Gas-Liquid Chromatographic Separation of Diastereoisomeric Amides of Racemic Cyclic Amines Barry L. Karger, Robert L. Stern, and Wiuiam Keane Department of Chemistry, Northeastern University, Boston, Mass.
B. Halpern and J. W. Westley Department of Genetics, Stanford Medical School, Palo Alto, Gal$
THESEPARATION of diastereoisomers by gas-liquid chromatography is an area currently under active investigation (1-5). Karger et al. (6) have been concerned with the mechanism of separation of diastereoisomeric esters4.e. the underlying structural factors of both solute and solvent required for separation. In this work to date, it has been found that given two optical centers, flanking a polar ester linkage, the more conformationally immobile the groups attached to the asyrnmetric carbon atoms, the better the separation. It has been suggested that this conformational immobility assists in the creation of an asymmetric environment surrounding the central ester linkage. This asymmetry provides for nonequivalent accessibility of the ester linkage for interactions with the stationary liquid phase by each of the diastereoisomeric pair. In this paper, we wish to report gas chromatographic studies on racemic cyclic amines as their N-trifluoroacetyl-L-prolyl derivatives. These studies will be shown to support the importance of conformational immobility of the groups attached to the asymmetric centers in the separation of diastereoisomers. For quantitative measures of separation, the following parameters have been used: a,the relative volatility, and A(AGo) = -RT In a the free energy differences in the gas-liquid partition behavior of both diastereoisomers. EXPERIMENTAL
For the cyclic amines studied in this work, the diastereoisomeric amides were synthesized from N-trifluoroacetyl-Lproline, the structure of which is given below:
0 pN 4-0l-I
I
c=o 1
C F3
In a typical preparation the redistilled cyclic (i) amine (1 mM) was added to a solution of N-TFA-L-prolylchloride (7)in methylene chloride (1 m M in 10 ml) at 0" C, and the solution was neutralized with triethylamine (0.14 ml; 1 mM). After washing with acid, sodium bicarbonate, and water, the solu(1) B. Halpern and J. W. Westley, Ckem. Comm., 2, 34 (1966). (2) E. Gil-Av. R. Charles-Sider, . G. Fisher, and D. Nurok, J. Gas Chromatog., 6, 51 (1966). (3) G. E. Pollock, V. I. Oyarna, and R.D. Johnson, J. Gas Chroma tog., 5,174 (1966). (4) Y.Gault and J. Felkin, Bull. SOC.Chim. France, 1968,742, (5) H. C. Rose, R. L. Stern, and B. L. Karger, ANAL.CHEM.,38,469 (1966). (6) B. L. Karger, R. L. Stern, H. C. Rose, and W.Keane, Sixth \-,
International Symposium on Gas Chromatography and Associated Techniques, September 20-23, 1966, Rome (preprints, Paper No. 16). (7) F. Weygand, P. Klinke, and I. Eigen, Chern. Ber., 90, 1896 (1957).
228
ANALYTICAL CHEMISTRY
tion was dried (Na2S04)and the solvent was evaporated in vacuo to give a crude mixture of the two diastereoisomers. In a similar manner, the N-TFA-L-prolyl derivatives were prepared from optically pure amines obtained by resolution via the (+) amine-d-tartrate (8) or the (-) amine-d-a-bromowcamphorsulfonate (9). All compounds were characterized by mass spectrometry and by coupling a gas chromatograph to an EA1 Quad 300 laboratory mass spectrometer. Each set of diastereoisomers was shown to have identical mass spectra. Gas chromatographic conditions necessary for separation may be found at the bottom of Table 1.
RESULTS AND DISCUSSION Table I presents the amines examined and the gas chromatographic results obtained for the separation of the diastereoisomeric amides. The A(AG ") values for the diastereoisomers in Table I are in general considerably higher than any observed in the acyclic ester series studied previously by Karger et al. (6). Indeed 'to our knowledge the A(AG') value of -298 cal/mole for the amide prepared from 2-methylindoline is one of the highest values ever obtained in the separation of diastereoisomers by gas chromatography. The compounds in Table I were also chromatographed on a '/*-inch X 5-foot column packed with 0.5% EGA on 8C-100 mesh Chromosorb W, AW (Varian Aerograph). In general, the free energy differences were higher on this column than those obtained in Table I. For example, the amide prepared from 2-methylindoline, chromatographed at 185' C, gave a A(AG") value of - 355 cal/mole. The higher values obtained with the low liquid loaded column, relative to those obtained in Table I, may be caused by three factors : the lower operating temperature, a different stationary liquid phase, and solid support adsorption contributing to selective retention on the lightly loaded column. As Karger et a / . ( 5 ) have previously pointed out, an understanding of the mechanism of separation of diastereoisomers is most easily developed when one process of retention-Le., partition-clearly over-shadows all others. Thus in Table I a heavily loaded column and, as a consequence, higher operating temperature have purposely been used, at the sacrifice in A(AGa), in order to permit a better understanding of the mechanism of separation through a possibly simpler retention process. It is interesting to examine the large free energy differences obtained in Table I in the light of the previous separation mechanism suggested for diastereoisomeric esters by Karger et al. (6). It seems reasonable to assume that the gas chromatographic behavior of the diastereoisomeric amides might parallel that of the esters, at least in general terms. It can first be seen that in all compounds studied in Table I both asymmetric carbon atoms are part of cyclic systems. (8) A. Landenburg, Ann. 247, 85 (1888). (9) W.J. Pope and G . Clarke, J. Chem. Soc., 85,1334 (1904).
Amines 2-Methylpiperidine
Table I. Separation Data of N-Trifluoroacetyl-L-ProlylCyclic Aminesa Uncorrected retention of Amine Molecular ion diastereoisomers (min)* structure (mass spect.) L-(+) L+-)
a
WG"), cal/molec
292
52.73
58.00
1.101
-95
3-Methylpiperidine
292
61.60
57.05
1.081
- 78
2-Ethylpiperidine
306
57.28
65.95
1,153
- 142
320
61.50
70.92
1,153
- 142
332
149.40
123.38
1.213
- 193
326
192.33
258.80
1.341
- 298
O C H a
2-propyl piperidine
trans-Decahydroquinoline 2-Methylindoline
a W
C
H
,
The a GLC analyses were pxformed on a Barber-Colman Model 5000 gas chromatograph equipped with a flame ionization detector. column ('/a inch by 10 feet) was packed in the usual manner with 2 0 z w/w HI-EFF 4B (Applied Science Labs) on 80-100 mesh Chromosorb P, AW, DMCS (F & M !Scientific Corp.). The inlet pressure was maintained at 28 psi, and this resulted in an outlet velocity of 6.5 cmjsec ( f a = 47 sec.). The column temperature was 230" & 0.3"C. * The sign refers to optical rotation of the pure amine. c ( f2 cal/mole).
Since free rotations about bonds on the ring are not possible, the groups attached to the asymmetric centers will be immobile, in particular those groups that are part of the ring. This conformational :.mmobility in the piperidine series can be better understood by an examination of the following amide structure derived from 2methylpiperidine :
where R is equal to N-TFA-L-prolyl. It can be seen in this structure that free rotation about bond Y is not possible, as this bond is part of the 6-membered cyclic system. There remains the possibility of conformational mobility about bond X. Examination of Fisher-Hirschfelder models reveals, however, that the size of the N-TFA-L-prolyl group, and the methyl group in the alpha position of the cyclic amine both tend to make this bond (X) also immobile. Indeed this total conformational immobility should be even greater than that in the previously studied acyclic esters. In effect the conformational immobility of the groups attached to the asymmetric carbon atoms should impart for each of the diastereoisomers a different accessibility to the amide linkage for interaction with the stationary liquid phase. The larger, statistically, the l~opulationof the preferred conformer, the more difference there will be between the two diastereoisomers in the accessibility of this amide linkage, resulting in higher absolute A(AG")values. Previously Karger let al. (5) found that in the ester series the distance between optical centers was a critical factor in determining separa1:ion. Indeed, moving the alcoholic asymmetric center one carbon away from the ethereal oxygen in this series-i.e., changing from the 2-hexyl to the 3-methyl1-pentyl ester-decreased the resolution to zero. However, as
seen in Table I, the diastereoisomeric pair derived from 3methylpiperidine has a A(AG ") value of -78 cal/mole. That this pair is separable may be a result of the fact that the asymmetric center on the amine side of the molecule is part of a rigid ring system. As a consequence, the groups attached to this asymmetric center must remain immobile to some extent. A comparison of the A(AG") values obtained for the diastereoisomers derived from 2-ethylpiperidine and 2-propylpiperidine reveals that the addition of a methylene group, two carbons removed from the asymmetric center on the amine portion of the molecule, results in no change in A(AG"). However, the further addition of a carbon atom, as in the transdecahydroquinoline system, significantly increases A(AGo) (- 51 cal/mole), relative to the 2-propylpiperidine diastereoisomer. This increase is readily understood in terms of the formation of second ring, fused to the piperidine ring, with the generation of a system closely related to trans-decalin. The trans-decalin system is known to be structurally incapable of undergoing chair inversion (IO). As a result, the groups attached to the asymmetric center on the amine side of the molecule for trans-decahydroquinoline, must be immobile. This immobility should be the cause of the increased A(AGo) value. Examination of Table I further indicates that the A(AG") value of the N-TFA-L-prolyl derivative of 2-methylindoline is much larger than the values obtained for the piperidine derivatives. This result may also be understood in terms of conformational effects. In the 2-methylindoline derivatives, the asymmetric carbon atom is part of a five-membered ring, while in the piperidine derivatives the optical center is part of a sixmembered ring. Five-membered rings are known to be conformationally more immobile than six-membered rings. The
(10) E. L. Eliel, N. L. Allinger, S. J. Angyal, and G. A. Morrison, "Conformational Analysis," p. 71, Interscience, New York, 1965. VOL. 39, NO. 2, FEBRUARY 1967
e
229
fusion of a planar benzene ring onto a cyclopentanoid moiety, as in the indoline, must make this system even more immobile and in fact force the system into a rigid planar arrangement. As a consequence of this high rigidity, the N-TFA-L-prolyl derivative of 2-methylindoline would be expected to have a larger A(AG ") value than the corresponding derivatives for the piperidines. It does not seem to us to be appropriate to discuss in detail what the preferred conformations in both the liquid and gas phases may actually be on the acid and amine sides of the molecule. Indeed the conformations of six-membered rings containing one heteroatom are not well understood at the
present time (11). A better understanding of these conformations and studies of gas chromatographic behavior of structurally related diastereoisomeric amides would be necessary for a detailed description of the mechanism of separation. RECEIVED for review November 14, 1966. Accepted December 12, 1966. Work supported by the National Science Foundation under Grant Number G P 5742 and by the National Aeronautics and Space Administration under Grant Number NsG 81-60. (11) Zbid., p. 244.
Controlted-Current Bipotentiometric Titration of Uranium(lV) with Iron(lll) Roy A. Whiteker and Donald W. Murphy Department of Chemistry, Haroey Mudd College, Clurernont, Calif. RECENTLY SEVERAL STuDrEs have been reported in which various electrometric techniques have been used as end-point methods in the oxidimetric titration of uranium with iron(II1). Sympsonet al. ( I ) obtained excellent results for the titration of mixtures of uranium(II1) and uranium(1V) in 0.15F H2S04 with standard iron(II1) using amperometry at a rotating platinum electrode (RPE) as the end-point method. Florence and Shirvington ( 2 ) surveyed a number of end-point methods including amperometry at a dropping mercury electrode (DME), amperometry at a RPE, biamperometry, potentiometry, and controlled-current bipotentiometry for the titration of uranium(1V) in 0.1F HlS04with iron(II1). They concluded that amperometry at a DME was the best method, reporting that they had found amperometry at a RPE to be less reliable and less precise, even though they had used the same electrodeconditioning treatment as Sympson et ul. In addition they observed no end point in a controlled-current bipotentiometric titration, finding that the potential difference between two platinum indicator electrodes remained essentially constant over the range from 50 to 150% titrated. In spite of these negative findings we decided to try to find conditions under which controlled-current bipotentiometry would be feasible as the end-point method for the uranium(1V)-iron(II1) titration. EXPERIMENTAL
Reagents. A stock iron(I1I) solution, approximately 0.1F in FeNH4(S04)2. 12 HzO and O S F in HzS04, was standardized by passing a portion through a Jones reductor, and then titrating the reduced sample with standard K2Cr207in the presence of H3P04 using sodium diphenylamine sulfonate as an indicator. A stock uranium solution, approximately 0.05F in uranium (VI) and 0.5F in H2SO4, was prepared from Bureau of Standards U308which had been ignited in a furnace at 900" C (1) R. F. Sympson, R. P. Larsen, R. J. Meyer, and R. D. Oldham, ANAL.CHEM., 37,58 (1965). (2) T. M. Florence and P. J. Shirvington, ANAL.CHEM., 37, 950 (1965).
230
ANALYTICAL CHEMISTRY
for 1 hour. A 25-gram sample of the U308was dissolved in the minimum amount of concentrated H N 0 3 ,concentrated H S 0 4was added, and the resultant solution was evaporated to dense white fumes. The sides of the flask were rinsed with H 2 0 and the evaporation process was repeated twice. The appropriate additional amount of H2S04was added and the solution diluted to volume in a 2-liter volumetric flask. The standardization of the solution was checked by passing a portion through a Jones reductor, bubbling filtered air through the reduced solution to oxidize uranium(II1) to uranium(IV), and then titrating with standard K2Cr207 according to the method of Kolthoff and Lingane (3). Apparatus. Two platinum electrodes, each of 26-gauge wire and approximately 1 cm long, were used as indicator electrodes. A Beckman Zeromatic pH meter served both as the source of the 10-Ma polarizing current applied to the electrodes and as a vacuum-tube voltmeter to measure the potential difference between them as a function of titrant added. Standard Procedure. A 25-ml portion of the uranium stock solution was passed through a Jones reductor producing a mixture of U(II1) and U(1V). The column was rinsed with 30 ml of 1.5F H2S04and three 30-ml portions of H 2 0 to produce a solution 0.4F in H2S04,and the solution was blanketed with an atmosphere of nitrogen. The two platinum indicator electrodes were conditioned prior to each titration by placing them in 0.05F H2S04, connecting them to a 6-volt source, and electrolyzing the solution for 45 minutes. In attaching the electrodes to the pH meter, the anode during electrolysis was connected to the reference electrode terminal (negative terminal of the polarizing circuit) and the cathode was connected to the glass electrode terminal, which was in turn connected to the positive terminal of the polarizing circuit. The initial potential reading between the electrodes was set near the upper end of the 0-1400 mv scale using the asymmetry control. Standard iron(II1) solution was added as the titrant and the potential reading remained near its initial high value until almost all of the uranium(II1) had been oxidized. Just before the first end point the observed potential dropped sharply and it was necessary to wait about 3 (3) I. M. Kolthoff and J. J. Lingane, J. Am. Chem. Soc., 55, 1871
(1933).
