

FMOCHYPHydroxyproline21

OPALYSLysine20

OPALEULeucine19OPAILEIsoleucine18

OPAPHEPhenylalanine17

OPATRPTryptophan16

OPANVANorvaline*15

OPAMETMethionine14

OPAVALValine13

OPACYS-CYSCystine12

OPATYRTyrosine11

OPAALAAlanine10

OPAARGArginine9

OPATHRThreonine8

OPAGLYGlycine7

OPAHISHistidine6

OPAGLNGlutamine5

OPASERSerine4

OPAASNAsparagine3

OPAGLUGlutamic Acid2

OPAASPAspartic Acid1

Derivative TypeAA AbbreviationAA NamePeak #

* Internal Standard





Scalability of AAA on various RRHT Eclipse PlusC18, 50mm long, 1.8µ Columns, DAD

mi0 1 2 3 4 5 6 7

mAU

0

10

20

30

40

50

4.6 mm I.D.

3.0 mm I.D.

2.1 mm I.D.

125 pMoles on column

12

3

45

68 9 10 11

1314

15

1617

18 19

22

23

20

217

12





Scalability of AAA on various RRHT Eclipse PlusC18, 50mm long, 1.8µ Columns, FLD

4.6 mm I.D.

3.0 mm I.D.

2.1 mm I.D.

min0 1 2 3 4 5 6 7

LU

0

100

200

300

400

500

600

700

800

12

34 56 8

910

1113

14

15

1617

1819

22

232021

7

50 pMoles on column





Quantitative Reproducibility using RRHT EclipsePlus C18, 2.1x50mm, 1.8µ Columns

ProlineSarcosineHydroxyprolineLysineLeucineIsoleucine

PhenylalanineTryptophanNorvalineMethionineValineCystineTyrosineAlanineArginineThreonineGlycineHistidine

GlutamineSerineAsparagineGlutamic acidAspartic acid

2.22.31.70.80.71.5

0.91.12.30.80.91.1

11.31.90.91.42.1

2.1*1

1.6†2.30.8

RSD (n=12)

DAD

3.01.91.84.42.10.9

11.33.32.71.5NA

10.9

11.2

10.8

2.9*0.91†0.81.1

RSD (n=10)

FLD

50pMole FLD; 125pMole DAD; Raw Data Areas

Detector Reproducibility

PROSARHPALYSLEUILE

PHETRPNVAMETVALCYS-CYSTYRALAARGTHRGLYHIS

GLN*SERASN†GLUASP

AbbreviationAA Name

†Asparagine is somewhat unstable in solution * Glutamine is very unstable in solution

Average RSD = 1.7 1.4





Linearity of AAA on RRHT Eclipse Plus C18, 2.1 x 50mm, 1.8µusing an Agilent 1200SL HPLC System, no Internal Standard

† Asparagine is somewhat unstable in solution* Glutamine is very unstable in solution

0.99690.9981PRO

0.99600.9975SAR

0.99240.9980HYP

0.99570.9857LYS

0.99940.9944LEU 0.99930.9934ILE

0.99960.9925PHE

0.99870.9923TRP

0.99910.9989NVA

0.99970.9944MET

0.99980.9952VAL

NA0.9818CYS-CYS

0.99970.9936TYR

0.99960.9965ALA

0.99980.9960ARG

0.99960.9942THR0.99960.9931GLY

0.99960.9951HIS

0.9937*0.9966*GLN*

0.99970.9933SER

1.0000†0.9928†ASN†

0.99950.9976GLU

0.99920.9949ASP

Coefficients of Linearity (r2)Coefficients of Linearity (r2)Amino Acid

FLDDAD

Using Raw Data Areas





Linearity of AAA on RRHT Eclipse Plus C18, 4.6 x 50mm, 1.8µ using anAgilent 1200SL HPLC System with Internal Standards

† Asparagine is somewhat unstable in solution* Glutamine is very unstable in solution

+ I.S. at 250pMole on column for DAD and25pMole on column for FLD

0.999790.99998PRO

I.S.+I.S.+SAR0.997510.99991HYP

0.999790.99956LYS

0.999320.99986LEU

0.999100.99973ILE

0.999430.99988PHE0.996870.99997TRP

I.S.+I.S.+NVA

0.999960.99992MET

0.998860.99992VALNA0.99992CYS-CYS

0.999460.99991TYR

0.999240.99984ALA0.999460.99993ARG

0.999410.99996THR0.996810.99982GLY0.999930.99978HIS

0.99549*0.99996*GLN*

0.998800.99982SER

0.99581†1.00000†ASN†

0.998790.99916GLU0.998870.99995ASP

Coefficients of Linearity (r2)Coefficients of Linearity (r2)Amino Acid

FLDDAD





Amino Acid Linearities by FLD on RRHT EclipsePlus C18, 3.0 x 50mm, 1.8µ, no ISs

0

200

400

600

800

1000

1200

0 20 40 60 80 100 120 140

pMoles on Column

A r e a C o u n t

s

ASP

GLU

SER

HIS

GLY

THR

ARG

ALA





Amino Acid Linearities by FLD on RRHT EclipsePlus C18, 3.0 x 50mm, 1.8µ, no ISs

0

200

400

600

800

1000

1200

1400

0 20 40 60 80 100 120 140

pMoles on Column

A r e a C o u n t s

TYR

VAL

MET

PHE

ILE

LEU

LYS

PRO





Reproducibility of 5.0 pMoles on column using an RRHTEclipse Plus C18, 2.1 x 50mm, 1.8µ, FLD, no ISs

min0 1 2 3 4 5 6 7

LU

0

20

40

60

80

100

120

140

160

1

24 6

8

9

10

11 1314 17

18 19

23

20

7





Preparation of Low-Sensitivity Amino Acid StandardSolutions. Prepare the three low-sensitivity standards bymixing together stock solutions in the volumes shown.

1 mL 1 mL 1 mLFinal AA Solution with

EAA and 500 pMoles/µL ISTD

— — 900µLAdd 100 pMolesAA standard

— 900µL —Add 250 pMoles AA standard

900µL — —Add 1000 pMoles AA standard

100µL 100µL 100µLTake 100 µ L EAA-ISTD mix

10mL 10mL 10mLEAA-ISTD mix

5mL 5mL 5mLAdd 10 nMoles ISTD solution

5mL 5mL 5mLTake 5 mL diluted EAA mix

5mL 20mL 50mLDiluted EAA mix

― 15ml 45mlDilute with 0.1NHCl

5ml 5ml 5mlTake 5 mL 18 nMoles EAA

Concentration of Final AA Solutions

(pMoles/µL)

900 225 90





Preparation of High-Sensitivity Amino Acid StandardSolutions. Prepare the three high-sensitivity standards bymixing together stock solutions in the volumes shown.

1 mL 1 mL 1 mLFinal AA Solution with

EAA and 50 pMoles/µL ISTD

— — 900µLAdd 10 pMoles AA standard

— 900µL —Add 25 pMoles AA standard 900µL — —Add 100 pMoles AA standard

100µL 100µL 100µLTake 100 µ L EAA-ISTD mix

10mL 10mL 10mLEAA-ISTD mix

5mL 5mL 5mLAdd 1.0 nMoles ISTD solution

5mL 5mL 5mLTake 5 mL diluted EAA mix

5mL 20mL 50mLDiluted EAA mix

― 15ml 45mlDilute with 0.1NHCl

5ml 5ml 5mlTake 5 mL 1.8 nMoles EAA

Concentration of Final AA Solutions

(pMoles/µL)

90 22.5 9





Comparison of a variety of Beers available in theUSA using the 1200SL, DAD & I.S.

min1 2 3 4 5 60

mAU

0

20

40

60

80

100

120

140

English Beerbrewed for US Market

German Beer brewed for US market

Typical American Beer

American Beer brewed to German purity laws

900pMole/ul AAA std + 500pMole/ul I.S.1

2 3 4 5

6 89 10 11 13 14

15

1617 18 19

22 23

20

21

7

12

Column: RRHT Eclipse Plus C18, 4.6x50mm, 1.8µ





Comparison of the Amino Acid Content of VariousBottled Beers

36.034.862.727.9Total =

10.2915.8834.848.65PROISISISISSAR

1.261.171.740.20HYP

0.900.080.320.30LYS1.750.551.711.02LEU0.940.340.970.54ILE

2.071.232.211.33PHE0.670.801.070.58TRP

ISISISISNVA

0.160.110.270.17MET2.831.933.351.92VAL0.050.080.100.08CYS-CYS

1.771.652.861.36TYR4.604.565.144.28ALA1.831.290.940.48ARG

0.200.740.340.30THR1.571.612.001.54GLY0.891.071.000.68HIS

0.330.380.760.67GLN0.260.120.400.45SER0.540.140.981.19ASN

1.980.800.931.07GLU1.150.260.731.06ASP

µMoles /mlµMoles /mlµMoles /mlµMoles /ml

English Beer brewed forUS market

German Beer brewed forUS market

American Beer brewed toGerman purity laws

Typical American BeerAmino Acid



Group/Presentation TitleAgilent Restricted

Month ##, 200X

Amino Acid Analysis on RRHT Eclipse Plus C18, 2.1, 3.0 or4.6 x 50mm, 1.8µ Column: Experimental Conditions

AAA on production 1200SL using Eclipse Plus-C18, 2.1, 3.0, or 4.6 x 50mm, 1.8uMobile phase A: 10mM Na2HPO4 – 10mM Na2B4O7, pH 8.2 – 0.5mM NaN3

5.6gm anhydrous Na2HPO4 + 15.2gm Na2B4O7· 10H2O in 4L water + 32mg NaN3

Adjust to ~pH 9 with 6ml concentrated HCl and then small drops until pH 8.2. Be cautious with strong acids!

Filter through 0.45µ regenerated cellulose membranes (Agilent P/N 3150-0576)

Stable for ~1.5 weeks at room temperature

Mobile phase B: ACN: MeOH: H2O 45:45:10 by volume

Injection diluent: 1ml Mobile phase A + 15µl concentrated H3PO4 in a 1 ml vial. Make this in 100ml batches.

Instrument config:

Pump: no mixer, no pulse dampener, bypass used (at 0.1min after inject command), compressibility settings used: A= 35, B= 80

Flow rate: 0.420ml/min for 2.1mm ID; 0.85ml/min for 3.0mmID 2.00ml/min for 4.6mm ID

Gradient Timetable: Time (min) %B0.0 2.0

1.0 2.0

7.0 57.0

7.1 100.0

8.4 100.0

8.6 2.0

Stop time 8.7

DAD: PW 0.01min; slit 4nm; Stop time 7min (adjust as needed) Cell = 5µl, 6mm flow path (Agilent P/N G1315-60025)

338, 10nm; Ref 390, 20nm

262, 16nm; Ref 324, 8nm

338, 10nm; Ref 390, 20nm230, 16nm; Ref 360,100nm

Timetable Signal C): 0.00 min 338, 10nm; Ref 390, 20nm

5.53 min 262, 16nm; Ref 324, 8nm (adjust as needed; 4.6mm ID transition at ~5.4min)

FLD: PW 0.01min, Stop time 7 min (adjust as needed), never use this detector before another due to fragility of flow cell

Ex 230nm; Em 450nm; Filter 295nm (Default filter)

Timetable Signal: 0.00 min Ex 230nm, Em 450nm; PMT Gain 9 (as needed)

5.53 min Ex 266nm, Em 305nm; PMT Gain 9 (as needed; 4.6mm ID transition at ~5.4min)




Month ##, 200X

Amino Acid Analysis on RRHT Eclipse Plus C18, 2.1, 3.0 or4.6 x 50mm, 1.8µ Column: Conditions continued

TCC: used with low dispersion kit installed; T = 40°C for column side, 30°C for exit side. Low dispersion kitused on both sides.WPS: Def. vol set to 0.5ul, def speed used throughout injector program is 200ul/min

Injector program:1) Draw 2.5µl from Borate vial (Agilent P/N 5061-3339)2) Draw 0.5µl from Sample vial3) Mix 3µl in washport 5X4) Wait 0.2min

5) Draw 0.5µl from OPA vial (Agilent P/N 5061-3335)6) Mix 3.5µl in washport 6X7) Draw 0.4µl from FMOC vial (Agilent P/N 5061-3337)8) Mix in 3.9µl in washport 10X9) Draw 32µl from Diluent vial10) Mix 20µl in washport 8X11) Inject12) Wait 0.10 min13) Valve bypass

All tubing is 0.12mm ID throughout. Maximum sensitivity is obtained with 2.1mm columns. Toproperly integrate the first peak, set the integrator to detect negative peaks. If you wish to minimizethe degradants in the DAD chromatogram use new reagents and mobile phase. OPA and FMOC arenot stable left open to the atmosphere at room temperature. All modules are 1200SL whereavailable; the autosampler is a WPS. Typically use 54 position plate in front for reagents andsamples and 15 position plate in back for 6ml diluent vial. Gives enough for 24 hour sequence.




Month ##, 200X

Conclusions: We can Squeeze More into Less!

1. Derivatization and Analysis are still totally Automated2. Cycle time more than cut in half (~13.5 min.)

3. Peak shapes of early eluters Asp and Glu muchnarrower4. Reproducibility of secondary AAs improved5. Linearity by linear regression is still almost 1.06. Average area reproducibility is below 2%

7. Scalability is greatly improved (2.1→

4.6mm id)8. Number of useable columns (12) and phases (2) isgreatly expanded

New State:




Month ##, 200X

Bibliography

• Rainer Schuster and Alex Apfel, Hewlett-Packard App. Note, Pub.#5954-6257 (1986)

• Rainer Schuster, J. Chromatogr., 431, 271-284 (1988)• Herbert Godel, Petra Seitz, and Martin Verhoef, LC-GC International,

5(2), 44-49 (1992)• John W. Henderson, Robert D. Ricker, Brian A. Bidlingmeyer, and Cliff

Woodward, Agilent App. Note, Pub.# 5980-1193E (2000)• S. Moore and W.H. Stein, J. Biol. Chem., 192, 663 (1951)• M. Roth, Anal Chem, 43, 880-882 (1971)• S.B. Einarsson, B. Josefsson, and S. Lagerkvist, J. Chromatogr., 282,

609-618 (1983)• I. Betnér and P. Földi, LC-GC International, 2(3), 44-53 (1989)• B. Gustavsson and I. Betnér, J. Chromatogr., 507, 67-77 (1990)




Month ##, 200X

Supplies list:ZORBAX Eclipse Plus-C18 HPLC Columns

Description Size Particle Agilent(mm) Size (µm) Part No.

Eclipse Plus-C18 4.6 x 50mm 1.8µm 959941-902



Derivatization Reagents

Description AgilentPart No.

Borate Buffer: 0.4 M in water, pH 10.2, 100mL 5061-3339

F MOC Rea ge nt, 2.5 mg/mL in ACN, 10 x 1 mL a mpoule s 5061-3337

OPA Reagent, 10mg/mL in 0.4M borate buffer and

3-mercaptoproprionic acid, 6 x 1mL ampoules 5061-3335

DTDPA Reagent for analysis of cysteine, 5g 5062-2479

Mobile Phase and Injection Diluent Components

Description Mfgr.'sManufacturer Mfgr.'sPart No.

Na2HPO4, Sodium Phosphate, Dibasic Sigma S 7907Na2B4O7·10H2O, Sodium Tetrborate Decahydrate Sigma S 9640NaN3, Sodium Azide Sigma S 2002H3PO4, ortho Phosphoric Acid Sigma 79617

Vials

Description AgilentPart No.

100 µL Conical insert with polymer feet, 100/pk 5181-1270Amber, wide-opening, write-on, screw-top vial, 2mL,100/pk 5182-0716Blue polypropylene cap, PTFE/silicone septum, 100/pk 5182-0721Clear glass screw cap vial, 6ml,16mm cap size, 100/pk 9301-1377Screw caps, 16mm, 100/pk 9301-1379PTFE/silicone septa, 16mm, 100/pk 9301-1378

StandardsDescription Agilent

Part No.

Amino Acid Standards in 0.1 M HCl, 10 x 1mL ampoules

1 nmol /ml 5061-3330250 pmol /ml 5061-3331100 pmol /ml 5061-3332

25 pmol /ml 5061-333310 pmol /ml 5061-3334

Supplemental Amino Acids:

Nva, Sar, Asn, Gln, Trp, Hyp, 1g each 5062-2478




Month ##, 200X

Availability of this talk

A new App note has just been published 2/23/2007:

“High-Speed Amino Acid Analysis (AAA) on 1.8µmReversed-Phase (RP) Columns”

Agilent Pub. # 5989-6297EN

theanine 2

Documents

x ophthalaldehyde opa

nm dad

x linearity of aaa

nm fluorescence

x scalability of aaa

agilent technologies

x names

x introduction