Resolution by Gas-Liquid Chromatography of Diastereomers of Five Nonprotein Amino Acids Known to Occur in the Murchison Meteorite G . E. Pollock Ames Research Center, NASA, Moffett Field,Calif. 94305 The Murchison meteorite contains several protein and nonprotein amino acids which were identified previously by gas-liquid chromatography-mass spectrometry. The derivatives used in the identification were the N-trifluoroacetyl-(N-TFA)-amino acid-R(+)-2-butyl esters. With these derivatives, it was possible to demonstrate that the meteorite amino acids were racemic and, hence, probably of abiogenic origin. Of the nonprotein amino acids, however, diastereomeric derivatives of isovaline, p-amino-n-butyric acid and Nmethyl-alanine were completely unresolved, and pipecolic and p-amino-isobutyric acids were incompletely resolved. This paper describes the synthesis of model derivatives of these compounds and their resolution by gas-liquid chromatography. In the process of resolution and peak identification, two exceptions (isovaline and p-amino-n-butyric) to the usual elution pattern of diastereomers were found.
THE RESOLUTION of diastereomers of amino acids by gasliquid chromatography has been reported by several investigators in past years (1-12). The method which we originally developed has been useful in medical (13, 1 4 , soil ( 1 3 , and geochemical research (16-18). Kvenvolden and coworkers (19-21) recently applied the method in their analysis of the ( I ) F. Weygand. A. Prox, L. Schmidhammer, and W. Konig, Argebv. Chcm., Ifit. Ed. B i g / . , 2, 183 (1963). (2) R . Charles, G. Fischer, and E. Gil-Av, Zsr. J . Cliem., 1, 234
(1963). (3) E. GII-Av, R. Charles. and G . Fischer, J . Clironzatogr., 17,408 (1965). (4) B. Halpern and J. W. Westley, Biocliem. Biopliys. Res. Comm m . , 19, 361 (1965). ( 5 ) G. E. Pollock, V. I. Oyama, and R. D. Johnson, J . GUSCliromafogr.. 3, 174 (1965). (6) B. Haloern and J. W. Westlev. Cliem. Commun.. 12,246 (1965). (7; S. V. V i t t , M. B. Saparovskaya, I. P. Gudkova,’and’V. M. Belikov, Termliedroti L e f f . ,30, 2,575 (1965). (8) G. E. Pollock and V. I. Oyama, J . Gas Cliromatogr., 4, 126 (1966). (9) B. Halpern and J. W. Westley, Tefraliedron Lett., 21, 2283 (1966). (10) E. Gil-Av, R. Charles-Sigler, G. Fischer, and D. Nurok, J . Gas Chroniatogr., 4, 51 (1966). i l l ) E. Gil-Av and B. Feibush, Terraliedroii Left., 35, 3345 (1967). (12; G. E. Pollock and A. H. Kawauchi, ANAL.CHEM.,40, 1356 (1968). (13) R. Charles-Sigler and E. Gil-Av, Tetnrhedron Lett., 35, 4231 (1966). (14) B. Halpern and G. E. Pollock, Bioclieni. Med., 4,352 (1970). (15) G. E. Pollock and L. H. Frommhagen, Ami. Biocliern., 24, 18 (1968). (16) K. A. Kvenvolden, E. Peterson, and G. E. Pollock, Nature, 221, 141 (1969). (17) G. E. Pollock, A. K. Miyamoto, and V. I. Oyama, “Life Sciences and Space Research VIII.” North-Holland Publishing Co., 1970, p 99. ( 1 8 ) K. A. Kvenvolden. E. Peterson. and F. S. Brown, Scieme. 169, 1079 (1970). (19) K. A. Kvenvolden, J. G. Lawless, and C. Ponnamperuma, Proc. Nc,!. Acud. Sci.. 68. 486 (1971). 2368
organic constituents in the Murchison meteorite. They identified 18 of the amino acids which were extracted from the meteorite; nine of them were shown to be racemic. Some of the meteoritic amino acids were the same as those found in abiogenic chemical evolution experiments. In addition t o six protein amino acids, 12 nonprotein amino acids were identified by gas-liquid chromatography-mass spectrometry. Of these nonprotein amino acids, identifications revealed that several had to exhibit optical asymmetry. They should, therefore, have produced double peaks if they were present as racemic mixtures. However, the N-trifluoracetyl-(SR)amino acid-(R)-2-butyl esters of three of these nonprotein amino acids were completely unresolved, two of them were poorly resolved, and only two were well resolved (a-amino-nbutyric acid and norvaline). The amino acids which had optical centers, but gave n o doublets were isovaline, p-aminon-butyric acid, and N-methylalanine. It was, therefore, impossible t o determine conclusively whether they were racemic or not. Naturally, it was assumed that they were racemic since the five protein amino acids and two nonprotein amino acids identified in the meteorite were racemic. Furthermore, two additional amino acids, 6-amino isobutyric acid a n d pipecolic acid, were poorly resolved. Two peaks each were obtained, and they appeared t o be racemic since the patterns of control racemic derivatives were similar. With the discovery of these deficiencies in the method employing 2-butyl ester derivatives, it was decided to investigate other esters in the hope that with the resolutions of these amino acids a more generally applicable approach could be developed for optical asymmetry determinations. Diastereomeric esters from secondary alcohols higher than 2-butanol gave better resolution of single a-amino acids than did 2-butanol (10, 2 2 ) ; however, judicious choice of gas chromatographic parameters and adequate column technology had, until this time, enabled us t o resolve most compounds of interest (except arginine, histidine, and cystine). Since this technique is valuable in numerous terrestrial applications, and since it also has great potential as a n extraterrestrial, life detection and/or chemical evolution space probe, further investigations of 2-alkanols were carried out t o determine which, if any, of the alcohols from 2-pentanol through 2-octanol might form esters which would accomplish the resolution of these particular amino acids. In addition, different acyl groups were investigated because some of our evidence (23)
(20) K. A. Kvenvolden, J. G. Lawless, K. Pering. E. Peterson. J. Flores, C. Ponnamperuma, I. R. Kaplan, and C. Moore. N n t i ~ r e . 228, 923 (1970). (21) K. A. Kvenvolden. E. Peterson, and G. E. Pollock. “Advances in Organic Geochemistry,” Proc. Int. Meeting. 5th, Hanover, Germany, 1971. Pergamon Press, Oxford-Braunschweig, 1972. pp 387-401. (22) B. Halpern and J. W. Westley, Aust. J . Clieni., 19, 1533 (1966). (23) G. E. Pollock, ANAL.CHEhi., 39, 1194 (1967).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1 9 7 2
as well as that of others (24) indicated that acyl groups other than trifluoroacetyl might offer some time and resolution advantages. This is a report of a study on the resolution of the nonprotein amino acids, isovaline, N-methylalanine, p-amino-nbutyric acid, ,R-aminoisobutyric acid, a n d pipecolic acid as 2-butyl through 2-octyl esters! with different acyl groups, i.e., trifluoroacetyl (TFA), pentafluoropropionyl (PFP), heptafluoro-butyryl (HFB), and chlorodifluoroacetyl (CDF). The principal objectives were t o improve the ester route for amino acid diastereomer resolution so that it could be more generally applicable t o terrestrial studies as well as t o extraterrestrial space probes, and t o determine the order of elution of the diastereomeric compounds studied. EXPERIMENTAL
Apparatus. Perkin-Elmer gas chromatographs, Models 880 and 900 modified for capillary columns, were used. Capillary columns, 0.02 in. x 150 ft, coated with Dexsil 400 GC (66 mg) and Ucon 75H 90,000 (25 mg) were employed a s liquid phases. Reagents. Anhydrous rt 2-alkanols, 3 4 N in anhydrous HCl were used in preparing diastereomeric esters of S or S R amino acids. F o r the study involving peak elution order, RR or SR, optically active R-( +)-Z-alkanols were obtained from Norse Laboratories, Inc., Santa Barbara, Calif. 93103. The anhydrides for acylation were obtained from PCR, Inc., P. 0. Box 1466, Gainesville, Fla. 32601. To determine peak elution order of diastereomers, a reasonably optically pure 2alkanol and a pure R or S amino acid, are required. The Nmethyl-S-alanine was obtained commercially from Cyclo Chemical Corp., 1922 East 64th St., Los Angeles, Calif. Spipecolic acid was obtained from Calbiochem, P. O. Box 12087, San Diego, Calif. 92112. A mixture of isovaline, sarcosine, and P-amino-n-butyric acids (mixture 1) a n d another of N-methyl-alanine, &aminoisobutyric acid, and pipecolic acid (mixture 2) were prepared such that 50 p1 of each mixture contained 10 p n o l e s of each amino acid in 3 N HC1. Since isovaline, B-amino-n-butyric acid and /I-aminoisobutyric acid were commercially unavailable in optically active forms, it was necessary t o prepare them. Procedures. Optically active S-(+)-isovaline was prepared using hog acylase resolution of chloroacetyl-SR-isovaline as described by Baker et al. (25, 26). The R-(-)-p-amino-nbutyric acid was prepared by the method of Fischer and Scheibler (27). Optically active R-( -)-P-aminoisobutyric acid has been identified as a constituent of human urine (28, 29) and Wedgewood iris bulbs (30). Resolution of this compound has been reported by Balenovic (31) and Kakimoto and Armstrong (32); however, it was decided to try (24) W . Parr, C. Yang, J. Ptelerski, and E. Bayer, J . Ckromutogr., 50, 510 (1970). (25) C. G. Baker, S.-C. J. Fu. S. A. Birnbaum, H . A. Sober, and J. P. Greenstein, J . Amer. Chem. Soc., 74, 4701 (1952). (26) J. P. Greenstein and M. Winitz, “Chemistry of the Amino Acids,” Vol. 3, John Wiley and Sons, New York, N.Y., 1961, p 2575. (27) E. Fischer and H. Scheibler, A m . , 383, 337 (1911). (28) H. R . Crumpler and C. E. Dent, Notiire (London), 164, 441 (19491. (29) H. R. Crumpler. C. E. Dent, H. Harris. and R . G. Westall, ihid.. 167, 307 (1951). (30) S. Asen, J. F. Thompson, C. J. Morris, and F. Irreverre, J . B i d . Chem.. 234, 313 (1959). (31) K . Balenovic, in “Amino Acids and Peptides with Antimetabolic Activity,” G. E. W. Wolstenholme and C . M. O’Conner, Ed.. Little, Brown and Co., Boston, 1958, p. 5. (32) Y . Kakimoto and M. D. Armstrong. J . B i d . Chem., 236, 3283 ( I 961).
Table I.
Resolution of 2-Alkanol Derivatives on Dexsil400 GCa
N-TFAb 22222Amino acid Butylc Pentylc Hexyl‘ Heptyld Octyld Isovaline 66.7 66.3 62.4 0 78.8 N-Me-alanine 97.3 86.9 94.5 0 52.9 75.0 55.3 72.9 P-”2-n-butyric 0 42.1 98.1 99.2 P-NHz-isobutyric 97.7 93.2 91.1 Pipecolic 99.1 95.7 97.6 0 50.7 86.6 80.5 85.9 Av resolution 18.6 64.3 N-PFP 0 75.8 60.2 54.1 45.0 Isovaline 0 88.7 98.3 98.5 98.2 N-Me-alanine 0 37.0 61.2 82.6 80.0 6-NHz-x-butyric 92.9 96.1 98.3 98.1 98.6 P-NH1-isobutyric 99.7 45.0 96.3 99.4 100.0 Pipecolic 27.6 78.8 83.5 86.5 83.4 Av % resolution N-HFB 7.0 0 34.6 Isovaline 0 68.4 96.0 99.0 99.0 0 88.2 N-Me-alanine 0 45.7 73.0 92.0 91.8 P-NH*-n-butyric 93.2 97.6 98.8 P-NH2-isobutyric 98.2 98.8 30.6 95.7 99.0 99.6 99.6 Pipecolic 24.8 79.1 80.2 79.3 77.8 Av resolution N-CDF 0 72.3 37.3 ,.. ... Isovaline 0 66.6 90.3 ... ... N-Me-alanine 77.1 ... ... p-NH2-rz-butyric 0 58.3 93.5 96.6 96.7 ... ... P-NH2-isobutyric 97.5 ... ... 0 60.9 Pipecolic 18.7 70.9 80.0 . .. ... Av % resolution Column: 0.02 in. X 150 ft (66 mg phase). Helium, ( 3 ml/ min). N-TFA = N-Trifluoroacetyl, N-PFP = Ai-Pentafluoropropionyl. N-HFB = N-Heptafluorobutyryl, N-CDF = N-Chlorodifluoroacetyl. c Program for these derivatives was 100-200 ’C at 2’:min. Program for these derivatives was 100-225 ‘C at 2”/min. Note: This program was used for this survey only and is not optimum. A rate of l”/min rise gives better resolution values (cn. 5-20z); however, patterns and conclusions are unchanged.
Ehrlich’s (33) procedure using Saccharomyces cerevisiae in the form of Fleischmann’s bakers yeast. Reasonably pure R(-)-P-aminoisobutyric acid was obtained a n d verified by comparing it with 6-aminoisobutyric acid isolated from urine. The methodology for the resolution using bakers yeast will be reported elsewhere. Derivatization Procedure. To a Teflon (Du Pont) lined screwcap tube (10 ml) was added 50 pl of mixture 1 a n d t o another tube was added a n equal amount of mixture 2. Each tube was heated a t 100 “C in a stream of dry nitrogen t o remove solvent. To each tube was added 3 ml of the R, S(*)-2-alkanol (ca. 3-4N in anhydrous HC1) under investigation. After addition of the 2-alkanol, the tube was capped and esterification carried out for 3 hours a t 100 “ C in a silicone oil bath. The tube was removed and the 2-alkanol and HCI were evaporated in a stream of dry nitrogen a t 100 “ C . The tubes were cooled t o room temperature and t o each tube was added 1.8 ml of methylene chloride and 0.2 ml of the anhydride under investigation. The tubes were recapped and placed i n a n oil bath a t 100 “ C for 15 minutes, cooled, and the solvent removed. The T F A , PFP, and C D F samples were evaporated o n a rotary evaporator a t room temperature. The 2-butyl and 2-pentyl derivatives were evaporated in a n (33) F. Ehrlich and A. Wendel, Biochem. Z., 8,438 (1908).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
2369
Resolution of 2-Alkanol Derivatives on Ucon 75H90,000a
Table 11.
N-TFAb
20
2Pentyl 0
N-Me-alanine 3-NH2-ri-butyric
0 0
0 Very
/3-NH?-isobutyric Pipecolic Av 7; resolution
74.1 28.1 20.4
Amino acid Isovaline
Butyl
2Hexyl Very slight 42.5 25.5
22Heptyl Octyl Very 24.3 slight 65.1 92.3 35.7 63.6
100.0 91.5 51.9
96.1 100.0 96.8 100.0 58.7 76.0
slight
94.7 56.0 30.1
N-PFP
Isovaline
0
Very slight 53.1 Very
N-Me-alanine /3-NHs-/i-butyric
0 0
$-NH?-isobutpric Pipecolic Av resolution
86.0 60.6 29.3
38.0
45.7
52.0
82.6 36.2
91.4 66.6
97.8 81.8
slight
100.0 92.1 49.0
100.0
98.0 71.0
100.0 100.0 95.3 100.0 79.8 86.3
N-HFB I sovaline
0
N-Me-alanine P-NH?-rz-butyric 13-NHZ-isobutyric Pipecolic Av :{ resolution
0 0 90.0 43.6 26.7
Very slight 60.8 30.9 99.8 89.0 38.3
Very slight 78.5 64.5 100.0 100.0 68.6
Slight
14.0
94.4 96.5 82.6 86.4 100.0 100.0 100.0 100.0 75.4 79.4
N-CDF
Very 24.3 ... ... slight 0 40.0 37.5 ... ... N- Me-alanine 0 51.5 70.4 $-NHL-rz-butyric ... ... 90.6 100.0 97.5 P-NH,-isobutyric ... ... 32.3 56.8 95.0 Pipecolic ... ... Av resolution 24.6 49.7 64.9 ... ... a Column: 0.02 in. X 150 f t (ca. 25 mg phase). Helium. 2.7 ml/min. Program for these derivatives was 100-170 "C at 1 "/min. * N-TFA = N-Trifluoroacetyl, N-PFP = N-Pentafluoropropionyl, N-HFB = N-Heptafluorobutyryl, N-CDF = N-Chlorodifluoroacetyl. Isovaline
0
butyric acid, are difficult t o resolve. The studies of Rose et al. ( 3 4 , Stern et al. ( 3 3 , Feibush et al. (36), and Feibush (37) may be pertinent to these results. Rose and Stern investigated the separation of diastereomeric esters, and Feibush and coworkers investigated the separation of stereoisomers o n a n optically active phase. Both of these groups of investigators were interested in the factors which were involved in determining resolution of either stereoisomers or diastereomers. All investigators found that the bulk of the substituents associated with the asymmetric carbon affected resolution. Feibush (37), commenting o n the subject of his research, said: "The separation factors of RC*(H) (HNCOCF3) CO? R1 depend o n the relative size of the substituents of the asymmetric carbon (H, R and C 0 2 R1). While the hydrogen is always the smallest substituent, the relative size of the R and COY R1 depends o n the compound considered; when their effective size is equal, no separation is observed;. . . " Stern et al. (35) obtained similar results with the diastereomers of methyl alkyl carbinyl a-halopropionates and a-butyrates. They found in the class of compounds having the general structure X O H il R-C*-C-O-C*-R 1 ~
~
I
I
H
CH, that when R1 was ethyl and R was methyl, the separation factor, a , was 1.022. When R1 and R were both ethyl, a was only 1.008. This and other information in the same paper supported the idea that if the effective bulk of R and R 1were equal, very little, if any, separation of diastereomers occurred. This is a possible explanation for the lack of resolution of isovaline as the 2-butyl derivative. The structure of the isovaline derivative is CH3O H I C~H~-C*-C-O-C"-C~H j
I I 1
NH
1
I
CH,
c=o ice bath as a precaution against loss of some low molecular weight amino acids. The H F B derivatives were evaporated in a stream of dry nitrogen a t room temperature (ca. 100 ml/min). The samples were dissolved in 2 ml of methylene chloride and 2 p1 were chromatographed in a Perkin-Elmer 900 or 880 gas chromatograph. This procedure appears to give reasonable derivatization of the amino acids investigated ; however, quantitative studies have yet t o be done for the 2alkanols higher than 2-butanol. This study reports only the degree of resolution of diastereomeric derivatives and retention times o n two different capillary columns as well as the peak elution order. RESULTS AND DISCUSSION
The resolution of these derivatives o n Dexsil 400 G C and o n the more polar phase Ucon 75H 90,000 are shown, respectively, in Tables I and 11. The respective retention times are given in Tables 111and IV. I t is seen from Tables I and I1 that the best resolutions of the compounds of interest are obtained on the lower polarity Dexsil400 G C column. This was expected since the isovaline and N-methylalanine have methyl groups substituted for hydrogens, which increases the interaction of the derivatives in less polar substances and decreases the interaction in polar phases. Only isovaline, N-methylalanine, a n d 6-amino-n2370
CF3 Bulk is possibly not the total answer t o resolution as indicated by the variation in bulk which exists between 2-butyl and 2octyl and from N-TFA t o N-HFB. The resolution pattern on the Ucon column (Table 11), for isovaline is somewhat anomalous o n the basis of bulk since the PFP derivatives uniformly resolve best, while on the basis of bulk, one might expect 2-octyl-N-TFA t o resolve best. The Ucon column is, however, a polar column and this would cause the amideester interaction with this phase to play a more prominent role than o n the less polar Dexsil column; consequently, probable polarity influences in the amide-ester region may be dominant here. In the case of N-methylalanine which does not resolve as the 2-butyl derivative, electronic charge in the amide bond may also be more significant than bulk. The p-amino-n_ _ _ _ _ _ ~ _ _ .
-
(34) H. C. Rose: R. L. Stern, and B. L. Karger, AKAL.CHEM..38, 469 (1966). ( 3 5 ) R . L. Stern, B. L. Karger. W. J. Kcane, and H. C. Rose, J . Chrormrtogr.. 39, 17 ( I 969). (36) B. Feibush, E. Gil-Av, and T. Tamari. Zsr. J . Chem.. 8, 50 (1970) supplement. (37) B. Feibush, ANAL.Ctjmi.. 43, 1098 (1971).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
Table 111. Retention Times of Amino Acids on Dexsil400 GC" N-TFAd 2-Butylb 2-Pentyl* 2-Heptylc 2-Octvlc Ester .2 1 2 1 i 1 I Amino acid 2 1 2 26.21 ... 31.94 32.28 39.56 Isovaline 39.82 44.18 44.48 50.53 50.85 30.43 ... 34.72 34.93 42.02 N- Me-alanine 46.74 32.37 47.17 52.51 53.02 ... 36.66 30.84 @-NH?-rz-butyric 36.86 44,48 44.81 48.71 54.87 49.06 55.18 31.39 $-NH?-isobutyric 31.82 35.94 36.51 43.45 44. GO 48.22 53.92 48.87 54.57 ... 52.17 48.53 Pipecolic 52.45 60.30 60.93 63.37 69.08 63.88 69.80 IV-PFP 25.49 Isovaline ... 30.90 31.25 38.14 38.44 44.28 44.54 48.47 48.71 ... 32.98 28.44 33.36 40.84 N- Me-alanine 41,33 46,94 47.49 51.46 50,64 ... 35.17 35.42 42.57 $-NH1-ri-butyric 29.38 42.86 48.41 48.75 52.41 52.74 ... 34.14 29.13 34.70 42.36 /3-NHi-isobutyric 42.95 48.35 48.89 52.35 53.04 45.46 49.63 45.26 50.05 57.86 Plpecollc 58.61 62.70 63.45 66.22 67.11 N-HFB 27.41 ... 32.37 Isovaline 32.67 39.66 39.81 46.80 46.90 50.48 ... 29.72 ... 34.34 34.73 42.55 N-Me-alanine 48.14 42,84 48.71 51.66 52.29 ... 36,37 30.94 +"?-butyric 36.60 43.74 50.54 41.02 50.93 53.92 54.32 30.41 30.84 35.62 36.15 44.08 44.67 49.65 6-NHy-isobutyric 50.34 53.27 53.98 46.05 46,25 50.38 Pipecolic 50.83 59.12 59.91 63.47 64.16 67.31 66.52 N-CDF Isovaline 38.69 ... 43.14 43.73 51.27 51.46 ... ... ... ... 41.66 N-Me-alanine ... 46.92 47.17 54.59 ... 54.99 ... ... ... 43.01 , . . 48.12 $-NH?-ri-butyric 48.35 55.99 56.36 ... ... .,. ... 42.45 42.90 48.12 $-NH2-isobutyric 48.65 56,03 56.74 ... ... ... ... 60.55 ... 67.50 Pipecolic 67,99 82.62 ... 83.80 ... Void volume, CH,, 4.03 min (subtract to obtain corrected R.T.). * Program for these derivatives was 100-200 T at 2"jmin. Program for these derivatives was 100-225 ' C at 2'/min. "FA = N-Trifluoroacetll. N-PFP = !V-PentaflLioropropionyl, N H F B = hr-Heptafluorobutyr\.l, N-CDF = A'-Chlorodifluoroacetyl. for /i-methylalanine, @-amino-ri-butyric,and pipecolic acids. Peak I - S R ( I+ak - R R , for isovaline and 3-amino-isobutyric acid.
-
zk1
[
Table IV.
Retention Times of Amino Acids on Ucon 75H90,000a N-TFAb
2-Pentyl
Ester Amino acid Isovaline
15.35
...
N-Me-alanine fl-NH,-rz-butqric B-NHy-isobutyric Pipecolic
19.78 35.42 33.95 40 64
...
Isovaline
11.81 17.32 27.16 27.06 34.64
N- Mc-alanine
j3-NH2-/7-butyric 6-"?-isobutyric Plpecollc Isovaline
2-Heptyl 1 2 32 57 32.79
2-Octyl 1 2 40.64 40.90
31.68 50.48 49.44 56.03
39.66 58.39 56.97 62.20
40.19 58.84 58.15 65.28
47.66 67.07 65.10 72.86
48.31 67.54 66.36 73.13
20.37 27.47 41.19 40.97 49,08
26.57 34.58 49.10 48.35 56.68
26.90 35.36 49.55 49.51 58.35
33.61 42.55 57,07 56.70 65.63
34.05 43.53 57.66 57.98 67.11
2-Hexyl ~-~ ~
L
12.20 17.16 26.57 26,23 33.65
...
34.54 41.03
... ... ... 27.65 35.07
...
1
2
1
i
19.62 Shoulder 25.45 41.82 40,84 47,35
...
25.19
...
...
31.29 50.18 47.47 55.20 20.13 26.90 40.82 30.05 47.86
I
42.02 41.72 47.82 Ai-PFP 15.86 21.25 ... 33.36 41.03 N-HFB
15.74 20.90 33.95 32.61 40.25 14.56 21.22 31.53 31 .98 39.46
... 20.00 20.17 25.49 25.78 33.57 33.85 21.57 26.67 33.69 27.20 34.60 41.47 42.63 2-NH?-n-butyric 31.81 39.60 40.05 40.88 47.53 56.38 55.69 $-NH2-isobut)ric 26.82 32.81 38.65 46.58 39.60 47.82 56.03 54.61 Pipecolic 34.11 40.19 46.39 47.47 54.95 56.19 64.65 63.25 IV-CDF Isovaline 28.73 ... 34,44 34.64 42.31 ... 42.55 ... ... N-Me-alanine 38.04 ... 44,08 44.52 52.09 52.43 ... ... ... ... d-NH2-r?-butqric 53.04 ... 59.47 59.89 68.19 ... 68.72 ... ... ... $-NH?-isobutyric 52 49 53.20 59.20 60.26 67.15 68.29 ... ... ... ... Pipecolic 63 1 3 63.82 69.77 70.26 78.53 ... ... 79.57 ... ... Void volume. CH,, 4.33 min (subtract to obtain corrected R.T.). Program for these derivatives was 100-170 T at 1 ';min. N-TFA = N-Trifluoroacetyl. N-PFP = N-Fentafluoropropionll, .V-HFB = N-Heptafluorobutyryl, M-CDF = N-Chlorodifluoroacetyl. [.for rr-methylalanine, $-amino-ri-butyric, and pipecolic acids. N- Me-alanine
... ...
. . I
;:$ 1izi;
Peak I - S R I Peak - R R {for isovaline and 2-amino-isobutyric acid.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBER 1972
rn
2371
2- PENTYL-N- PFP 100-200°CAT 2’/min He 4 Y - 8 0 x DEXSIL 4 0 0 4 , 0.02 x 150
”e r
m
I
1
45
40
35
30
1
I
,
I
45
40
35
30
I
50
i -
60
55
50
-
,
I
I
35
30
25
i
1-I
45
40
35
Figure 1. Chromatograms showing resolutions obtained for 2-pentyl and 2-hexyl derivatives butyric acid derivative resolves poorly, probably because of effective bulk and the fact that hydrogen bonding between the active sites (amide and ester) and the phase will be affected by the greater distance between the two optical centers of the diastereomers. The elution order of the diastereomers of N-methylalanine, (3-amino-n-butyric acid, and pipecolic acid (Tables I11 and IV) is the same that has been normally encountered previously with this method of resolution; i.e., the RR peak elutes first and the S R peak second. Isovaline and 8-amino-isobutyric acid, however. show a reversal of elution pattern, the S R peak elutes first and the RR peak second. U p t o this time, only one case of reversal of elution order has been reported. Charles-Sigler and coworkers (10, 13) report that N-TFARS-phenyl-glycine-R(+)-2-octyl ester has a reversed elution order while the R(+)-2-butyl ester is normal. In this study, the 2-butyl derivative of isovaline is not sufficiently resolved o n these columns t o make any peak order assignment. In the case of the isovaline, when low gas flow and program rates are used, a slight indication of separation is possible. but determination of peak elution order would require a longer column, e,g.. perhaps 300 ft or more. The 2-pentyl. 2-hexyl, and 2-octyl derivatives all show the same reversed pattern. Optically active 2-heptanol was not obtained for this study, but in view of the uniform elution patterns of the derivatives 2372
of the other three alcohols, it is probable that elution patterns are the same. No readily discernible reason for the reversals is obvious from molecular models of isovaline and (3-amino-isobutyric acid, if hydrogen bonding concepts (38) and cis-trans type conformation (39) ideas are considered. It is suggested that a study of a-methyl. a-amino acids and a-methyl, P-amino acids might show that these classes of compounds all reverse their elution pattern, but at present no structural reason is suggested for reversal. While (3-amino-n-butyric acid does not show a reversed elution pattern, its optically active carbon is in the 6 position and is, therefore, not a n analog of (3-aminoisobutyric acid which has its optical center in the a-carbon atom. The main purposes in this investigation were to develop a method capable of resolving model compounds of the unresolved nonprotein amino acids identified i n the Murchison meteorite, and t o determine their diastereomeric elution patterns. The data in Tables I and I1 show that reasonable success was obtained in this study. The ultimate aim is t o separate and resolve as many amino acids as might possibly be present in a given sample, such as a mixture containing both meteoritic and protein type amino acids. Resolution, however, is not the only concern. Since time and power are limitations in a space probe, the speed of analysis becomes a n important consideration as well. The nature of the acyl groups will change retention time. The C D F group causes long retention times o n both the Dexsil and Ucon columns studied, while it does not materially offer any great advantage in resolution except for (3-amino-nbutyric acid. We have therefore ruled out further investigation of this particular acyl group beyond 2-hexyl derivatives. Tables 111 and IV also show that of the acyl groups studied, the PFP group derivatives have shorter retention times o n the Dexsil column, while the H F B derivatives have shorter times on the Ucon column. The retention times of the P F P derivatives on the Ucon column are, however, very close t o the H F B times while the T F A and C D F times are significantly longer. When this information is considered along with the average resolution values shown for each class, the best choices appear to be the 2-pentyl and 2-hexyl-N-PFP derivatives. The 2-heptyl and 2-octyl N-PFP derivatives, while giving slightly better resolution on the average, begin t o have very long retention times when the total analysis time is considered for these acids and the common protein amino acids (not reported here). Examination of Tables I11 and IV reveals that conflicts in retention time exist between some of these amino acids, e.g., 6-amino-n-butyric acid and (3-aminoisobutyric acid (see Figure 1). Elimination of these conflicts, and others which exist between certain low molecular weight, common protein and nonprotein a-amino acids, e.g., P-alanine, valine, glycine, norvaline, and a-amino-n-butyric acid, will require a n extensive column technology study which is presently being carried out. From previous experience with protein a-amino acids, it is felt that it will be possible, using various phase combinations and column lengths, t o obtain reasonably good resolution and separations, with a minimum of retention time conflicts. RECElVEDfor review April 21, 1972. Accepted July 10,1972.
~ _ _ _________ _______~ (38) E. Gil-Av. B. Feibush, and R . Charles-Sigler. “Cras Chromatography 1966.” A . B. Littlewood. Ed., Institute of Petroleum, London. 1967. p 227.
(39) J. M. Cross. €3. F. Putney, and J. Bernstein. J . Chroniarogr. Sci., 8 , 39 (1970).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 14, DECEMBE:R 1972
