This article was downloaded by: [Carnegie Mellon University]On: 22 October 2014, At: 03:23Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK

Analytical LettersPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lanl20

A ChemiluminesClentPrecolumn Labelling Reagentfor High-Performance LiquidChromatography of AminoAcidsStan R. Spurlin a & Melanie M. Cooper aa Department of Chemistry , Clemson University ,Clemson, SC, 29634-1905Published online: 05 Dec 2006.

To cite this article: Stan R. Spurlin & Melanie M. Cooper (1986) A ChemiluminesClentPrecolumn Labelling Reagent for High-Performance Liquid Chromatography of AminoAcids, Analytical Letters, 19:23-24, 2277-2283, DOI: 10.1080/00032718608064553

To link to this article: http://dx.doi.org/10.1080/00032718608064553

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all theinformation (the “Content”) contained in the publications on our platform.However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness,or suitability for any purpose of the Content. Any opinions and viewsexpressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of theContent should not be relied upon and should be independently verified withprimary sources of information. Taylor and Francis shall not be liable for anylosses, actions, claims, proceedings, demands, costs, expenses, damages,and other liabilities whatsoever or howsoever caused arising directly orindirectly in connection with, in relation to or arising out of the use of theContent.

This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan,sub-licensing, systematic supply, or distribution in any form to anyone isexpressly forbidden. Terms & Conditions of access and use can be found athttp://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014

A CHEMILUnINESCENT PRECOLW LABELLING REAGEUT FOR HIGH-PERFORUANCE LIQUID CHROMATOGRAPHY OF AWINO ACIDS

Stan R. Spurlin and Melanie PI. Cooper* Department of Chemistry

Clemson University Clemson, SC 29634-1905

Keywords: Chemiluminescence, amino acids. HPLC.

ABSTRACT

A chemiluminescent tag for precolumn derivatization of amino acids

has been developed. The tag, 4-isocyanatophthalhydrazide, couples with

17 amino acids tested, including proline and hydroxyproline, in less than

ten minutes. Twelve derivatized amino acids have been separated and

detected with an average detection limit of 10 femptomoles per 20 pL

inject ion.

The qualitative and quantitative analysis of amino acids has been

the subject of a vast amount of research since the early work of Moore

and Stein on ion exchange procedures.' Improvements in instrumentation

and column design have led to improved detection limits but the procedure

is still essentially the same as developed in their original work.

nore recently, the rapid development of reverse phase HPLC

techniques has offered an alternative to ion exchange procedures.

However, direct detection of many amino acids by standard UV detectors is

2 2 7 7

Copyright 0 1986 by Marcel Dekker, Inc. 0003-27 191861 1923-2277 $3.50/0

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014

2 2 78 SPURLIN AND COOPER

difficult owing to the lack of a suitable chromophore, and standard

refractive index detectors lack sufficient sensitivity at picomole

levels. To overcome these problems a variety of pre- and postcolumn 2 labelling agents have been developed. Those include ninhydrin.

~-phthalaldahyde,~ dansyl ~hloride,~ 1-f luor0-2,4-dinitrobenzene,~ and

dimethylaminoazobenzene-4'-sulfuryl chloride,6 a11 of which form W

absorbent or fluorescent species with amino acids. All of these however

suffer from one o r more of the following disadvantages: lack of

quantitative reaction with certain amino acids, complex coupling

procedures o r instability of the products.

Harry chemiluminescence (CL) techniques have appeared in the

literature in the last several years, primarily because of the tremendous

sensitivity available through this method. Imao and Toshika have applied

an indirect chemiluminescence technique to the determination of

fluorescent amino acid derivatives such as dansyl derivatives.' In this

procedure a high energy intermediate is formed which transfers energy to

the fluorescent product resulting in emission. Tsuji et el. have

recently reported a direct chemiluminescence method for aromatic and

aliphatic amines using a derivative of 4-aminophthalhydrazide(isolumino1)

as a precolumn labelling reagent. However, the precolumn labelling

procedure required a two step process with a 4 hour reaction time. 8

We report here on a new precolumn CL labelling reagent for amino

acids which reacts in 5-10 minutes at room temperature. Our compound.

4-isothiocyanatophthalhydrazide (I), possesses both the chemiluminescent

isoluminol group and an isothiocyanate moiety. Thus the coupling

procedure is essentially the same as that recently reported by Heinrikson

and Heredith in which phenylisothiocyanate (Bdman reagent) was used as a

w absorbant tag for amino acids.' The coupled compounds are then

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014

CHEMILUMINESCENT PRECOLUMN LABELLING REAGENT 2 2 79

separated by RP-HPLC and reacted postcolumn with H202 and Pe(CW)i- to

produce chemiluminescence.

Synthesis of 4-isothiocyanatophthalhydrazide (isoluminolisothiocyanate,

ILITC) :

Thiophosgene (0.6 mL, 7.8 manol) is added to a stirred suspension of

isoluminol (1 g. 5.6 m o l ) in water. The reaction should be carried out

in an efficient fume hood since thiophosgene is extremely toxic, and HCl

fumes are generated by the reaction. The slurry is stirred for 1 hour,

and then filtered yielding the ILITC as a white powder in quantitative

yield (mp )3OO0C).

Coupling Procedure:

The amino acids (Sigma Chemical) are 99% pure and are used as

received. Stock solutions of these are prepared in water or

water-methanol as necessary and a1 1 subsequent samples are prepared from

these stocks. Usually a second stock is prepared by subsequent dilution

so that the appropriate sample levels can be obtained in a solution

volume between 10 and 100 ~ 1 . This volume range can be accurately

measured while still allowing for rapid solvent removal under vacuum.

A typical standard preparation is as follows. A 100 pl sample of a

1 x 10-4M glycine solution is placed in a small flask. The solvent is

removed under vacuum end then 1 mg of isoluminol isothiocyanete (I) is

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014

2280 SPURLIN AND COOPER

added. 100 p1 of the coupling solution, 5% triethylamine:95% H 0, is

added and the solution is sonicated for 1 min and swirled. After 9-10

minute reaction time the solvent is removed under high vacuum and the

resulting product refrigerated until analysis. All standards, mixtures,

and blind unkowns are prepared in the same way and coupled in the same

manner.

Chromatography Procedure:

2

All separations are performed on an in-house built, HPLC

chromatograph consisting of a Milton Roy piston pump, a Rainin pressure

gauge and pulse dampner, a Rheodyne 7125 injection valve with 20 p l and

40 p l sample loops, and a Bio-Sil ODS-5s 150 nwn C column ( 5 pm). The

mobile phase consists of a -05 M ammonium acetate, pH 6.8. solution with

varying degrees of methanol added as an organic modifier. The

derivatized amino acid standards are injected in quantities corresponding

to 10 picomoles to 100 nanamole quantities for calibration. both as

individuals to identify peaks, and as mixtures to investigate any overlap

or memory effects. The blind unknowns are prepared in the same manner as

the unknowns and then injected in quantities thought to be within the

working range. Concentration adjustments to stay within the working

range are then made and samples rerun if necessary.

18

The detector cell was machined in-house from Lexan and has a volume

of 50 p l . It is similar in design to many reported previously and has

an inlet for ferricyanide-KOH solutions. The H 0 is added upstream €rom

the detector in a mixing tee. These solutions are added by means of

piston pumps. The detector is an Amperex 2273XB photomultiplier tube,

the output of which is fed to an amplifier and then directly to a strip

chart recorder. The optimum concentrations of the various reagents are

0.3 W H202 and 1 x 10-2W Fe(CU)6 in a 2.5 W UaOH solution.

2 2

3-

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014

CHEMILUMINESCENT PRECOLUMN LABELLING REAGENT 2 2 8 1

0 10 20 30 40 T i m e ( m i n u t e s )

Fig. 1 Chromatogram of ILITC derivatives of some amino acids (1 picomole of each). Peaks: aspartic acid, serine, glycine. histidine. alanine, arginine, proline, tyrosine, valine, isoleucine, leucine, lysine. Bio-Sil 005 column 15Onrm x 4 mm; mobile phase O . l l 4 ammonium acetate in water (15 min). 15% UeOHI85X 0.lM ammonium acetate in water (remainder).

2ml min -1

Optimization of the CL detector conditions was carried out using the

ILITC itself, the ILITC-glycine derivative and the ILITC-histidine

derivative. In all cases the optimization was carried out with both the

column removed and in the ca$e of the amino acid derivatives, with the

column in place. Under all circumstances the conditions for maximum

emission intensity are similar. The optimum peroxide and ferricyanide

concentrations are 3 x 10-'l4 and 1 x 10-211, respectively.

The coupling procedure was tested with a variety of amino acids.

All were found to couple efficiently in 5 minutes both separately and in

mixtures of amino acids, as monitored by BP-TLC. Figure 1 is a

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014

2282 SPURLIN AND COOPER

chromatogram of 12 derivatized amino acids separated on a CI8 column.

Our separation results are comparable to those obtained using PITC

derivatives separated under similar conditions. The detection limit for

most amino acids chromatographed was 10 femptomoles per injection

(291). Histidine and isolencine detection limits were somewhat higher,

around 20 femptomoles. Several other amino acids besides those shown in

Figure 1 were also derivatized but could not be chromatographed on our

simple isocratic system in complex mixtures. Therefore, each of these

was chromatographed individually and the detection limits were found to

be comparable to those above. These compounds include glutamic acid,

threonine, methionine. phenylalanine and hydroxyproline. A more detailed

paper will follow using a gradient HPLC system. The derivatized amino

acids appear to be stable for 2 o r more weeks when refrigerated but may

be somewhat light sensitive over long periods of storage.

In conclusion, we have demonstrated a new CL precolumn

derivatization reagent for amino acid analysis. Detection limits are

comparable o r better than those obtained with previously reported CL

coupling reagents and the coupling procedure is much quicker and decidedly

simpler . Also, our preliminary detection limits are an order of

magnitude better than those reported using the PITC derivatives with W

detection. This is a preliminary cornnicetion of our results and a more

detailed paper will follow in the near future.

ACMlOWLEDQIENTS

This work partially supported by American Association of Clinical

Chemists Endowment Fund and by e Bristol Heyers Grant from Research

Corporation.

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014

CHEMILUMINESCENT PRECOLUMN LABELLING REAGENT

1.

2. 3. 4. 5. 6.

7. 8. 9.

REFEREUCES

Moore, S., Spaclanan, D.H., Stem, W.H. Anal. Chem. 30 (1958)

Rawn, J.D. Biochemistry, Harper and Row, New York, 1983. Lindroth, P., Mopper. K. Anal. Chem. 51 (1979) 1167-1169. Schomer, G., Dreil, G. J. ChromatoRr. a (1967) 458-461. Ram, J.D. Biochemistry. Harper and Row, New York, 1983. Chang, J.Y.. Knecht, R., Braun, D.G. Methods Enzymol. 91 (1983)

Miyaguchi, K., Honda, K., Imai, K. J. Chromatogr. 303 (1984) 173-6. Kawasaki, T., Hacda, M., Tsuji, A. Chromatography 17 (1985) 676-681. Heinrikson. R.L., Meredith. S.J. Anal. Biohcem. 136 (1984) 65-74.

1185-1190.

41-45.

Received August 26, 1986 Accepted October 5, 1986

Dow

nloa

ded

by [

Car

negi

e M

ello

n U

nive

rsity

] at

03:

23 2

2 O

ctob

er 2

014