ligands and report values in the 3 X 108 M-1 sec-' range-Le., nearly diffusion controlled. The calculation of dissociation rate constants for these complexes from the equilibrium formation constants and the formation rate constants yields values in the 108 sec-1 range for the most stable complexes. Valinomycin is thus seen to be a n ideal carrier for such ions as potassium and rubidium in membrane electrodes because it
offers both attractive equilibrium selectivity and adequate interfacial kinetics to yield a rapidly responding electrode. RECEIVED for review March 19, 1971. Accepted April 27, 1971. We gratefully acknowledge the generous support of the Environmental Protection Agency, the National Science Foundation, and the National Institute of Health.
I
NOTES
Gas Chromatographic Resolution of OpticalIy Active Alcohols as 3 p-Acetoxy-A5-Etienates M. W. Anders a n d M. J. Cooper Department of Pharmacology, University of Minnesota, Minneapolis, Minn.55455
GASCHROMATOGRAPHY has been employed for the determination of absolute optical purity. Two approaches have been followed. Alcohols or amines may be converted into diastereomeric esters and amides, respectively, which are then separated on conventional packed gas chromatographic columns. Resolving agents used for the optical analysis of alcohols and amines include N-trifluoroacetyl-(S)-prolyl chloride ( I ) , N-trifluoroacetyl-(S)-phenylalanyl chloride (2), 2-acetoxypropionates (3), and (R)-(-)-menthyl chloroformate (4). Alternatively, derivatives of amino acids may be separated by chromatography on capillary columns coated with a n optically active liquid phase such as N-trifluoroacetyl-(5')-valyl-(S)-valine cyclohexyl ester (5). 3/3-Acetoxyd5-etienic acid has been employed for the resolution of asymmetric alcohols by fractional crystallization
(6). This suggested the possibility that etienate esters of
optically active alcohols could be resolved by gas-liquid chromatography. This report describes the gas chromatographic separation of diastereomeric esters formed from asymmetric alcohols and 3&acetoxy-AS-etienic acid. EXPERIMENTAL
Chemicals. 3P-Acetoxy-A5-etienic acid (Mann Research Laboratories, New York, N. Y., [alD-20 "C [c = 1, acetone]) was converted to the acid chloride as described by Djerassi and Staunton (6). Etienic acid has the S configuration at -)-phenylmethylcarbinol were carbon 17. (R)-(+)-and Q-( prepared in this laboratory by the pathway: mandelic acid + phenylethylene glycol + phenylethylene glycol-Ztosylate + phenylmethylcarbinol. Polarimetric examination of (R)-(+)and (a-( -)-phenylmethylcarbinol thus prepared showed [a]g +45.9" (c = 3.32, methanol) and -45.6' (c = 1.48, methanol), respectively. Literature : [a]2go - 44.2 O (neat) (7). Rotations were measured on a Bendix model 969 polarimeter in a 0.2-dm cell. (S)-(+)-and (R)-(-)-methyln-hexylcarbinols (Aldrich Chemical Co.) had [a]:: +9.5 " (neat).
(1) B. Halpern and J. W. Westley, Biochem. Biophys. Res. Commun., 19, 361 (1965). (2) J. W. Westley, B. Halpern, and B. L. Karger, ANAL.CHEM.,40, 2046 (1968). (3) R. L. Stern, B. L. Karger, W. J. Keane, and H. C. Rose, J. Chromatogr., 39, 17 (1969). (4) J. W. Westley and B. Halpern, J. Org. Chem., 33, 3978 (1968). (5) S.Nakaparksin, P. Birrell, E. Gil-Av, and J. Or6, J. Chromatogr. Sci., 8, 177 (1970).
(6) C. Djerassi and J. Staunton,J. Amer. Chem. Soc., 83,736(1961). ( 7 ) E. Downer and J. Kenyon,J. Chem. SOC.,1156 (1939).
Table I. Gas Chromatographic Resolution of Optically Active Alcohols as 3/3-Acetoxy-A5-Etienatesand Menthyl Carbonates. 3!3-Acetoxy-A5-etienates Menthyl carbonates f R of Diastereomers, min t R of Diastereomers, min DC 200
OV-17
Temp., Carbinol "C (94-1( R ) - ( + ) (SI-(-) Phenylmethylcarbinol 260 16.0 17.5 1.09 Phenylethylcarbinol 260 19.0 21.5 1.13 Phenyl-n-propylcarbinol 260 22.0 24.0 1,09 Phenyl-n-butylcarbinol 260 27.5 29.0 1 ,05 Phenylcyclohexylcarbinol 250 93.5 98.5 1.06 Methyl-n-hexylcarbinolb 260 9.0 9.5 1.05 a Configuration and rotation refer to the alcohol. (S)-methyl-n-hexylcarbinol is dextrorotatory.
Temp., O C
230 230 230 230 250 230
OV-17
Temp., @)-(+I ( R ) - ( + ) (94-1 "C (94-1( R ) - ( + ) (94-1 (R)-(+)
694-1 73.5 91.0 110.0 140.0 110.0 58.5
83.0 102.0 120.0 150.0 110.0 62.0
1.13 1.12 1.09 1.07 1.00 1.06
150
160 160 160 170 140
11.0 9.5 12.5 19.0 38.5 9.0
12.0 11.0
14.0 20.5 41.5 10.0
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
1.09 1.16 1.12 1.08 1.08 1.11
1093
W v)
PHE NYLMETHY LCARBINOL (R)-(t) / ( S )--(-I
2
2 v)
Figure 2. Relationship between entio of peak heights of 3P-acetoxy-A5-etienates of (I?)-(+) and (S)-(-)-phenylmethylcarbinol and enantiomeric composition
w
a
Column: 3% OV-17; column temperature: 250 "C
1 . I
I
I
I
IO
20
x)
40
I 50
TIME (min)
Figure 1. Chromatogram of 3/3-acetoxy-A5etienates of (I+(+) and (S)-(-)-phenylmethylcarbinol a t 250 "C on 3 % OV-17 Diastereomer Synthesis. Carbinol (1 8 pmoles), 3P-acetoxyAb-etienic acid chloride (50 pmoles), and pyridine (10 ~ 1 ) were dissolved in 1.0 ml benzene and refluxed for 1 hr. The reaction mixture was diluted with 2.0 ml of benzene and an aliquot injected directly into the gas chromatograph. Derivatives of phenylmethylcarbinol were characterized by mass spectrometry on a Finnigan 1015 quadrupole mass spectrometer. Menthyloxycarbonyl derivatives were prepared as described by Westley and Halpern (4). Gas Chromatography. Analyses were performed on a Varian 1200 gas chromatograph using stainless steeel columns, l/&. 0.d. X 6 ft long; the injector was a 6-in. medium wall glass tube filled with column packing material. Column packings employed were 3 % OV-17 on 100/120 mesh Gas Chrom Q and 5 % DC 200 on 80/100 mesh Gas Chrom Q. Column temperatures are given in Table I. Nitrogen flow rate was 20 ml/min. RESULTS AND DISCUSSION
Table I shows the separation of diastereomers formed from various carbinols and 3P-acetoxyd6-etienic acid. Results obtained with menthyloxycarbonyl derivatives are included for comparison. In the series of phenylalkylcarbinols studied, it can be seen that maximum separation on the OV-17 column is obtained with esters of phenylethylcarbinol. Shortening or lengthening of the alkyl side-chain progressively lowers separation. Similar results were obtained with both 3P-acetoxy-A5-etienates and menthyl carbonates. The magnitude of the separations obtained with esters of etienic acid and menthyloxycarbonyl derivatives
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
(Table I) are approximately the same as those reported previously for the latter compounds (4). Table I also shows the results obtained with both a nonpolar (DC 200) and a moderately polar (OV-17) liquid phase. It is of interest to note that when DC 200 is employed, maximum separation between diastereomers is obtained with phenylmethylcarbinol. Furthermore, the DC 200 liquid phase, in contrast to OV-17, fails to resolve the steroidal esters of phenylcyclohexylcarbinol. The effect of temperature on the separation of diastereomeric esters, using OV-17 as the liquid phase, has also been examined. The ratio of retention times [(I?)-(+)/(,!+( -)] of 3P-acetoxyds-etienates of phenylmethylcarbinol at 270, 260, 250, 240, and 230 "C was 1.10, 1.08, 1.10, 1.11, and 1.12, respectively. Thus it appears that no marked differences in diastereomer separations are produced by altering column temperature. The separation of 3/3-acetoxyd5-etienates of phenylmethylcarbinol at 250 " C is shown in Figure 1 . Inasmuch as these studies were undertaken to provide a more sensitive alternative to polarimetry for metabolic studies, it was of interest to examine the quantitative reliability of the procedure. For this purpose, known mixtures of (I?)-(+)and (S)-( -)-phenylmethylcarbinols were prepared and analyzed. The results are shown in Figure 2. It can be seen that the ratio of peak heights of 3P-acetoxy-A5-etienates of (R)-(+)- and (S)-(-)-phenylmethylcarbinol are linearly related to the ratio of enantiomers present. The deviation seen at ratios of ( R ) - ( + ) / ( S ) - ( - ) of 3 and 4 is probably attributable to the unreliability of peak height measurements when peaks differ considerably in size. It is anticipated that electronic integration would remove this deviation. The techniques described in this report are currently being employed in a study of the stereochemistry of ethyl benzene metabolism in the rat. It has proved feasible to quantitatively determine the absolute optical purity of less than 10 pg of phenylmethylcarbinol isolated from tissue homogenates incubated with ethylbenzene. RECEIVED for review January 18, 1971. Accepted March 17, 1971. This \vork was supported by U.S.P.H.S. Grant No. G M 17699.
