Effect of Solute Structure on Separation of Diastereoisomeric Esters and Amides by Gas-Liquid Chromatography J. W. Westleg and B. Halpern Department of Genetics, Stanford Medical Center, Palo Alto, C a l f . 94304 B. L. Karger Department of Clieniistry, Northeastern UniGersity, Boston, Mass. 021 1.5 THESEPARATION of diastereoisomers by gas-liquid chromatography (GLC) is an area currently being actively investigated (1-4, since the method can be successfully used for the determination of steric purity of asymmetric compounds ( 5 ) . We have been concerned with the underlying solute structural features necessary for separation to occur (6, 7), in order t o provide guidelines for the selection of suitable resolving agents and chromatographic conditions. In this paper we wish t o report results on the separation of a series of systematically structurally related N-trifluoroacetyl-S-phenylalaninef alkyl esters and a series of N-trifluoroacetyl-S-prolylamides derived from asymmetric amines and amino acid esters. EXPERIMENTAL
Diastereoisomeric esters were separated on a Varian Autoprep gas chromatograph using a 15-foot X '/(-inch column packed with 0.75/0.25 W/W (DEGS)/(EGSS-X) o n Chromosorb W, with a nitrogen flow of 65 ml/min (Table I). The amide and peptide derivatives were chromatographed on a Varian Aerograph 1200 chromatograph using 5-foot X lia-inch stainless steel columns packed with 5x (QF-I) on Aeropack 30 and 0.5% ethylene glycol adipate (EGA) on Aeropack 30, with a nitrogen flow of 30 ml/min (Table 11). All compounds were prepared by procedures previously described, and the configurational assignment under each chromatographic peak was made by synthesis of the optically pure diastereoisomers (7, 8). All derivatives were characterized by mass spectrometry using a Beckman Time of Flight mass spectrometer and a Varian Aerograph 600D gas chromatograph coupled to a Finnigan 1015 Quadropole mass spectrometer. RESULTS AND DISCUSSION
Tables I and I1 present the results of the separation of the series of diastereoisomeric esters and amides. The relative volatility, CY, is a ratio of corrected retention times (net retention) and A(AG") is the difference in standard molar free energies of gas-liquid partition behavior for the diastereoisomeric pairs (6). The retention times are reproducible t o (1) B. Halpern and J. W. Westley, Chem. Cornmiin., 1966, 34. (2) E. Gil-Av, R. Charles-Sigler, G. Fischer, and D. Nurok, J . Gus Cht.ornutogr., 6, 51 (1966). (3) G. E. Pollock, V. I. Oyama, and R. D. Johnson, ibid., 5, 174 (1 966). (4) Y . Gault and J. Felkin, Bull. SOC.Clzim. France, 1965, 742. ( 5 ) M. Raban and K. Mislow, "Topics in Stereochemistry," Vol. 2, N. L. Allinger and E. L. Eliel, Eds., Interscience, New York, 1967, p 211. (6) B. L. Karger, R. L. Stern, H. C. Rose, and W. Keane, "Gas Chromatography, 1966," A. B. Littlewood, Ed., Institute of Petroleum, London, 1967, p 240. (7) B. L. Karger, R. L. Stern, W. Keane, B. Halpern, and J. W. Westley, ANAL.CHEM., 39, 228 (1967). (8) B. Halpern, J. Ricks, and J. W. Westley, Aust. J . Chem., 20, 389 (1967). 2046
ANALYTICAL CHEMISTRY
1 0 . 0 5 min which results in a reproducibility ofA(AG") of + 5 calimole. In Table I we show the data for structurally related (+) alkyl esters of N-TFA-S-phenylalanine. Increases in the size differential of groups attached t o the alcoholic asymmetric carbon atom of esters of acetylated lactic acid had been shown previously to cause corresponding increases in the degree of separation of diastereoisomeric pairs (6). The data in Table I confirm that this effect also makes an important contribution in the separation of the diastereoisomeric N-TFA-phenylalanine (f) alkyl esters. For example, in compounds 1, 4, and 5 , R is maintained constant as methyl, and R' is varied in size. As expected, the largest A(AG") value occurs for R' = t-butyl and the smallest for R' = ethyl. In compounds 5 , 6, and 7 , R ' is maintained constant as isopropyl, and R is varied in size. The largest size difference in the groups attached t o the alcoholic asymmetric center occurs when R is methyl and accordingly, compound 5 gives the largest value of A(AG"). Note that the change in A(AG") is significant when R is varied from methyl to ethyl, but the change is only slight when npropyl is substituted for ethyl. Another series for comparison is compounds 2, 8, and 9. Here, R is maintained as methyl, and R ' is varied from npropyl to iso-butyl to sec-butyl. Note that the chain length of R' is maintained fairly constant in this series while branching affects the size of the group. Branching improves the value of A(AGo); however, in compound 8, the effect is slight whereas in compound 9 it is significant. This trend is the expected one, since the closer branching is to the asymmetric center, the bigger will be the change in size from the straight chain group. It is clear then that increased asymmetry at the alcoholic asymmetric center aids in separation of these diastereoisomers. Finally, in Table IB, compounds 12, 13, and 14 illustrate the case in which the alcoholic asymmetric center is part of a ring system. The values of A(AGo) are fairly large and result from the hindrance t o free rotation about the carbon-oxygen bond on the alcohol side of the molecule. This hindrance is caused by the interaction of the CY-alkyl substituent on the cyclohexane ring with the carbonyl group. As the alkyl group increases in size, the hindrance to free rotation also increases, with a resultant improvement in A(AG"). These large values are in agreement with the trend previously obtained for diastereoisomeric amides of racemic cyclic amines (7). In Table I1 we show the gas chromatographic separation data for a series of diastereoisomeric N-TFA-S-prolylamides derived from asymmetric amines and amino acid esters. The results are shown for two columns: a relatively nonpolar trifluoropropyl methyl silicone phase (QF-1) and a relative polar ethylene glycol adipate phase (EGA). The first point to note in Table I1 is that the diastereoisomeric amides are in general better resolved on the polar (EGA) column. This result indicates that hydrogen bonding between the amide hydrogen and the carbonyl groups of the ester
Table I. Separation Data of N-Trifiuoroacetyl-S-Phenylalanine-(&)-Alkyl Esters $
R
0
I
II
A. TFA-NH-CH-C-0-CH-R' Uncorrected retention" of diastereoisomers (min)
Compound No.
s-
R'
R
(--)
S-(+)
a
NAG "), cal/mole
T,b 'C
2 3
-CH3 -CH3 -CHI
-CHz-CH3 --CHr--CHz-CH, --CHZ-CHZ-CH~-CH~
15.3 19.4 25.5
15.9 19.8 26.3
1.04 1.02 1.03
- 35 - 18 - 27
138 138 138
4
-CHI
-C-CH,
FH3
9.0
10.3
1.17
- 134
155
8.5
9.2
1.10
- 83
155
10.6
10.9
1.03
-27
155
11.7
12.0
1.03
- 24
155
9.2
9.4
1.025
-22
155
11.0
11.7
1.07
-60
155
12.5
13.7
1.11
- 90
170
18.8
20.7
1.11
-90
170
ff
NAG ">, cal/mole
T> "C
1
\
CH3 CH3
5
/
-CH
-CH3
\
CH3
6
/CH3
CH
--CHz--CH3
\
CH3
/
CH
7
\
CH3
8
-CH3
-CHZ-CH
9
-CH3
-CH
/CH3
/CH3
\
CHz-CHB
10
-CH3
11
-CHa
-0
-0 $
I
Compound No.
0
Ii
B. TFA-NH-CH-C--O-R '' Uncorrected retentiona of diastereoisomers (min) S-(-) S-(+)
R"
13.4
14.6
1.10
- 83
170
13
14.2
15.8
1.12
- 103
170
14
16.7
19.4
1.175
- 142
170
12
C
H
3
9
a GLC analyses were carried out on a Wilkens Autoprep Gas Chromatograph using a 15-foot X l/a-inch column (0.75/0.25 w / w z of DEGS/EGSS-X on Chromosorb W). During the analyses, the NZflow was 65 ml/min. b Inert gas peak times, T = 138 "C 1.5 min, T = 155 "C + 1.4 min, T = 170 "C + 1.3 min. -f
VOL. 40, NO. 13, NOVEMBER 1968
0
2047
Table 11. Separation Data of N-Trifluoroacetyl-S-Prolylamides
Compound No.
Asymmetric compound
R'
R"
Columna
15
1-Amino-1-phenylethane
CH3
5zQF- 1 A 0.5ZEGS B
16
1-Amino-1-cyclohexylethane
CHI
17
Amphetamine
CYCH2-
5zQF - 1 A 0.5zEGA B
CH3
18
Desoxyephedrine
CYHz-
5zQF- 1 A 0.5ZEGA B
19
Phenylglycine ethyl esters
20
Cyclohexylglycine ethyl ester
21
Phenylalanine methyl ester
22
Cyclohexylalanine methyl ester
5zQF- 1 A 0.5zEGA B
CHa -COOC2HS
0wo-"-
5%QF - 1 A 0.5zEGA B
-COOC*H5
5zQF- 1 A 0.5zEGA B
-COOCHa
5%QF - 1 A 0.5zEGA B
-COOCHa
5zQF- 1 A 0.5ZEGA B
Uncorrected retention Compound No. 15
of diastereoisomers (min.) SR ss
Relative retention a 1.20 1.37 1.13 1.27 1.15 1.25 1.12 1.11 1 .OO 1.10 1.08 1.15 1 .08 1.03 1.07 1.14
3.80 4.50 5.90 8.00 16 3.70 4.15 2.70 3.35 17 5.15 5.90 3.10 3.80 18 8.80 9.80 4.80 5.30 19 10.20 10.20 7.90 8.70 20 10.10 10.90 4.20 4.80 21 12.00 13.00 10.40 10.70 22 10.25 10.90 5.10 5.80 a Column A = 5-foot X 1i8-inchof 5z w/wQ F - 1 on Aeropack 30. CoIumn B = 5-foot X 1/8-inchof 0.5% w/w ethylene glycol adipate on Aeropack 30. In both cases, N2flow rate was 30 ml/min. * Inert gas peak times T = 180-210 "C -P 0.25 min.
stationary phase is probably involved in the separation, since in the case of desoxyephedrine, 18, where this hydrogen is replaced by methyl, there is poorer separation of the diastereoisomers on EGA than on QF-1. Since the amide hydrogen is flanked by the two asymmetric centers, it is reasonable to expect differences in hydrogen bond strength for the diastereoisomeric pair. Differences in the extent of interaction of the center ester linkage for the isomeric pair was also thought to be involved in the separation in the previously studied diastereoisomeric esters (6). 2048
ANALYTICAL CHEMISTRY
A(AG ") cal/mole - 175 - 284 -117 -211 -134 -209 - 108 - 98
- 89 - 74 - 131 - 74 - 28
- 65 - 122
Tb
210 180 210 180 210 200 210 200 210 200 210 200 210 200 210 200
Second, it is to be noted that in all cases, the SS diastereoisomer elutes after the SR form. It is suggested that the open chain structure of the SS diastereoisomer (9) makes the amide hydrogen more available for interactions with the solvent than the SR form. Presumably this same increased accessibility in the SS form results in stronger amide molecule intermolecular interactions such that the vapor pressure of the SS form should be lower than that of the SR form at a given temperature. (9) T. Wieland and H. Bende, Ber., 98, 504 (1965).
In general, it is noted that separation is better for the amides in Table I1 in contrast to the esters in Table I. This result is probably due to the hydrogen bonding capacity of the amide hydrogen along with the rigidity imparted by the partial double bond character of the C-N bond in the amide linkage (IO). It is also probable that the use of different acids in Tables I and I1 contributes to separation differences for the esters and amides in this case. However, recent results in which the groups attached t o both asymmetric centers remain constant does reveal that diastereoisomeric amides separate better than esters (11). It is next of interest to compare the aromatic compounds 15, 19, and 21 with their corresponding alkyl derivatives 16, 20, and 22, respectively. While the retention times of the phenyl and cyclohexyl derivatives are approximately the same on the nonpolar QF-1 phase, substantial increases in retention occur for the aromatics relative to the alkyls o n the polar EGA phase, indicative of a solute-solvent interaction of the aromatic ring with the EGA ester phase. Comparison of the 01 and A(AGo) values for compounds 15 with 17 and 19 with 21 o n the polar EGA phase further indicates that separation is better the closer the aromatic ring is to the amine asymmetric center.
While much more work remains t o be done for an understanding of the separation of diastereoisomers, several conclusions may be drawn from the work of this paper and the previous ones ( I , 6, 7). In general, a polar phase, such as a polyester, produces better separation for diastereoisomeric esters and amides than a nonpolar phase. The resolving agent should be a readily available, low molecular weight optically pure compound containing a suitable functional group close to the asymmetric center. This last requirement results from the necessity of having the two asymmetric centers close t o one another in the diastereoisomeric molecule. If acyclic resolving agents are used, the three groups attached to the asymmetric center (along with the functional group) should have a large size differential. Alternatively, cyclic compounds with a functional group adjacent t o the asymmetric center, such as proline (7), also serve as excellent resolving agents. In general, the more rigid the diastereoisomeric molecule close t o the asymmetric centers, the larger will be the separation. Finally, if polar groups (along with the functional group) are attached t o the asymmetric center, it is difficult t o predict the effect o n separation.
(10) J. R. Dyer, “Applications of Absorption Spectroscopy of Organic Compounds,” Prentice-Hall, Englewood Cliffs, N. J., 1965, p 113. ( 1 1 ) B. L. Karger, R. L. Stern, S . Herliczek, unpublished results, Northeastern University, June 1968.
RECEIVED for review June 10, 1968. Accepted July 29, 1968. Work supported by National Aeronautics and Space Administration under Grant NsG 81 and National Science Foundation under Grant No. 8572.
Spectrophotometric Determination of Some Organic Acids with Ferric 5-NitrosaIicylate Complex Kil Sang Lee and Dai Woon Lee Department of C/iemistry, Yonsei Unicersity, Seoul, Korea Jae Young Hwang Chemical Research Laboratories, AGTL Inc., Natick, Mass. A SPECTROPHOTOMETRIC STUDY of the colored complex produced by the interaction of a solution of ferric salt with salicylic acid and its derivatives, such as 5-chloro, 5-bromo-, 5-nitro-, and 3-nitrosalicylic acid, has been extensively made by many authors. The spectrophotometric determination of the stability constants of some ferric salicylates was reported by Ernst and Menashi ( I , 2). They also found that the complexes were formed by 1 mole of ferric ion and 1 mole of each reagent. The authors have found that the reddish-orange complex which is obtained by the interaction of Fe3+ion with 5-nitrosalicylic acid within the p H range of 2.5 to 3.0, can be used for the determination of iron within a concentration range of 5 t o 30 ppm. Molar absorptivity of ferric 5-nitrosalicylate was previously found to be 2253 mole-’ cm-ll-l at 492 m p (3). The absorption of the ferric 5-nitrosalicylate in a n aqueous or acetate medium at 492 m p is diminished considerably by the addition of small quantities of some water-soluble organic acids. This phenomenon is employed as the basis of a simple (1) 2. L. Ernst and J. Menashi, Trans. Faraday SOC.,59, 2838
spectrophotometric technique for the determination of organic acids. A properly diluted portion of the organic acid is added to a known quantity of the ferric 5-nitrosalicylate. The absorption is measured at 492 m p and compared to a previously prepared plot of absorbance cs. concentration of the organic acid. Kovalenko and Petrashen (4) determined some organic acids, such as citric, tartaric, and oxalic acid, by a spectrophotometric method with chromium (VI)-diphenylcarbazide. A method is described for oxidimetric determination of tartaric acid with potassium cupri-3-periodate in alkaline medium (5). This method could be used only for determination of tartaric acid in the presence of other plant acids after separation of tartaric acid by using ion exchanger Dowex 1-X 10. All methods which use permanganate in alkaline or acid medium have the disadvantage of spontaneous decomposition of permanganate a t elevated temperatures and after a prolonged reaction time. The present study, when compared with the methods mentioned above, is much less tedious, far simpler in the operation, and less time-consuming than the other methods.
(19631.
( 2 j I & i , p 1794. (3) D. W. Lee, M. S. Thesis, Yonsei University, Seoul, Korea, 1966.
~
(4) E. V. Kovalenko and V. I. Petrashen, Tr. Nooocherk, Politekhn, Znsf., 141, 59 (1964). (5) N. Velikonja, Arhiu Za Kemi/rr, 27, 161 (1955). VOL. 40, NO. 13, NOVEMBER 1968
0
2049
