Indian Journal of Chemi stry Vol. 43A, May 2004. pp. 1095 -1 098

Determination of amino acids separated by micellar electrokineti c chromatography

after pre-column deri vatization with o-phthalaldehyde

A Durgbanshi , S Sharma, M Shelke, D Bose, A Asthana & S K Sanghi *

Micro Separat ion Unit, Regional Research Labora tory (CS IR ), Hoshangabad Road. Bhopa l (M.P) 462026, India.

E- mail: sanghi sk@sancharnet.in

Receil'ed 10 } al/llar v 2002; revised 29 }mlllary 2004

A simple, sensitive and rapid analyti cal method has been developed for the determination of ami no acids separated by micellar electrokinetic chromatography after pre-co lumn de ri vat ization with o-phthala ldehyde. The separated amino ac ids have been de tected using ultrav iolet as well as electrochemica l detection modes. The OPA deri va tized am ino acids have been separated on a 75 11 m (intcrnal diameter) fu sed silica capill ary. UV de tec tion has bec n performed at 220 and 335 nm respecti vely, while electrochemi cal detec ti on has been done at 500 mV against Ag/AgC I reference electrode. Optimum separati on has bcen achieved usi ng an app lied voltage of 28 kV. Enhanced sensiti vity is achieved with the electrochemi cal de tec ti on mode. The standard curve of amino acid s is fo und to be linear in the range of 10-5 to 5x I 0-7 M. The % relati ve standard dev iati on for retention time is fo und to be less th an 0.4 while for peak height and peak area it is within 1%.

IPC Code: Int.Cl.7 BO ID 15/08

Most of the applicatio ns in volving amino ac ids analys is are perfo rmed using liquid chromatography (LC)1.2 and gas chromatography (GC)3-5 methods. In LC sample extracti on and c lean-up methods are necessary as columns can easily contaminate. GC with mass spect rometry (GC- MS) is ve ry sensitive detection techni queo.7 but requires cumbersome dcri vati zati o n and ex tractio n steps. Due to mo re effi ciency and sensiti vity of capill ary e lectro phores is (CE) methods it has proven extremely useful in the analys is of many bio logical substances8

.9

. A subs tanti al proportio n of analyti cal methods based on HPLC and CE re ly on the de ri vati zation of the analyte for improved de tectability w ith ultrav io let (UV) and fluorescence detectors, which are most w idely used detectors fo r CE ' O. " . The cl ass ical method o f amino ac id analys is , first proposed by Moore and Ste in 12 in vo lves separati on by ion exchange chromatography,

fo llowed by post-column deri vati zati o n with ninhydrin. But the lack of sensiti vity led to the development of o ther, more sensiti ve, deri vati z ing

13- 19 A' 'd h I b d reagents ' . mll10 aCI s ave a so een converte into UV-absorbing o r fluo rescent deri vatives for the purpose o f HPLC analys is by a vari ety of approaches2o

.21. The most well kno wn reagents for the

homogeneous deri vatizati on of amino ac ids are dansy l hI 'd 2223 fl . 247 5 I I ' c o n e " , uorescamll1e '-, p leny -ISO-

thiocyanate (PITC)26-28 and o-phtha laldehyde (OPA)2'!-3 1.

In the case of fluorescent de ri vati z ing agents e .g . dansy l chloride, the inev itable presence o f excess reagent in the reactio n mi xture causes hi gh background response. Thi s results in an inadequate detectio n limit fo r the analyte . Introducti on of steps to remove excess reagent o ften makes the method mo re tedious and prone to error. Non-fluorescent o r poorl y flu orescent reagent such as fluo rescamine and o-phthalaldehyde, w hich reacts w ith amino gro up to g i ve sensi ti ve deri vati ves are much better than a fluo rescent reagent in thi s respect. Use of OPA is more advantageous because it is water soluble whil e fluorescamine requires organic solvents and since OPA forms small es t de ri vati ve w ith amino group it has wider utility e.g. in MS analys is . The OPA deri vati ves w ith amino ac ids have been shown to be e lec trochemically ac ti ve as well as UV absorbing3:!·D.

El ec trochemical detectors fo r CE, tho ugh not co mmo nly used , ut ili ze the inherent sensitivity of hydrodynamic e lectroche mi stry to a llow de tec tio n and quantitati on of trace amounts of e lectro-act ive materi als. They are the o nly commercially available CE detectors capable o f competing with fluo rescence de tectors in te rms o f analyte detectabilit/ 4-39

.

The princ ipa l obj ec ti ve o f thi s work was to develop an analyti cal method that could be used fo r amino ac ids in aqueous samples . In th is note, the use of o-phthalaldehyde as a pre-column de ri vatizing reagent fo r primary amino ac ids has been evaluated . An attempt has been made to improve the analyti cal technique fro m the po int of view o f separatio n and fl exibility. The separati on was achieved by using Mi cellar Electrokinetic Chro matography (M EKC) and detectio n by UV as well as e lectroche mical modes. CE has been evaluated as a comple menta ry o r alte rnative technique to assess amino ac ids.

1096 INDIAN J CHEM, SEC A, MA Y 2004

Experimental Chemi cal s used were of anal ytical grade purity.

They were obtained from standard suppliers and used as received. Hi stidine, Serine, Glycine, Alanine and Tyrosine were obtained from BDH (Poo le, UK). Glutamic acid and aspart ic acid were obtained from Fluka (Buchs, Switzerl and). The standard solution of am ino ac ids was prepared in deioni zed warer and HPLC grade methanol. A working compos ite standard so lution was prepared by combining an aliquot of each of the solution and diluting the mixture with buffer. The stock solutions were stored at - 4°C as a precautionary measure.

OPA (Aldri ch, Mil waukee, WI , U.S .A.) 10-2 mM (milimolar) was prepared with water and HPLC grade methanol.

Prince CE inst rument with programmable inj ector (Lauer Labs, Em men , Netherlands) including hi gh voltage source was used for all the experimen ts. Fuscd silica capi llary with in ternal diameter of 75 ~lJn

was purchased from Composi te Metal Service (Hallow, England). A capi ll ary of 85 cm total length was used as separation col umn for UY detection. While for electrochemical detecti on the total length of the capi Il ary used was 95 cm.

For electrochemical detection, the elect rochemical ce ll used was a unijet cell (Bioanaiytical Systems, West Lafayette, I , U.S.A.) with a custom made I mm diameter glassy carbon work ing electrode and an (intlow) Agi AgCl reference electrode. The stai nless steel hal f of the cell body served as the aux ili ary electrode. Spacer gaskets were 19 ~lm thick.

For UY detect ion, a UY-visibl e detector (UY-vis 200, Linear Instruments , Fremont, CA, USA), was operated at 220 and 335 nm. Signals were transferred to a strip chart recorder model BD 112 (Kipp & Zonen , Delft, Netherlands) and to a data processing system with Caesar 40.0 so ftware (Lauer Labs, Emmen, Netherlands). Solutions were filtered through 0.45 ~m filter and degassed with heliu m before use.

The outlet of the capil lary was connected to the electrochemical detector cell through a connecti ve junction in the form of a palladium decoupler. The connection between the capillary and the cell was done by a polyether ether ketone (PEEK) (Upchurch, Oak harbour, WA, USA) coupling tubing of 6 cm length and 63 ~m 1.0. A bioanalyticaI systems petit ampere LC-3D potentiostat was used to apply potential on the electrode.

Derivatizatioll procedure

In order to derivatize amino acids wi th OPA. a reagent solution was used containin g 7 mM OPA. 3 mM 2-mercaptoethanol (2-M E), 5 111M borax and 20lfc (v/v) methanol. The reagent was mi xed wi th the amino acid so lution in the rati o of I: I. After 3 min . or reaction time, the mixture was injected. One major source of vari ability was fo und out to be th e instabil ity of th e derivati zatio n reagent. So it was necessary to prepare fresh derivat ization reagen t from OPA , 2-ME, methanol and borax butTer each day .

Separation conditions

Samples 'were introduced hydrody namica ll y. by applying pressure of 40 mbar for 6 sec. The buffer used for the separation of amino acids was of pH 9.5 and was preparcd by mi xing 14 mM boric acid and 10 111M sod iul11 hydroxide . 20 mM sod iul11 dodccy l su lphate (SDS) was added to the bu ffer as surfactant. All bu ffer solution used contained I 111M sodium ch loride to stabili ze the in-now reference electrode of the cell. The potenti ostat was sel at 500 mY aga inst Agi AgCl reference electrode. The vo ltage applied for all the se paration was 28 kY . In order to achieve a fl at flow profi Ie of electro-osmotic f low a compcnsation pressure of 20 mbars was applied in all the ex peri ments.

The capillary was etched with 100 111M sodium hydrox ide for 10 min before use, da ily . Thi s was done in order to regenerate the capi ll ary or in simple words to create a double layer at the wall of the capillary.

Results and disclission The reaction of OPA with amino acids is very fast.

If the OPA/RSH is present in concentrati on exceeding that of the amino acid by 2-3 orders of magnitUde, the reaction is usually complete with in 1-2 min at room temperature. OPA is esse ntiall y non-absorbing species until reacted with primary amino group in the presence of thiol i.e. 2-ME. Separate reaction mixtures were incubated with different reaction times. Derivatization was found to increase from I to 3 minu tes and then slowly decreased by further increasing the reaction time. A reaction time of 3 minutes was selected as optimum reaction time to permi t adequate time for the amino acids to react to its maximum .

Amino acids react with OPA in presence of thiol to fo rm hi ghl y sensitive l -alkylth io-2-alky l substituted isoi ndol e deri vati ves4U

•

NOTES 1097

S H

f" S H F~hJH2 --

c Pt>. Iso ndole

Therefore presence of th iol (mercapto n) is must for the above reacti o n to take place, The poss ibility of enha.nc ing OPA derivative stability is in vest igated by varying the concentrati on o f thi ol co mpo nent. A number of combinations were tri ed and it was found th at the stability properti es of the de ri vative were markediy dependent o n the concentrati o n o f thi o l. It was fo und that if the conce ntration of 2-ME was reduced then the sens iti vity of the derivati ves increased but the concentrati o n of 2-M E cannot be

10,wered be low 5 /11/25 ml. It is seen that the response of glyc ine is increased as we lower the vo lume of 2-

M E fro m 10 /11 to 5 ).11. The o ther condi tions were kept constant.

The effect o f surfactant concentration on the separati on was studi ed using 28 kY appli ed vo ltage, It was observed during pre liminary experiments that without addition o f SDS , the reso lutio n o f OPA de ri va ti ves was unsati sfac tory. The mi grati on and re,~o lut i o n of OPA derivatives are s ig nificantly affected by the SDS concentration. The separation o f 8 amino acids was achieved using 20 mM SDS . Comp le te separat ion at any o ther SDS concentrati on was not possib le. Addition of sur factant in separation bu ffer at concent ration above the cri ti cal micellar concentration forms micelles and interact io n between mi ce ll es and the so lu tes causes separati on. In thi s case 20 mM is the optimum concentration the refore the mobility of the OPA derivatives first increased with an increase in the SDS concentratio n till 20 mM. Further increase in SDS concentrati on results in decreased mobi lity of the io ns due to increased viscosity o f solvent. Hi gher concent ration of SDS leads to excess ive current result ing in joule heating problem.

The effect of p H on the separation of ei crht amino acids was studied using 28 kY as the app li :d voltage on the carillary . The pH effect was studied over the range of pH 8- 10. 80l'are buffer with different concentrat ion of sodium hyd roxide was used to attain diffe rent pH values. The retention time of all the amino ac ids vary at different pH clue to the ir different pKa values e .g. Hi stidine (6.0) , Serine e 13), Lysine ( 10), Glutamic acid (4.3) etc. pH 9.5 was found out to

be opti mum for the separati on o f aU e ight OPA deri vatized amino acids as the retention ti me was not too lo ng and all the amino acids were resolved.

The opt i mi zati on o f the separation was do ne at 335 nm. After complete optimization o f all the conditions the wave length was changed. I t was found th at at 220 nm there was a drastic increase in the sensiti vity ,

The iso indole derivative is a lso respo nsive to Electrochemical Detector (ECD) . An alte rn ati ve to UY and fluorescence detection with comparabl e sensitivity is provided by the use of ECD, Thi s app licati on was chosen because it enabl ed us to compare the performance of various detecti on principles in CE: UY detection and EC D, The OPA derivatives were separated with the same condit ions as for UY detec ti o n with only an increase in the capillary length . The detec ti o n potential was opt imi zed and 500 mY was found to be the optimu m detecti on potenti a l.

The excess of 2-M E in the reaction mixture caused a hi gh peak in the e lectropherogram due to whi ch only 6 OPA deri vatized ami no ac ids were separated with ECD. T he no ise level under these conditi ons was considerably hi ghe r. T hi s was probably re lated to the combination of a high pH and a hi gh conduct ivi ty of the background e lectrolyte (BGE).

The linearity o f a ll the OPA derivati zed am ino ac ids was verifi ed by dete rmining four differenr concentrations of each a mi no ac id . The standard curve of amino ac ids was found to be linear in the range of 10'5 to 5x lO,7 M . The reproducibility of the method was inves ti gated by repeated inj ect ion o f derivatized amino acids and measuring the peak area. The % relati ve standard deviatio n for retention ti me was less th an 0 .4% while for peak height and peak area It was within I %. Excellent plate numbers in the range of 80,000-130,000 were achieved .

The limit of detec ti o n for the OPA de ri vati ves was assessed using UY as well as e lectrochemi cal detection . T he UY de tection was performed at 335 and 220 nm. It was found that at 220 nm there was a ten-fold inc rease in the sensiti vity . In case o f UV, the detectio n limits were in the range of 5xlO,7- 1Clxl O,7 M. While in the case of ECD the detection limi ts were in the range of I x I 0·7-2x I0~7 M .

Acknowledgement Thi s work was financiall y supported by the

European Commiss ion as a joint research project with the University of Amsterdam, Amsterdam, The Netherlands eCI 1 *-CT93-0087). Acknowledgement i ~

1098 INDI AN J CHEM, SEC A, MA Y 2004

due to Council of Scientific and Industrial Research, New Delhi for providing seni or research fe llowship.

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