liquid chromatographic separation of some pth-amino acids

Liquid Chromatographic Separation of SomePTH-Amino Acids

Ravi Bhushan* and Rachna AgarwalDepartment of Chemistry, University of Roorkee, Roorkee, 247 667, India

Liquid chromatographic studies on the separation of ten PTH-amino acids were carried out using normalphase untreated silica gel plates, C-18 RP precoated plates and RP-HPLC. Resolution of a complex mixtureof PTH-amino acids was achieved using all the three types. Certain new successful solvent systems have beenworked out in each case. HPLC was carried out with Lichrosphere 100 RP-18 (5mm) column. Acetonitrileand sodium acetate buffer of pH 4.0 was used for reversed phase chromatography while for normal phaseTLC combinations of chloroform–acetonitrile and chloroform–tetrahydrofuran were applied. # 1998 JohnWiley & Sons, Ltd.

INTRODUCTION

Sequencing of peptides and proteins is genarallyaccomplished by Edman degradation (Edman, 1950)coupled with identification of phenyl thio hydantoin(PTH-) derivatives of amino acid from each step. HPLCand TLC, besides other techniques, have been used forthe purpose of identification. TLC of PTH-amino acidshas extensively been reviewed by Rosmus and Deyl(1972), Niederwieser (1972), Allen (1981), and Bhushanand Reddy (1987). Some of the reported problemsinclude a long time period for the development of thechromatogram, difficulties in quantitation and discrimi-nation between derivatives of Leu and Ile (Kulbe, 1971,1974), and resolving complex mixtures such as PTH-Phe/Val/Met without two-dimensional chromatography(Brener et al. 1967). The excellent resolution andreproducibility with high sensitivity of UV absorbancedetectors have made HPLC a very successful method foridentification and quantitation of PTH-amino acids usingisocratic elution (Kin, 1982), binary gradient (Somack,1980) and ternary gradient (Cunicoet al.,1984). Presentpaper describes separation and identification of ten PTH-amino acids from their mixture by TLC and HPLC. Theeffect of heating time for the activation of home-madeTLC plates on the separation was also studied.

EXPERIMENTAL

HPLC equipment from Merck-Hitachi (Darmstadt,Germany) was used, consisting of an L-6200 intelligentpump, L-4250 UV–vis variable wavelength absorptiondetector and a D-2500 chromato integrator. The columnwas Lichrosphere 100 RP-18 (5mm)

Operating conditions. column temperature ambient, chartspeed 5 cm/min, injection mode 25mL Hamilton syringe (Mode

no. 802 N) U6-K universal injector (Syringe-loop injector),recorder range 10 mV. Filtration assembly, Millipore (Milford,MA, USA) assembly with filters of pore size 0.45 and 0.50mmporosity, solvent compatible.

Reagents/Solvents.Acetonitrile and water were of HPLCgrade from E. Merck. Sodium acetate, buffers etc. wereanalytical grade from E. Merck. All solvents were degassed bysimultaneous warming (gently) and evacuation for 15 min.Double distilled water was filtered through a 0.45mm Milliporeassembly and degassed before use.

Selection of chromatographic variables.Buffer A, 10%acetonitrile in 0.01M sodium acetate buffer (pH 4.0); Buffer B,90% acetonitrile in 0.01M sodium acetate buffer (pH 4.0); wereused as solvent systems. The time programme used was 0–100% B for 15 min, at a flow rate of 1.5 ml/min with pressureranging from 100–150 bar.

TLC. All solvents and reagents were from Merck (India) Ltd.Bombay, and amino acids were from SISCO Research Lab.(Bombay, India). The standard PTH amino acids were pre-pared, recrystallized and their purity was ascertained (Bailey,1967). Solutions of PTH amino acids (2� 10ÿ3) were preparedin acetonitrile and were applied as tight spots, 1–2 cm from thebottom of the silica plate, using a graduated glass capillary at aconcentration of 800 ng.

Silica gel G for tlc was from Spectrochem, Bombay (India),with 13% calcium sulphate as binder, having chloride, iron andlead impurities up to 0.02% and showing pH 7.0 in aqueoussuspension. TLC plates were prepared by spreading a slurry ofsilica gel (50 g) in double distilled water (100 ml) with a Stahltype applicator. The plates were dried at 80°C for 2 h or over-night in an oven. The chromatograms were developed at 15, 25and 33� 2°C in preequilibrated rectangular glass chambers.

Reversed phase (Alugram, RP-18 W/UV254) precoated TLCplates (20 cm� 10 cm� 0.15 mm) were generously donatedby Macherey-Nagel, Du¨ren, Germany. The chromatogramswere developed at 15� 2°C in pre-equilibrated rectangularglass chambers using different combinations of 0.01M sodiumacetate buffer (pH 4.0) and acetonitrile. The chromatograms

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr.12, 322–325 (1998)

CCC 0269–3879/98/010322–04 $17.50# 1998 John Wiley & Sons, Ltd.

Received 18 August 1997Accepted 29 September 1997

*Correspondence to: R. Bhushan, Department of Chemistry,University of Roorkee, Roorkee, 247 667, India.

wereair driedandexposedto iodinevapoursto locatethespotsaslight browncompactzones.

RESULTS AND DISCUSSION

Normal PhaseTLC. A large numberof solvent systemsatdifferent temperaturesweretried andonly thesuccessfulonesarediscussedhere.Two typesof normalphaseplates,activatedfor 2 h or overnight,wereusedandthe resultsarecompared.Threesolventsystemsviz; N1: chloroform–acetonitrile(15:0.5,v/v); N2: chloroform–tetrahydrofuran(15:0.48,v/v); and N3:chloroform–tetrahydrofuran(15:0.45,v/v), arementionedherefor 2 h activatedandovernightactivatedplatesrunat15,25and33� 2°C.

The hRf valuesof the PTH amino acids in thesesolventsystemsfor normalphaseTLC arereportedin Tables1–4.ThehRf valuesare basedon at least five identical runs. Typicalchromatogramsfor 2 h activatedandovernightactivatedplatesareshownin Fig. 1 and2; theseclearly showseparationfromsevencomponentmixtures.

Therewasno separationat 33°C on both 2 h activatedandovernight activated plates using any of the three solventsystems.In N1 (Table1) the separationwasbetterat 25°C on

both kind of plates except that PTH-asparagineand PTH-glutamic acid did not show any movement.The hRf valueswerecomparableat 25°C for both 2 h activatedandovernightactivatedplatesand similarly at 15°C. N2 showed(Table 2)goodseparationonovernightactivatedplatesat25°C aswell asat 15°C. On the 2 h activatedplatesPTH-val/ile/pro did notresolveat 15°C, while theygot separatedat 25°C. N3 showed(Table3) betterseparationon 2 h activatedplatesexceptthathRf valuesfor PTH–ile/prowererelatively closerat 25°C, buttherewas a betterseparationfor thesetwo at 15°C. The hRf

values were higher at 15°C in all the three solvents incomparisonto thoseat 25°C. Thus,out of thesethree,N1 wasconsideredto be thebestoverall solventsystem.

RP-TLC. Of the severalcombinationsof acetonitrile–sodiumacetatebuffer used,the following arereported:R1, 4.56:15.4;R2, 5.0:15.0;R3, 6.0:15.0;R4, 8.0:15.0.

ThehRf valuesarerecordedin Table4. Thesolventsystemsdiffer in increasingamountof acetonitrile.As the volume ofacetonitrilewas increased,Rf valuesincreasedfor PTH-Ala,Gly, Val, Phe,Leu, Ile andMet, but therewasno changeforPTH-Ala,Met, Val andIle on goingfrom R2 to R3.PTH-Phe/

Table 1. hRf valuesof PTH-amino acids on normal phaseplatesin N1: chloroform–acetonitrile (15:0.5,v/v)

Overnight activated plate 2 h activated platePTH-amino acid 25°C 15°C 25°C 15°CMethionine 45 50 45 57Isoleucine 72 76 71 73Phenylalanine 53 60 49 60Valine 63 70 59 66Glycine 26 30 25 32Alanine 40 48 39 48Tyrosine 10 13 11 14Proline 85 88 87 88Asparagine 00 00 00 00Glutamic acid 00 00 00 00Time (min) 40 40 30 25

Solvent front: 10 cm for all. Spots located in iodine chamber.

Table 2. hRf valuesof PTH-amino acids on normal phaseplates in N2: chloroform–tetrahydrofuran(15:0.48,v/v)

Overnight activated plate 2 h activated platePTH-amino acid 25°C 15°C 25°C 15°CMethionine 55 57 65 84Isoleucine 77 81 86 91Phenylalanine 60 64 69 75

Valine 71 76 76 91Glycine 28 32 41 80Alanine 44 44 53 72

Tyrosine 15 16 23 19Proline 85 89 94 93Asparagine 00 00 00 00Glutamic acid 00 00 00 00Time (min) 45 40 50 30


Table 3. hRf valuesof PTH-amino acids on normal phaseplates in N3: chloroform–tetrahydrofuran(15:0.45,v/v)

Overnight activated plate 2 h activated platePTH-amino acid 25°C 15°C 25°C 15°CMethionine 59 61 69 71Isoleucine 77 84 93 91Phenylalanine 60 63 79 80

Valine 72 74 90 84Glycine 24 25 30 56Alanine 45 48 66 57

Proline 87 89 94 95Tyrosine 14 16 24 30Asparagine 00 00 00 00Glutamic acid 00 00 00 00Time (min) 50 40 45


Table 4. hRf valuesof PTH-amino acidson C18 RP platesat 15� 2°C

Acetonotrile±sodium acetate bufferPTH-amino acid R1 R2 R3 R4

Phenylalanine 07 08 09 20Leucine 07 09 11 17Isoleucine 09 11 11 20Methionine 13 14 14 25

Valine 13 16 16 28Alanine 26 27 27 42Glycine 35 36 39 49

Asparagine 49 48 40 51Glutamic acid 86 71 56 72Time (min) 105 90 75 65

Solvent front: 10 cm for all. Spots located in iodine chamber.R1, 4.56:15.4; R2, 5.0:15.0; R3, 6.0:15.0; R4, 8.0:15.0, combina-tion (v/v) of acetonitrile±sodium acetate buffer. (0.1 M, pH 4.0)

LIQUID CHROMATIC SEPARATION OF PTH-AMINO ACIDS 323

# 1998JohnWiley & Sons,Ltd. Biomed.Chromatogr. 12, 322–325(1998)

Leu/Ile did not resolvein all the four combinations;however,PTH-Leu/Ile or PTH-Leu/Phecan be consideredto haveresolvedin R4 astherewasa differenceof at leastthreeunits.Similarly, PTH-Met/Val resolvedonly in R4. Thus,out of thenine PTH derivatives,an averageof six canbe consideredtohaveresolvedin R1,R2andR3,while R4provedto bethebestaseightderivativesresolvedin it. An increasein thevolumeofacetonitrilealsoresultedin decreasein developmenttime; R4(containingeight volumesof acetonitrile)took only 65min asagainst105min by R1 (containingfour volumesof acetoni-trile). The initial solventsystemi.e. R1 (acetonitrile–sodiumacetatebuffer with a compositionof 4.56:15.4)was selectedfrom the RP HPLC datawhereat a retentiontime of 6.0 thecalculation for buffer compositiongives a result of 18% ofbuffer-B and92%of buffer-A (thus,a ratio of 4.56:15.4);at ornear this composition there should have been a maximumresolutionon RPTLC, but the resultsshowa needof gradientsystemfor reversedphasechromatography.

HPLC. The conditionsare alreadydescribedin the experi-mentalsection.The columnwasequilibratedwith buffer A-B(100= 0) at a flow rate of 1.5mL/min at room temperature.Detection was carried out by UV at a fixed wavelengthof254nm. First the samplesof standardPTH-aminoacidswererun followed by their mixture underidentical conditions.Allthe nine derivativesgot separated.The retentiontimesfor the

standardsandfor theseparatedonesaregivenin Table5. PTH-asparaginewaselutedfirst andPTH-methioninewaselutedlastasbroadshouldercomparableto thestandard.

Asparagineand glutamic acid (the most polar), which didnot movein solventsystemsN1, N2, andN3 on normalphaseTLC had the highestRf valueson RP TLC plates,and alsoelutedfirst in theRPHPLC.PTH-IlehasahigherRf valuethanPTH-Phein all the three normal phaseTLC systemswhichmeansthe former elutes first, while PTH-Ile has a higherretentiontime in RP HPLC, i.e. it is eluting after PTH-Phe;similar to the behaviourof PTH-Val andPTH-Gly. Thus,the

Figure 1. Chromatogram showing separation of PTH-aminoacids on normal phase thin layer silica gel plates activated for2 h using chloroform±acetonitrile, 15:0.5 v/v. PTH-aminoacids from left to right : PTH-Ala, Met, Val, Phe, Ile, Pro, Tyrand the mixture.

Figure 2. Chromatogram showing separation of PTH-aminoacids on normal phase thin layer silica gel plates activated forovernight using chloroform±acetonitrile, 15:0.5 v/v. PTH-amino acids from left to right : PTH-Ala, Met, Val, Phe, Ile,Pro, Tyr and the mixture.

Table 5. Retention times for PTH-amino acids on Lichro-sphere100RP-18(5 mm) column

PTH-amino acid Standard sample From the mixture

Asparagine 3.68 3.52Glutamic acid 3.94 3.94Alanine 4.16 4.37Glycine 4.85 4.85Valine 5.54 5.06Phenylalanine 5.70 5.44Leucine 5.97 6.24Isoleucine 6.29 6.45Methionine 7.41 (broad) 7.41 (broad)

324 R. BHUSHAN AND R. AGARWAL


behaviouris in agreementwith thenormalandreversedphases.It wasinterestingto notethat thederivativesin thegroupof

PTH-Phe,Leu, Ile, and Met, elute in increasingorder ofretentiontimesandshowincreasingRf valuesonRPTLC in allthefour solventsystems.Similarly, thePTH-Ala,Gly, andValelutein theincreasingorderof retentiontime in RPHPLC andshow an increasing trend of Rf values (PTH-Val beingdifferent) in the solvent systemsR1 to R4 on RP TLC;probablydueto thegradientmodeappliedin RPHPLCstudies.

The separationsystemspresentedhere are reliable andreproducible and are being used for the identification ofunknownPTHaminoacidsobtainedduringEdmandegradationof certain polypeptides in this (Roorkee) laboratory. The

locationof spotsonTLC platesby iodinevapoursdoesnotaltertheir chemicalnatureandthesecouldbeelutedfor furtheruseby markingthe zonesandcutting thegel from theplate.

Acknowledgements

Thanksaredueto AlexandervonHumboldt-Stiftung,Bonn,Germany,for awardingafellowship(duringJuly–August,1997)andfor donatingthe Merck–Hitachi HPLC equipment; to Macherey-Nagel,Duren,Germany,for generouslydonatingtheRPTLC plates(to R. B.); andtothe University GrantsCommission of India, New Delhi for SeniorResearchFellowship(to R. A.).

REFERENCES

Allen, G. (1981). In Laboratory Techniques in Biochemistryand Molecular Biology (Burdon, R. H. and Work, T. S.eds.), p. 192, Elsevier, New York.

Bailey, J. L. (1967). Techniques in Protein Chemistry p. 192.Elsevier, New York.

Bhushan, R. and Reddy, G. P. TLC of phenylthiohydantoinamino acids: a review. (1987). J. Liq. Chromatogr. 10,3497.

Brener, M., Niederwieser, A. and Pataki, G. (1967). In ThinLayer Chromatography (E. Stahl, ed.), p. 696. Springer,Berlin.

Cunico, R. L., Simpson, R., Correia, L. and Wehr, C. T. Highsensitivity phenylthiohydantoin amino acid analysisusing conventional and microbore chromatography.(1984). J. Chromatogr. 336, 105.

Edman, P. Preparation of phenylthiohydantoin from somenatural amino acids. (1950). Acta Chem. Scand. 4, 277±282.

Kim, S. M. Isocratic separation of phenylthiohydantion-

amino acids by reversed-phase high performance liquidchromatography. (1982). J. Chromatogr. 247, 103.

Kulbe, K. D. Rapid separation of phenlthiohydantoin (PTH)amino acids by thin-layer chromatography on polyamideglass plates. (1971). Anal. Biochem. 44, 548.

Kulbe, K. D. Micropolyamide thin layer chromatography ofPTH amino acids at subnanomolar level. (1974). Anal.Biochem. 59, 564.

Niederwieser, A. (1972). In Methods in Enzymology (Hirs, C.H. W. and Timasheff, S. N., eds.) Vol. 25, (Part B), p. 60.Academic Press, New York.

Rosmus, J. and Deyl, Z. Chromatographic methods in theanalysis of protein structure. The methods for identi®ca-tion of N-terminal amino acids in peptides and proteins.(1972). Part B, J. Chromatogr. 70, 221±340.

Somack, R. Complete phenylthiohydantoin amino acidanalysis by high performance liquid charomatogarphyon ULTRASPHERE octadecyltrimethyloxysilane. (1980).Anal. Biochem. 104, 464.

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liquid chromatographic separation of some pth-amino acids

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