Glass Capillaries for Fast Gas Chromatographic Separation of Amino Acid Enantiomers Wilfried A. Koenig' and Graeme J. Nicholson Chemisches lnstitut der Universitaef, D 74 Tuebingen, Germany
The determination of the configuration of amino acids is an analytical problem of great interest. I t finds application in the investigation of peptide antibiotics (1-3) and other natural peptides (e.g., cell membrane), in the dating of amino acid-containing fossils ( 4 ) ,and in the determination of the retention of optical purity during peptide synthesis (5-7). The gas chromatographic separation of amino acid enantiomers on an optically active stationary phase was first demonstrated by Gil-Av et al. (8) using N-TFA-L-isoleucine lauryl ester coated on glass capillary columns. Improved separations were obtained by using N-TFAdipeptide cyclohexyl esters as stationary phases. Thus, N TFA-L-valyl-L-valine cyclohexyl ester (val-val) ( 9 ) , N TFA-L-phenylalanyl-L-leucinecyclohexyl ester (phe-leu) ( I O ) , and N-TFA-L.-n-aminobutyryl-L-a-amino butyric acid cyclohexyl ester (abu-abu) ( 1 1 ) have variously been used, whereby differences in separating ability and thermal stability were observed. A highly efficient stationary phase, N-Lauroyl-L-valyl-tert-butylamide, was proposed by Feibush ( 1 2 ) .Studies have been made to correlate the configuration of solute and stationary phase with degree of enantiomer separation (13-15). Upon inspection of the chromatograms of D-/L-amino acid enantiomer separations published, the extremely long analysis times and the low efficiency of the columns used is apparent. The reason for the long retention times is, to a large extent, the limited thermal stability of the stationary phases used. Val-val for example, can be used only between 100 and 110 "C (mp of val-val = 96-98 O C ) . T o shorten the time of analysis with all its consequent advantages, we have attempted to make highly efficient capillaries coated with phases of low volatility. We chose to use glass capillaries throughout our investigations because of the lower catalytic activity of the glass surface in relation to stainless steel (we have observed partial decomposition of cysteine, serine, and threonine derivatives during chromatography on steel capillaries), and because it appears that glass capillaries yield columns of higher efficiency than steel capillaries. Untreated glass capillaries coated with any of the dipeptide stationary phases deteriorated rapidly during usage (within one to two days). Surface modification proved to be necessary. Two new dipeptide stationary phases were synthesized and their properties in relation to enantiomer separation, temperature range of operation, and thermal stability investigated. These new phases are N-TFA-L-phenylalanylI,-phenylalanine cyclohexyl ester (phe-phe) and N-TFAI,-phenylalanyl-L aspartic acid bis(cyclohexy1) ester (pheasp).
EXPERIMENTAL Gas Chromatography. A Carlo E r b a gas chromatograph model Fractovap 2101 Milan, Italy) equipped with an all glass injection port. inlet splitter, and flame ionization detector was used. Hydrogen was employed as carrier gas. Preparation of Glass Capillary Columns. Glass capillaries Present address, Institut fur Organische Chemie und Biochemie der Universitaet, 112 Hamburg 13, Germany.
were drawn t o the dimensions 0.9-mm o.d. and 0.30-mm i.d. with t h e glass drawing apparatus from H u p e and Busch (KarlsruheGroetzingen, Germany). T h e length of t h e capillaries used varied between 14 and 40 meters. T h e following two methods were found t o improve the stability of t h e coating ( u p to two months constant usage). Method 1. A solution of 1% 3-aminopropyl triethoxy silane (hlerck, Darmstadt, Germany) in CH2C12 was forced through t h e column, the ends were sealed, and t h e remaining film of reagent was allowed to react a t 120-130 "C. Excess reagent was removed by washing with CHPC12, a n d t h e column subsequently coated by t h e static method as described by Bouche and Verzele (16) using 0.2% stationary phase dissolved in CH2C12. T h e column was then conditioned by slowly raising t h e temperature t o above the melting point of the stationary phase. In this way, we intended t o form a stable chemical bond between t h e glass surface (Si-OH) and the aminopropyl-triethoxy silane and t o link the stationary phase t o the reagent by means of a n aminolysis of the cyclohexyl ester. Method 2. Soda-lime glass capillaries were etched with dry HC1 gas according t o t h e method of Novotny a n d Tesarik ( 1 7 ) . Synthesis of Dipeptide Derivatives. N-TFA-L-valyl-L-valine cyclohexyl ester was purchased from Miles Laboratories, Elkhart, IN. T h e phe-leu. phe-asp, and phe-phe phases were analogously synthesized by t h e method described by Koenig e t al. ( 1 0 )and recrystallized from diethyl ether. (Melting points: phe-leu 113 " C , phe-phe 136 O C , phe-asp 96 "C.) Retention Indices. In order t o measure t h e relative volatility of the dipeptide phases, t h e retention indices were determined on a 2-m glass column packed with 3% OV 17 on Gas Chrom Q a t 280 O C . (Jphe-asp = 3545, Iphe-phe = 3342, Jphe-leu = 2825, Jiv-Lauroyl-L-Valteit-butylamide = 2400,Jvnl-vai = 2260). Preparation of Amino Acid Derivatives was carried out according to ( 1 0 ) .
ANALYTICAL APPLICATIONS The high efficiency of glass capillaries as compared to stainless steel capillaries (IO) is excellently demonstrated in Figure 1. A column coated with val-val yielded 100.000 theoretical plates for k = 23.3 (2570 theoretical plates/ meter). Figure 2 demonstrates the high temperature stability (low volatility) of the phe-asp phase. Its upper limit of around 165 "C is considerably higher than that found in all published phases of this type, and is accompanied by a low melting point (96 "C), which enables the column to be used over a larger temperature range than has hitherto been possible. T h e use of temperature programming, thus enabling the analysis of derivatives of greatly diverging volatility in a single injection is possible with phe-asp. This is of considerable interest for the analysis of micro amounts of complex amino acid mixtures as obtained from natural sources or from peptide or protein hydrolysates. In the practice of amino acid enantiomer analysis the following problems are encountered: 1) The separation of certain amino acids (e.g., pro, asp) requires phases with high a ( D / L ) factors. 2) Overlap of D- and L-enantiomers of different amino acid species is encountered (e.g., D-ileu with L-a-ileu, D-ser with L-leu). 3) Amino acid derivatives of low volality require very long retention times or are not eluted a t all. According to our experience, the val-val phase meets the requirements of problems 1 and 2 most adequately. Although N-lauroyl-L-val-tert-butylamide ( 1 2 ) displays sigANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
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Figure 1. Separation of TFA-amino acid isopropyl esters on 39-m glass capillary coated with KTFA-L-Val-L-val cyclohexyl ester. Column temperature, ii o "C
nificantly higher ~ ( D / Lvalues ) for most enantiomeric pairs, overlap of amino acid species is encountered to a greater extent (leu, pro, ser) than is the case with Val-Val. T h e dipeptide phase with lowest volatility and largest usable temperature range is phe-asp. Although ~ ( D / L values ) for phe-asp are considerably lower than those of Val-Val, phe-asp is capable of fully separating all amino acid enantiomers except proline, but overlapping is found in the case of various amino acid species (e.g., D-ileu, L-a-ileu). T h e high temperature stability of phe-asp enables the rapid elution of low-volatile amino acid derivatives as is shown in Figure 2. T o test the stability of phe-asp and phe-phe a t higher temperature, we sealed samples of 2 mg of each phase in a glass capillary and kept it a t 165 "C for 30 hours. Gas chromatography of a solution of the dipeptide phases showed no decomposition products in the case of phe-phe and two minor peaks of decomposition products in the case of pheasp. Optical purity of the heat treated phases was also investigated. After hydrolysis of the samples in 6 N hydrochloric acid, esterification with isopropanol (1.5N of HC1) and trifluoroacetylation, we found complete retention of configuration in the case of phe-phe but about 30% racemization in the case of phe-asp, a factor which must be considered when these phases are operated a t high temperatures. T h e phe-asp phase is more labile because of the chemical and thermal lability of the aspartic acid moiety. A small loss in separation efficiency of the phe-asp column after prolonged heating to 165 "C may be explained by the slow decomposition and racemization of the dipeptide derivative. The phe-phe phase may be used between 130 and 160 "C. Although its volatility is somewhat higher than that of
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Figure 2. Separation of pentafluoropropionyl amino acid isopropyl esters on 20-m glass capillary coated with KTFA-L-phe-L-asp bis(cyclohexyl) ester. Column temperature, 130 "C, temperature program, 1 "C/min to 165 'C
phe-asp, it is more stable against temperature degradation and racemization.
ACKNOWLEDGMENT T h e authors express their gratitude to Erba Science, Hofheim, Germany.
LITERATURE CITED (1) H. A. James in "Amino Acids, Peptides and Related Compounds". Organic Chemistry Series 1, Vol. 6, D. H. Hey and D. I, John, Ed., Butterworth University Press, 1973, p 213. (2) W. A. Koenig, W. Loeffler, W. H. Meyer. and R. Uhmann, Chem. Ber., 106, 816 (1973). (3) A. Hasenbohler, H. Kneifel, W. A. Koenig, H. Zahner, and H.-J. Zeiler, Arch. Microbioi., 99,307 (1974). (4)J. Bada and R. Protsch, Proc. Nat. Acad. Sci., USA, 70, 1331 (1973). (5) B. Halpern, L. F. Chew, and J. W. Westley, Anal. Chem., 39, 399 (1967). (6) F. Weygand, D. Hoffmann, and A. Prox, Z.Naturforsch., Teil 8, 23, 279 11 RRR\ L (7) E. Bayer. E. Gil-Av, W. A. Koenig, S. Nakaparksin, J. Orb, and W. Parr, J. Am. Chem. SOC.,92,1738 (1970). (8)E. Gil-Av, B. Feibush, and R. Charles-Sigier. in "Gas Chromatography 1966", A. E. Littlewood, Ed., Institute of Petroleum, London, 1967, p 227. (9) E. Gil-Av and 6. Feibush, Tetrahedron Lett., 3345 (1967). (10) W. A. Koenig, W. Parr, H. A. Lichtenstein, E. Bayer, and J. Orb, J. Chromatogr. Sci., 8, 183 (1970). ( 1 1) W. Parr and P. J. Howard, Angew. Chem., 84, 586 (1972). (12) B. Feibush, Chem. Commun., 544 (1971). (13) J. A. Corbin. J. E. Rhoad, and L. E. Rogers, Anal. Chem., 43, 332 (1971). (14) S. Weinstein. G. Jung, and E. Gil-Av, Proc. Annual Meetings lsrael Chem. Soc.. 1972, p 202. (15) E. Feibush and E. Gil-Av. Tetrahedron, 26, 1361 (1970). (16) J. Bouche and M. Verzeie, J. Gas Chromatogr., 6, 501 (1968). (17) M. Novotny and K. Tesarik, Chromatographia, 1, 332 (1968).
RECEIVEDfor review October 10, 1974. Accepted January 13, 1973.
