Structural Effects of Selected Dipeptides as Stationary Phases for the Chromatographic Separation of Enantiomeric Amino Acids Wolfgang Parr a n d P. Y. H o w a r d ' Department of Chemistry, University of Houston, Houston, Texas 77004
The gas chromatographic behavior of four systematically substituted optically active N-trifluoroacetyl (TFA)-L-Ldipeptide cyclohexyl esters used as stationary phases is described. N-TFA-L-alanyl-L-alanine cyclohexyl ester, NTFA- L-a-amino-n-butyryl-L-cu-amino-n-butyric acid cyclohexyl, ester, N-TFA-L- norvalyl-L-norvaline cyclohexyl ester, and N-TFA-L-norleucyl-L-norleucine cyclohexyl ester have been synthesized in good yield via conventional methods. The behavior of these peptides as optically active stationary phases for the separation of N-TFA-D,Lamino acid isopropyl esters has been examined with respect to separation factors ( a ) and thermodynamic properties of interaction. An increase in size of the alkyl substituent on the asymmetric centers of the dipeptide solvent produces greater solvent-solute interaction. However, the modification, when applied to the side chain on the N carbon of the solute, causes a decrease in interaction. Each phase has been investigated with respect to complete separability for a mixture of naturally occurring enantiomeric amino acid derivatives: and N-TFA-L-aamino-n-butyryl-L-a-amino butyric acid cyclohexyl ester has been found to effect complete resolution of the different amino acids as well as their respective enantiomers.
The separation of amino acid enantiomers by gas-liquid partition chromatography (GLC) can now be easily achieved. Principal methods of resolution involve the diastereoisomerization of D,L mixtures followed by chromatography on optically inactive stationary phases and the direct injection of derivatized enantiomeric mixtures onto optically active dipeptide stationary phases. Though the two techniques are clearly different, both give satisfactory results, and each has its own application depending upon the type of analysis desired, available materials, degree and accuracy of quantitation and analysis time. The use of optically active stationary phases for these resolutiops involves a complex solute-solvent interaction and, therefore, investigations into the behavior of such systems have been few. A working knowledge concerning the relationship between solute and solvent properties would be most advantageous, since an understanding of the nature of asymmetric solute-solvent interactions not only leads to the development of more efficient methods of separation for optical isomers, but also may allow a preparative scale resolution for enantiomers which can only be differentiated by the tedious and time-consumptive measures presently available. For most work, it has been shown that the N-trifluoroacetyl (TFA)-L-L-dipeptide cyclohexyl esters as stationary phases produce optimum GLC resolution for N-TFA isolPresent address, V a r i a n Associates, 611 Hansen Way, Palo Alto. Calif. 94303.
propyl ester derivatives of D,L amino acids (1-12). The mechanism by which this preferential solvation occurs has been postulated as involving the formation of a hydrogen bonded diastereoisomeric association complex (Figure 1) which contributes just enough stability to the L moietey to allow its retardation and subsequent longer elution time than the analogous D complex (7, 13,14). Recently, formation of the diastereoisomeric associatior complex (Figure LA) has been confirmed by Rogers e t a (9) as well as by Grohmann and Parr (13). In both cases, the presence of the amide portion of the solvent molecule was found to be the segment actually participating in complex formation. Similarly, Parr and Howard f l 5 ) have shown the proper spatial orientation within the solute between hydrogen bonded atoms to be that depicted in Figure lA,even though the H-substituted nitrogen in the solute is not essential to complex formation. It is additionally possible to determine the thermodynamic quantities associated with formation of the diastereoisomeric association complexes, since the separation factor, a , is related to the retention times for a given enantiomeric pair by the ratio:
where t i , is the retention time of the L enantiomer with respect to chloroform which was assumed to be nonretained, and t ' b i s measured in the same way. Furthermore, since t ' k and t k , are representative of the relative solubilities of the enantiomers in the same solvent system, the differential Gibbs free energy of solution [ A ( A G ) ] is expressed as:
A(AG)
=
-RT l n a
=
-2.303 RT logcv
(b)
E. Gil-Av, B. Feibush, and R. Charles-Sigler, in "Gas Chromatography. 1966," A. B. Littlewood, Ed., Institute of Petroleum, London, 1967, p 227. E. Gil-Av and B. Feibush, Tetrahedron Lett.. 1967, 3345. W. Konig. W. Parr. H. Lichtenstein, E. Bayer, and J. Oro. J . Chromatogr. Sci., 8, 813 (1970). B. Feibush, and E. Gil-Av, Tetrahedron, 26, 1361 (1970). S. Nakaparksin, P. Birrell, E. Gil-Av. and J. Oro, J. Chromatogr. Sci., 8, 183 (1970). J. A. Corbin and L. B. Rogers, Anai. Chem., 42, 1786 (1970) W. Parr. C. Yang, E. Bayer. and E. Gil-Av, J. Chromatogr. Sci., 8, 591 (1970). W . Parr, C. Yang, J. Pleterski, and E. Bayer, J . Chrornatogr.. 50, 510 (1970). J. A. Corbin, J. E. Rhoad. and L. B. Rogers, Anal. Chem., 43 327 (1971). W. Parr. J. Pleterski, C. Yang, and E. Bayer, J. Chrornatogr, Sci.. 9 , 141 (1971). W. Parr and P. Y. Howard, Chrornatographia, 4, 162 (1971). W. Parr and P. Y. Howard, Angew. Chem., Int. Ed., Engi., 11, 314 (1972). K. Grohmann and W. Parr, Chromatographia, 5 , 18 (1972). W. Parr and P. Y. Howard, J. Chromatogr., 67, 227 (1972). W. Parr and P. Y . Howard, J. Chromatogr., 71, 193 (1972)
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4 , APRIL 1973
0
711
Complex A H
H
I
I
-C-C- N-
H,,C,-0
II I
0
C-C-
R1 H 0, R,
,
C-
H
0
CF3
0
Ri
0
I I II HC -0-C-C-N-C-CF3 II
/
HllC6- 0-
Solvent
Solute
H
I I C -C-NC -C -N - C 1I I I It I I 1I
j
\ '\
d H k
CH,
N-
I II I I II
I
CH3,
H
CF3-C
I;I 0 R? I
;
t
O
H
H
CF,3
0
I
H
,
d
II I I 1I
CH3
-N- C Val > leu > aba nleu > nval > ala > t-leu. The differential enthalpies of solution [A( AH)] associated with ala-ala as the stationary phase have the order ile > Val > nval > aba = leu > ala > nleu > t-leu (Table V), but it should be noted that this order was determined from values obtained by plotting log a us. 1/T and, with only two temperatures, the result was naturally a straight line. For the rest of the phases studied for this project, a comparison of effects incurred by lengthening and branching the side chain of the a-carbon on the solute will be made. But for ala-ala, the small range of temperature operation does not allow accurate determination of thermodynamic characteristics associated with the phase. Those values which have been referred to in this section must be accepted with knowledge of the conditions from which they were derived. At 110 "C, ala-ala is not a liquid, hence factors determined a t this level are spurious a t best. They simply serve as guidelines to illustrate the methods used for obtaining the thermodynamic values for this phase, however dubious they might be, and will be supplemented with similar graphs for the other phases, which will be shown to behave well within a range of at least three temperatures. B. N-TFA-L-a-Amino-n-butyryl-L-a-aminon-butyric acid cyclohexyl ester (aba-aba). Separation factors for aba-aba (mp, 110-112 "C) were obtained a t temperatures ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, A P R I L 1973
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715
Table VI. Relative Retention Times and Separation Factors for N-TFA-D,L-Amino Acid Isopropyl Esters on N-TFA-L-a-Amino-n-Butyryl-L-cu-Amino-n-Butyric Acid Cyclohexyl Ester as the Stationary Phase Amino acid
D-ala L-ala D-aba L-aba D-Val L-val D-nval L-nval D-leu L-leu o-ile L-ile D-nleu 1,-n Ieu D-t-leu L-t-leu
100 "C r
0.246 0.268 0.341 0.372 0.366 0.399 0.563 0.61 1 0.764 0.839 0.535 0.591 0.91 1 1.000 0.362 0.381
llO°C 01
1.091 1.091 ,090 ,086 ,099 ,105 ,098 1.052
r
0.268 0.289 0.363 0.391 0.389 0.419 0.583 0.628 0.771 0.838 0.556 0.607 0.921 1.000 0.384 0.402
120 "C
1.079 1.079 ,078 ,076 ,087 ,091 ,085 1.046
Figure 3. Chromatogram of N-TFA isopropyl esters of D,L-ala and D,L-aba with N-TFA-L-aamino-n-butyryl-L-a-amino-n-butyric acid cyclohexyl ester as stationary phase Chromatographic conditions: 400-ft X 0.02-in. stainless steel capillary column: 110 "C isothermal operation: injector 180 "C; detector 275 " C ; carrier gas, He at 12 psig
of 100, 110, 120, and 130 "C. All are quite large and are indicative of the favorable utility of the dipeptide as a stationary phase for the separation of enantiomeric amino acid derivatives (Table VI). The (Y values show a general trend of the order t-leu < nval < Val < ala = aba < nleu < leu < ile, and the typical elution pattern is the same as for ala-ala (Figures 2, 3, and 4). Furthermore, since a plot of log a us. 1 / T shows .good linearity, the phase can be examined carefully with respect to both separation actors and thermodynamic properties. A typical graph is shown in Figure 5 . Aba-aba has side chains, R1 and Rz, which are ethyl (CHz-CH3) groups (see Table I). It is therefore of interest 716
r
0
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 4, APRIL 1973
0.290 0.310 0.380 0.406 " 0.412 0.440 0.603 0.643 0.787 0.844 0.572 0.616 0.931 1.000 0.400 0.416
130 "C cy
1.069 1.069 ,067 ,066 ,075 ,078
.0 74 1.040
r
0.31 2 0.330 0.409 0.434 0.436 0.463 0.623 0.661 0.788 0.838 0.600 0.639 0.941 1.000 0.428 1.442
cr
1.058 1.060 ,059 ,056 ,063 ,067 ,063 1.033
to study the influence of changing R3 (the side chain of the solute derivatives, Table 11) on the a and A ( A H ) values. If R3 is systematically modified by increasing its length, then the sequence ala (R3 = CHB),aba (R3 = -CHz-CHs), nval (R3 = -CHz-CHz-CH3) and nleu (R3 = -CHz-CHzCHz-CH3) lends itself to observation. For this homologous side chain modification, the separation factors behave in the fashion: nval < aba = ala < nleu. Or, the separation factors seem t o remain equal or grow slightly smaller until a four-carbon length in R3 is obtained, a t which time a becomes much larger. Comparison of the differential enthalpies of solution [ A ( A H ) ] for the homologs of increasing side chain length (Table V) obeys the sequence aba < nval < ala < nleu which shows no progressive relationship between solution enthalpy and chain length. A second study can be made by comparing the effect of branching in RS on the resolubility of aba-aba. Thus ala (R3 = CH3), aba (R3 = C H Z - C H ~ )Val , [R3 = -CH(CH&] and t-leu [R3 = -C(CH3)3] can be compared. Concerning separation factors, those for ala and aba are the same, while N for Val is slightly smaller and the factor for t-leu is very small. For these homologs, a t least, some predictable behavior emerges, in that an increase in bulkiness lowers the separability of a given amino acid. Calculation of solution enthalpies shows a not so similar trend, in that aba interacts less than ala, but val shows a higher enthalpy of solution than the former. The enthalpy for t-leu is by far the lowest and supports the possibility of an overall lack of solute-solvent interaction for derivatives with a bulky attachment on the asymmetric carbon; since difficulty in forming the complex in Figure 1 would certainly ensue if R3 were a large, unwieldy substituent. A final study was made in order to observe the effect of side chain isomerization in the solute on its separation by the stationary phase. An examination of leu [R3 = -CHzCH(CH3)2],ile (R3 = -CH(CH3)-CH2-CH3), t-leu [R3 = C(CH3)3] and nleu [R3 = -(CH2)3CH3] showed separation factors of the order ile > leu > nleu > t-leu. Solution enthalpies showed the same order, but for leu, ile, and nleu, deviation was small enough to disallow any concrete judgment concerning the effect of side chain isomerism on the separation of the three. A very low A(AH) was observed for t-leu, however (Table V). From the two groups of homologs as well as the four isomeric enantiomer pairs, it is difficult to provide a viable
Table V I I. Relative Retention Times and Separation Factors for N-TFA:D,L-Amino Acid Isopropyl Esters on N-TFA-L-Norvalyl-L-Norvaline Cyclohexyl Ester as the Stationary Phase
llo'c
100 "C Amino acid
r
D-ala L-ala D-aba L-aba D-Val L-val D-nval L-nval D-leu L-leu D-ile L-ile D-nleu I.-nleu D-t- le u L-t-leu
0.231 0.254 0.330 0.362 0.364 0.397 0.553 0.604 0.749 0.832 0.535 0.592 0.903 1.000 0.364 0.381
a
r
cy
0.253 0.275 0.354 0.384 0.386 0.41 6 0.573 0.619 0.760 0.833 0.558 0.61 0 0.920 1.000 0.389 0.404
1.100 1.097 1.083 1.092 1.110 1.108 1.108 1.044
120 "C
130 "C
r
(Y
0.273 0.293 0.362 0.388 0.409 0.437 0.592 0.633 0.752 0.813 0.567 0.612 0.924 1.000 0.401 0.414
1.087 1.086 1.069 1.081 1.096 0.093 1.096 1.040
1.075 1.071 1.050 1.070 1.082 1.079 1.083 1.034
r
cy
0.299 0.318 0.396 0.420 0.435 0.459 0.613 0.650 0.744 0.829 0.599 0.640 0.935 1.000 0.430 0.442
1.063 1.060 1.044 1.060 1.070 1.068 1.070 1.028
.34
X 0 0
I
.o i I
1
5G
120
I80
I
240
I
MIN
Figure 4. Chromatogram of N-TFA isopropyl esters of D,L-t-leU, D,L-ile, and D,L-leu with N-TFA-L-a-amino-n-butyryl-L-a-aminon-butyric acid cyclohexyl ester as stationary phase Chromatographic conditions: 400-ft X 0.02-in. stainless steel capillary column. 110 "C isothermal operation: injector 180 "C:detector 275 "C; carrier gas, He at 12 psig
relation between the size of R3 on the solute, and its effect on the separability of the enantiomeric pair. However, two inclinations do appear. First of all, there are factors which cause greater interactions and larger separation factors for solutes having a four-carbon composition, with the only exception being t-leu. And, second, factors which determine the separability of a given pair of enantiomers by aha-aba preclude the complete resolution of D,L-t-leU. These concepts may be approached with more certainty following discussion of the other stationary phases examined for this study. C. N-TFA-L-Norualyl-L-NorvalineCyclohexyl Ester fnoal-nLul). Groups R.1 and Rz on nval-nval ( m p = 88-90 "C) are n-propyl groups and the separation properties of this dipeptide phase may lend some insight into the steric configurations a t the asymmetric carbon on both solute and solvent necessary to achieve good resolution of D,Lamino acid derivatives. The separation factors for all derivatives were excellent and followed the sequence: leu > nleu > ile > ala > aba > nval > val > t-leu (Table VII). It should also be added
Ii
I L
-
-
-
-
-
2.5 2.5
I
r
26 26
I
j/Tx io3
Figure 5. Plot of the logarithm of the separation factor (0)vs. the inverse of the absolute temperature for N-TFA-D,L-amino acid isopropyl esters as solutes on N-TFA-L-a-amino-n-butyrylL-a-amino-n-butyric acid cyclohexyl ester as the stationary Dhase
that D,L-t-leU was completely resolved for the first time on this phase. Table V shows the differential enthalpies of solution for each enantiomeric pair on nval-nval, and these show behavior according $0 the pattern: leu = ile > nleu > ala > aba > val > nval > t-leu. The increase in the length of R3 on the solute shows more predictable trends on nval-nval than have been seen as yet. For instance, the separation factors for ala, aba, nval, and nleu are seen to decrease in that order, up to nleu a t which time the four-carbon size of the substituent brings about an increase in the separability of the solute. This last deviation. if it might be called such. has been encountered on all phases seen thus far; and nval-nval is not an exception. Solution enthalpies also support the earlier observations made upon straight chain homologs in this work, since an increase in the length of R3 shows an enthalpy relationship of nleu > ala > aba > nval. Again, the stability of the diastereoisomeric association complexes formed from the solute with a length of four carbons on the side chain is higher than that of smaller lengths, regardless of what pattern, if any, their relative solution enthalpies follow. When Rs of the solute is subjected to simple branching, rather than lengthening, the observations are slightly ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 4, APRIL 1973
0
717
Table V I I I. Relative Retention Times and Separation Factors for N-TFA-D,L-Amino Acid Isopropyl Esters on N-TFA-L-Norleucyl-L-Norleucine Cyclohexyl Ester as the Stationary Phase Amino acid
D-ala L-ala D-aba t-aba D-Val L-val D-nVd
L-nvdl
D-led L-leu D-ile L-ile D-nleu L-nleu
D-1-leu L-t-leu
100 "C r
0.228 0.249 0.327 0.356 0.366 0.396 0.551 0.600 0.748 0.828 0.541 0.595 0.904 1.000 0.368 0.380
1.096 1.091 1.080 1.090 1.107 1.101 1.107 1.033
r
0.249 0.270 0.351 0.378 0.389 0.416 0.571 0.616 0.759 0.831 0.565 0.614 0.914 1.000 0.390 0.402
1.078 ,069 ,079 ,094 ,087 ,094 1.030
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
cy
0.272 0.290 0.375 0.400 0.413 0.437 0.592 0.633 0.769 0.830 0.587 0.631 0.927 1.000 0.41 5 0.424
1.084
(17) P. Y . Howard, Dissertation, University of Houston, Houston, Texas. 1972.
*
r
N
more satisfying on nval-nval, since a definite pattern arises. For example, from ala, in which R3 is a methyl, through aba (primary), Val (secondary), on to t-leu, where R3 is tertiary, there is a steady decrease in separation factors (Table VII) as well as differential enthalpies of solution (Table V). Thus, for nval-nval, a t least, an increase in branching of the side chain on the solute is accompanied by less desirable separation of the enantiomers. When R3 is isomerically manipulated, as in the leucines, the behavior of the solute changes in about the same manner as seen on other phases. The separation factors are of the order: leu > nleu > ile >> t-leu and differential enthalpies of solution show, similarly: leu = ile > nleu >> t-leu. Again, except for t-leucine, in which R3 probably prevents facile solute-solvent complex formation, the position of the methyl group on the side chain seems to do little to change the separation properties of the solute. The determining factor seems to be the number of carbons attached to the asymmetric center of the solute rather than their orientation, except in extreme cases such as the tertiary group which is present in t-leucine. Indeed, there seems to be something characteristically suitable about the four-carbon size of the side chainwhich enables its interaction with the phase to proceed with such ease. D. N-TFA-L-Norleucyl-L-NorleucineCyclohexyl Ester (nleu-nleu). The efficiency of nleu-nleu as a stationary phase for the separation of enantiomeric amino acid derivatives is not as great as that for the other isomeric leucine dimers, but still compares favorably with the lower molecular weight dipeptides. Another desirable characteristic of nleu-nleu (mp = 86-88 "C) is the wide range of temperatures a t which it can be used. This was also found with Val-Val (7) and ile-ile ( I 7). Overall separation factors were acceptable, with D,L isomers of the non-tert.-leucine derivative showing the largest a values (Table VIII), while the smallest numbers were observed for t-leu. The quality of the separations was high enough to warrant the use of nleu-nleu as a stationary phase in any work where separation of D,L-aminO acid isomers is necessary. Differential enthalpies of solution between D and L isomers were small, on the whole, with the largest values again observed for the less bulky leucines (Table V ) .
718
130 " C
120°c
1lO0C N
1.067 ,059 ,069 ,079 ,075 ,079 1.024
-c*cI
-&X-
I
R,
1.070
PC
I
2 L C
I
dC y-methyl
1 I c I C
r
cy
0.293 0.31 1 0.397 0.420 0.435 0.458 0.612 0.647 0.777 0.829 0.608 0.646 0.937 1.000 0.446 0.446
1.060 1.057
,052 ,058 ,067 ,062 ,067 1.000
-c*easier complex formation t h a n
I
c\c I
C
C /3-methyl
-c*-
c*-c
I I C
/3-dimethyl
C 6-methyl Figure 6.
Facility of complex formation for D,L-leucine deriva-
tives An increase in length of the side chain (R3) is again accompanied by a decrease in the value for the separation factor until nleu, with a four-carbon side chain, has a higher a value than ala, which has only one carbon, and itself possesses a higher CY than solutes with two and three carbons in a straight chain a t R3. Differential enthalpies of solution show the same pattern, as the values for the D,L isomers decrease in the order: nleu > ala > aba > nval. These findings reinforce observations made on earlier phases, in which a length of four carbons in R3 produces better separation of the solute, while homologs of increasing side chain length show a decrease in separation factors up to that magnitude. When R3 is observed with respect to branching, rather than lengthening, the logical pattern observed on other phases is followed by solutes on nleu-nleu. Decreases in magnitude of both a and A(AH) follow the order: ala > aba > Val > t-leu, which indicates that increasing bulkiness a t R3 makes formation of the diastereoisomeric association complex in Figure 1 more difficult. As found with the other phases, isomerization of R3 has little effect on separation factors and differential enthalpies of solution for D,L-amino acid derivatives on nleu-nleu as the stationary phase. Leu, ile, and nleu all show high and only slightly different values. Summary. Four new optically active dipeptide stationary phases have been synthesized and demonstrated to perform well as solvents for the separation of derivatized enantiomers of amino acids by GLC. By holding R1 and RS constant, the steric effects of alkyl groups a t the a carbon of the solute have been observed. In general, increasing the length of R3 shows only a slight inclination toward decreasing the possibility of so-
J
a > -J
a J a
I
I
J
a J a I
0
L
I
I
1
I
2
3
I
I
I
1
4
I
1
5
6
I
HOURS
Chromatogram of N-TFA-D,L-amino acid isopropyl esters with N-TFA-L-a-amino-n-butyryl-L-a- amino-n-butyric acid cyclohexyl ester as stationary phase Figure 7.
Chromatographic conditions: 400-ft X 0.02-in. stainless steel capillary column; 110 "C isothermal operation; injector 180 "C: detector 275 "C; carrier gas, He at 12 psig
lute-solvent interaction, and a length of four carbons causes an unexpected facilitation of complex formation. Actually, careful consideration shows that these observations are not inexplicable, since lengthening of R3 does nothing to deter free rotation between the a and /3 carbons of the solute; and a long chain can be easily rotated so that it does not interfere significantly with the complex formation. This reasoning may warrant the higher interaction of the four carbon Rs than the three carbon R3 solute, but it does not account for the fact that when Ra has a length of four carbons, the solute shows a higher differential enthalpy of solution than when there is only one carbon a t R3. Changing the size of R3 by increasing bulky character of the solute side chain has been shown to decrease solutesolvent interaction. This is a simple matter of steric hindrance, and as the p carbon of the solute is changed from primary through secondary to tertiary, the factors indicative of complex formation diminish. Examination of isomerization in R3 yields somewhat nebulous results, but it appears that when the magnitude of R3 is four carbons, and the group is tertiary, a methyl group on the a or 6 carbon of the solute produces easier complex formation than substitution on the p carbon (Figure 6). The effects of changing groups on the solvent were determined by holding R3 constant and changing R1 and R:, so that they were always identical. When lengthened, R1 and Rz cause a general increase in ease of solute-solvent formation, with the three and four carbon lengths competing for the highest degree of interaction. Only t-leu shows a consistent decrease in interaction as R1 and R2 grow, and this is due to the tertiary nature of its side chain, which suffers increasing steric hindrance. Comparison of similar modifications in solute and solvent shows some striking dissimilarities which may be essential to a more complete elucidation of these types of separations. First of all, when the side chain of the solute is increased in length, a general diminution of interaction
is observed. But, when the side chains of the solvent are incremented in the same way, an increase in solute-solvent interaction is observed. Isomeric modifications show an analogous disparity of effect when solutes and solvents are compared. A solute having a methyl group a t the p position of a four-carbon side chain shows less interaction with the solvent than solute with a methyl on the y or 6 carbon. Conversely, the highest interaction of solutes on any solvent should be observed on ile-ile, a compound in which groups R1 and R2 have methyl groups on the p carbon as the stationary phase (1 7). Thus, identical modifications in the side chain of both solute and solvent produce opposite effects on thc'w respective abilities to form the diastereomeric complexes. Free energies of solution [ A ( A G ) ] were determined for all phases in order to calculate the differential entropies of solution [ A ( A S ) ] for each solute on every phase. Constant entropies of solution over a small range of temperatures indicate the accuracy of the method used for their derivation. These values, in good agreement, are found to be almost the same a t every temperature for a given solute on a single solvent. Applications. The separation factor, a, is widely used to determine enthalpies of solution for enantiomers on stationary phases, but it is erroneous to equate a high separation factor with good separation, since it is only relative to the differences between peak maxima and not to the degree of separation observed for a given pair of enantiomers. The resolution factor, 2 ( t ' R ~ - t ' )/WD ~ ~ i- WL, is indicative of good separation, where WD and WI, are the respective peak-widths for the D and L isomers of an enantiomeric pair. Each phase was used to separate a mixture of N-TFAD,L isopropyl esters of ala, Val, glycine (gly), D,L-threonine (thr), ile, leu, D,L-serine (ser) and D,L-proline (pro). These are common, naturally occurring amino acids which have not yet been completely resolved as a mixture, due to extensive overlap between gly and enantiomers of thr and ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 4 , A P R I L 1973
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719
Val; and between enantiomers of leu and ser. The elution pattern for five of the eight enantiomeric pairs as well as glycine is consistent from phase to phase, but retention times for optical isomers of thr and ser vary from solvent to solvent. When the separation for these seven enantiomeric pairs and glycine on nval-nval and nleu-nleu, which produce the largest separation factors, was attempted, overlap between leu and ser and thr and gly was evident. Nval-nval, which produces lower a values than nleu-nleu (Table VII) shows better separation of the mixture. The enantiomers of ser and leu were separated for the first time. However the time required, to achieve such an analysis is too long to make it practical. Figure 7 shows complete resolution and separation of all isomers by aba-aba in a reasonable time, which generates lower separation factors than nval-nval. Derivatives of less volatile natural amino acids such as D,L-aspartic acid, D,L-methionine, D,L-phenylalanine, D,L-glutamic acid, D,L-tyrosine, and D,L-lysine can be easily separated on a 100-ft column coated with the same dipeptide phase. However, aba-aba does not offer any advantages for the separation of the less volatile amino acid derivatives
720
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over N-TFA-L-phenylalanyl-L-leucine cyclohexyl ester (3,8). Parr et al. (3,8) operated the latter phase a t 140-145 "C and achieved excellent separation; aba-aba shows extensive bleeding a t this temperature. Thus, aba-aba now aids in complete separation of enmtiomers of 14 natural amino acid derivatives. It has also been found that nval-nval and aba-aba can completely separate derivatives of t-leu. Enantiomers of t-leu have not previously been separated by GLC on optically active stationary phases and a preparative scale separation would be desirable since conversion of t-leu into diastereoisomers followed by their separation and the process of enzymatic separation are difficult procedures to apply and do not always give complete resolution of t-leu. Received for review November 20, 1972. Accepted January 5, 1973. This paper was taken, in part, from a doctoral thesis submitted by P. Y. H. to the Department of Chemistry, University of Houston, Houston, Texas. The authors wish to thank the National Science Foundation (GP 26019) for financial support.
