Anal. Chem. 1988, 58, 1611-1617
resolution were increased (Figure 44. In addition, a significant change in selectivity was observed. The elution orders of the ribonuclease A-hemoglobin and lysozyme-cytochrome c pairs were reversed. Further work is under way to examine these selectivity changes. In addition to cation-exchange chromatography, immobilized protein A (40), reversed-phase, and anion-exchange columns have been prepared using carbodiimide coupling reactions.
ACKNOWLEDGMENT We thank Kevin Landgrebe and Margo Palmieri for their technical assistance. Registry No. Silica, 7631-86-9;succinic anhydride, 108-30-5; diglycolic anhydride, 4480-83-5;glucosamine hydrochloride, 6684-2; R, 9001-99-4;C, 9007-43-6;L, 9001-63-2. LITERATURE CITED (1) Hearn, M. T. W.; Grego, B. J . Chromatogr. 1983, 255, 125-136. (2) Stadalius, M. A.; Gold, H. S.;Snyder, L. R. J. Chromatogr. 1984, 296, 31-59. (3) Geng, X.; Regnler, F. E. J. Chromatogr. 1984, 296, 15-30. (4) Garcia, S.;Liautard, J.-P. J. Chromatogr. Scl. 1983, 27, 398-404. (5) Ekstrom, B.; Jacobson, G. Anal. Blochem. 1984, 742, 134-139. (6) Jennissen, H. P. J . Chromatogr. 1981, 275, 73-85. (7) Jennlssen, H. P.; Heilmeyer, L. M. G. Biochemistry 1975, 74, 754-780. (8) Jennissen, H. P. Biochemistry 1976, 75. 5683-5602. (9) Fausnaugh, J. L.; Kennedy, L. A,; Regnier, F. E. J. Chromatogr. 1984, 377, 141-155. (IO) Boardman, N. K.; Partridge, S . M. Biochem. J. 1955, 59, 543-552. (11) Kopaclewicz, W.; Rounds, M. A.; Fausnaugh, J.; Regnier, F. E. J. Chromatogr. 1983, 266,3-21. (12) Rounds, M. A,; Regnier, F. E. J . Chromatogr. 1984, 283,37-45. (13) Kowciewlcr. W.: Rounds, M. A.; Repnler. F. E. J. Chromatour. 1985, 316, 157-172. (14) Anderson, D. J.; Walters, R. R. J. Chromatogr. 1985, 337,1-10, (15) Koch-Schmidt, A . 4 . ; Mosbach, K. Biochemistry 1977, 76, 2105-21 00. (16) Eveleigh, J. W. J. Chromatogr. 1978, 759, 129-145.
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(17) Eveleigh, J. W.; Levy, D. E. J. SoM-Phese Blochem. 1977, 2 , 45-78. (18) Datta, R.; Ollls, D. F. Adv. Exp. Med. Blol. 1974, 42, 203-315. (19) Valentova, 0.;Turkova, J.; Lapka, R.; tima, J.; Coupek, J. Biochlm. BiOphy~.Acta 1975, 403, 192-108. (20) Manecke, G.; Vogt, H A . Makromoi. Chem. 1978, 177. 725-739. (21) Crowley, S.C.; Chan, K. C.; Waiters, R. R. J. Chromatogr., In press. (22) Bunton, C. A.; Fendler, J. H. J. Org. Chsm. 1987, 32, 1547-1551. (23) Cason, J. Org. Synth. 1955. 3 , 169-171. (24) Inman, J. K.; Dlntzis, H. M. Biochemistry 1989, 8 , 4074-4082. (25) Cuatrecasas, P. J. Bioi. Chem. 1970, 245, 3059-3065. (26) Gupta, S.; Pfannkoch, E.; Regnier, F. E. Anal. Biochem. 1983, 728, 196-20 1. (27) Walters, R. R. I n Affinity Chromatography: A Practical Approach; Dean, P. D. G., Johnson, W. S.,Middle, F. A,, Eds.; IRL: Oxford, 1985: DO 25-30. rr (28) Siggia, S.; Hanna, J. G. Quantltative Organic Analysis, 4th ed.; Wiley: New York, 1979; pp 172-183. (29) Robyt, J. F.; Ackerman, R. J.; Keng, J. G. Anal. Blochem. 1972, 4 5 , 517-524. (30) Fritz, J. S.;Gjerde, D. T.; Pohlandt, C. Ion Chromatography; Huthlg: Heidelberg. 1982; p 86. (31) Khorana. H. G. Chem. Rev. 1953, 53, 145-166. (32) Kurzer, F.; DouraghCZadeh. K. Chem. Rev. W87, 6 7 , 107-152. (33) Williams, A.; Ibrahlm, I.T. Chem. Rev. 1981, 87. 589-636. (34) Cuatrecasas, P.; Parikh, I.Biochemistry 1972, 7 7 . 2291-2298. (35) Lochmuller, C. H.; Colborn, A. S.; Hunnicutt, M. L.; Harris, J. M. Anal. Chem. 1983, 55, 1344-1348. (36) March, J. Advanced Organic Chemistry, 3rd ed.; Wlley: New York, 1085; pp 334-338. (37) Turkova, J. Affinity Chromatcgraphy; Elsevier: Amsterdam, 1978. (38) Peterson, E. A.; Sober, H. A. J . Am. Chem. SOC. 1958, 78, 751-755. (39) Inman, J. K. I n Affinity Chromatography: A Practical Approach; Dean, P. D. G., Johnson, W. S.,MMdle, F. A., Eds.; I R L Oxford, 1965; pp 53-59. (40) Hage, D. S.;Walters, R. R.; Hethcote, H. W. Anal. Chem. 1988, 58, 274-279.
--.
~~
~
RECEIVED for review October 30, 1985. Accepted March 10, 1986. This work was supported by Research Corporation, the donors of the Petroleum Research Fund, administered by the American Chemical Society, and National Science Foundation Grant CHE-8305057.
Theoretical Studies of a Chiral Stationary Phase Used in Column Chromatography Kenny Lipkowitz* and Jo M. Landwer Department of Chemistry, Indiana Purdue University at Indianapolis, Indianapolis, Indiana 46223
Thomas Darden Laboratory of Molecular Biophysics, National Institute of Environmental Health Science, P.O. Box 12233, Research Triangle Park, North Carolina 27709
The molecular stereodynamics of ionic Pirkle chiral stationary phases (CSP’s) and their parent carboxylic acids have been explored with semiempirical molecular orbital and empirical force field methods. These ionic CSP’s generally have two important conformations, syn and anti, that could play a role in enantiomer recognition. The multldimenslonai potential energy surfaces for the ionic CSP’s are somewhat flat allowing for rapid conformational Interconversion. Electrostatic attractions between amide C=O and pendant ammonium loris are found to be Important, and the correlated dynamics of substituent group movement are dlscussed.
There exists a need for optically pure organic molecules.
This need is being met in part by synthetic chemists who, in the last decade, have developed truly exceptional and clever methods for control of stereoselectivity ( I ) . Most of the asymmetric synthesesperformed to date have an enantiomeric excess less than unity, and so the unwanted optical antipode must somehow be removed. There are five methods of enantiomer resolution that we traditionally teach our students (2): (i) conversion to diastereomers, (ii) differential adsorption, (iii) biochemical digestion, (iv) differential reactivity (related to iii), and (v) mechanical separation (a l a Pasteur’s tartaric acid). It is the second method, we believe, that has the most potential for quick and efficient enantiomer resolution. Here one simply places a racemic mixture on a chromatographic column, which, if composed of suitable chiral substances,
0003-2700/S6/0358-1811$01.50/00 1986 American Chemical Society
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should in principle allow the enantiomers to migrate through the column at different rates and be individually collected a t the other end of the column. Although this technique has long been recognized as feasible (3-5) and chiral chromatographic columns have been developed 6-8, it is only recently that they have been marketed and incorporated into the bench-chemist’s ritual. Most chiral stationary phases have been designed by matching functional groups on the enantiomer to be resolved with functional groups on the chiral stationary phase in some complementary fashion. Exactly how a chiral stationary phase works is not known; clearly there is a difference between the two diastereomeric complexes formed upon adsorption, and from a series of studies there have evolved chiral recognition models that qualitatively seem correct (9-11). Yet, from a first principles approach, little is known about how one enantiomer is differentiated from the other enantiomer by a chiral surface. For simple examples, hand-held mechanical models can be used to rationalize how chiral stationary phases interact more selectively with one enantiomer than the other. For conformationally complex molecules and/or those with mixed functionality where multiple binding sites are available, mechanical models and simple bonding notions are not valid and may be misleading. Those research groups active in developing these new columns have traditionally designed chiral supports needed to resolve enantiomers with only a modest understanding of molecular interactions but with a keen sense for optimization of those factors deemed important for enantiomer recognition. This approach is usually empirical, time-consuming, and expensive. The need clearly exists to explore, from a first principles approach, how chiral stationary phases discriminate between enantiomers. We contend that with the aid of molecular graphics, molecular mechanics, molecular orbital theory, and some common sense we can pinpoint the important interactions in chiral recognition and, via computer simulation, help design improved versions of the chiral surface used for enantiomer separation. This application of computational chemistry will be called “column design” because it borrows tools and ideas used in the pharmaceutical industry for “drug design”. I t seems to us that one can not only analyze enantioselection qualitatively but, once those important terms in chiral recognition are defined, quantitatively assess the contribution of each interaction (attractions and repulsions) as well. With this knowledge one could, in a rational manner, orchestrate the design of enhanced chiral stationary phases. It is imperative that we fully comprehend, at the molecular level, the physicochemical properties of a chiral stationary phase before we can confidently develop a chiral recognition model. In this paper we begin our studies by focusing on the structural and electronic features of the Pirkle chiral stationary phases (CSP’s) (12-18). These CSP’s are known to separate a wide range of organic functional groups (19)and have been widely accepted as a useful tool for enantiomer resolution. Here a conformational analysis of the ionic Pirkle CSP is presented.
EXPERIMENTAL SECTION There are two principal approaches to calculating three-dimensional structure and other physicochemical properties of molecules; through a self-consistent-field molecular orbital (SCF-MO) method or by use of an empirical force field (EFF). Empirical force fields, as incorporated in molecular mechanics, are an attempt to formulate as reliable a recipe as possible for reproducing the potential energy surface for the movement of atoms in a molecule. The method as well as its underlying philosophy has been reviewed by us (20) and others (21, 22). SCF-MO theories are well-known and will not be discussed here. A thorough overview of SCF-MO and EFF methods used in computational chemistry exists and should be referred to (23).
,4
1.x=n 2. X
I
w
15
P,
;
i
l
l5
3.x=n
= H31h13
I A b
pa
0
= H3N-CH3
**
15
,n
5.x=n 6.
X
=H3&-~~J
B i”
7.x=n 8. X
9.x=n
t
= H3N-CH3
Figure 1. Numbering scheme of the Pirkle-like chiral stationary phases investlgated.
The molecular orbital methods used are the all valence semiempiricalMNDO (24)method as employed in MOPAC (25). All molecules were assumed to be ground-state singlets, and all internal degrees of freedom were fully optimized unless stated otherwise. The empirical force field employed is MM2 (26).
RESULTS AND DISCUSSION Several commerciallyavailable Pirkle columns are available. They are derived from N-(3,5-dinitrobenzoyl)aminoacids that are attached by a propylene chain to a silica surface
1FSl-Y
&”& R
EWG
O
where EWG = electron withdrawing group, e.g., NO,; R = alkyl or aryl groups; and Y = -NH- or -O-H3N+-. The systems studied are presented in Figure 1 along with the number scheme used to define torsion angles. Free Carboxylic Acids. We begin our study with the free carboxylic acids to gain some insight about the role R groups play in conformational biasing of the CSP. Knowledge of the possible stable conformations, the relative energies and populations of these states under chromatographic conditions, and the energy barriers separating these structures is especially important because chiral recognition models often assume a single conformation (the one which most conveniently explains enantioselection) for the templating effect. Presented in Figures 2-6 are reaction coordinates for the full 360° rotation around the N3-C2 bonds for carboxylic acids 1 , 3 , 5 , 7 , and 9, respectively. In Figure 2 we present slices through the multidimensional potential energy surface of 1 computed with the MNDO Hamiltonian and with the MM2 empirical force field. Our molecular orbital calculations restrict the nitro groups and the amide to remain in the plane of the benzene ring; all other degrees of freedom are fully optimized. The molecular mechanics calculations replace the NOz groups with CHO
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
6
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R
0
50
I00
150 w
200
250
300
350
3
(degrees)
OF!
50
I00
150
200
250
3 Y 3?0
u DEGREES
Flgure 2. Minimum-energy reaction pathway for clockwise rotation around C2-N3 in carboxylic acid 1: (0)MNDO, (+) MMPD, (0)MMX.
Flgure 3. Minimum-energy reaction pathway for clockwise rotation around C2-N3 in phenylglycine derivative 3: (X) MMPD, (+) MM2C.
functionality because nitro parameters do not exist. As in the molecular orbital calculations, both CHO groups and amide are constrained to be coplanar with the aromatic ring. The reaction coordinate involves rotation around the N 3 4 2 bond. All calculations begin with H21-C2-N3-C4 = 0'. This torsion angle has the methine C-H bond on the same side as the amide C=O bond and is heretofore referred to as the syn conformer. The conformation with H21-C2-N3-C4 = 180' is the anti conformer. N3-C2 bond rotation is in a direction such that the first encounter the amide C=O group feels is due to nonbonded repulsion with R (by using the enantiomers depicted in Figure 1, this involves a counterclockwise rotation along the N3-C2 bond). Within the framework of molecular mechanics the interaction of two polar bonds is typically expressed as two dipoles interacting by a classical Jeans expression (22). This calculation is heretofore called MM2D. Alternatively, a Coulomb expression of four point charges usually located on the nuclei of the corresponding atoms can be used. This type of calculation is called MM2C. Hence we also present in Figure 2 empirical force field results using MNDO derived charges. Qualitatively all computational procedures display similar N 3 4 2 torsional behavior. These results parallel expectations gleaned from mechanical models and intuition; as the N3-C2 bond is rotated, the amide C=O encounters destabilizing interactions first with the methyl group and then with the carboxyl group. All barriers to conformer interconversion are 6 kcal mol-' or less, implying rapid bond rotation under normal chromatographic conditions. Three minima are found by MNDO and MMZD, but only two minima are found with MM2C. All three techniques predict stable structures of w = 20-40' and w = 180-200'. MNDO predicts the most stable structure in the range w = 300-320'. MM2D has the most stable structure at w = 340'. MM2C predicts the most stable structure with w = 340'. All three methods qualitatively agree, however, that the methine hydrogen attached to the chiral center will not eclipse the amide C=O. The methine hydrogen will be skewed 20-40' on either side of the C=O. Additionally, the relatively flat surface in the region between w = -40' and w = +40° ensures a wide amplitude oscillating motion of the groups attached to the chiral carbon.
Unlike MNDO and MM2C, MMPD indicates a very low energy form at w = 200'. The energy difference between the minima found a t w = 200' and w = 340' (Figure 2) implies that the anti conformer could be significantly populated at room temperature. Furthermore we point out that the minima at w = 180' (anti) calculated by both MNDO and MM2C for carboxylic acid 1 are less than 1.5 kcal mol-' less stable than the global minima (syn). The implication of the results described above is that for carboxylic acid l , the methine hydrogen is effectively syn to the amide C=O but with wide amplitude oscillations and that the system can achieve the anti form where the C-H points away from the amide C=O. Replacing the methyl group in 1 with a phenyl ring results in carboxylic acid 3. This carboxylic acid, when ionically or covalently bound to a pendant amino propyl group, is one of the commercially available Pirkle columns. 3 is an N-33dinitrobenzoyl derivative of phenylglycine. Due to the size of this molecule, full MNDO reaction coordinates were not attempted. MMPD and MM2C results are presented in Figure 3. These calculations again use CHO rather than NO2 substituents and also hold fixed the CHO and amide functionality to be coplanar with the aryl ring to which they are attached. Again three minima are found at w = 40', w = 180-200°, and w = 300-320'. The global minimum is at w = 300-320'. Based on MM2C we find the minimum at w = 40' to be substantially populated (-35%) at room temperature; MM2D suggests this population to be more like 15% at 25 "C. In any event, as in 1, the system has the methine hydrogen syn to the amide C=O bond but with a wider amplitude of oscillation (f60') than 1. The energy of the 180' (anti) form is between 1.4 and 1.5 kcal mol-' less stable than the global minima computed by MM2D and MMPC, respectively. At 25 "C this state is populated less than 5 % . Nonetheless, the barrier separating the anti form from the syn form is under 5 kcal, and the anti form is stable enough to be considered important. MMPD calculations on carboxylic acids 5, 7, and 9 are presented in Figures 4-6, respectively. When R = isopropyl or tert-butyl we only find two minima located at w = 200° (anti) and 340' (syn). When R = isobutyl we again find three minima corresponding to the two syn conformers found in 1 and 3 and one minimum corresponding to the anti conformation. The latter is predicted to be the most stable structure, but all three forms will be populated a t room temperature.
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ANALYTICAL CHEMISTRY, VOL. 58,NO. 8, JULY 1986
7
14
6
12
5
-0
n -
10
n
0 E
E
2 4
1.8 0 u
0
s
Y
- 3
- 6
01 L
0
W
W
I
2
4
I
2
0
0 ~i
(degrees)
w
Figure 4. MM2D minimum-energy reaction pathway for clockwise rotation around C2-N3 in carboxylic acid 5. 5
(degrees)
F w e 6. MMPD minimum-energy reaction pathway for clockwise rotation aroun&C2-N3 in carboxylic acid 9.
all minima are easily accessible, and, because,the rotational barriers to interconversion are small, conformational interchange will be fast. Carboxylate Ions. The ionized forms of the carboxylic acids were next explored. In all cases a methylammonium ion was used as a suitable model for the pendant propylammonium ion; no attempt was made to investigate the ionized acid without a gegenion since this is unrealistic under chromatographic conditions. The addition of an ammonium ion significantly complicates the search for the lowest energy conformational structures. In addition to the individual rotations associated with the R groups we also had to search, at each 20' increment, for structures with the ammonium ion above the carboxylate
4
n
0
: -3 0
Y
-012 W
O ' C
1
' 0
0
50
100
150
200
250
300
350
400
u (degrees)
Figure 5. MM2D minimum-energy reaction pathway for clockwise rotation around C2-N3 in carboxylic acid 7.
The isopropyl and tert-butyl derivatives (5 and 9) display different conformational behavior than do the other carboxylic acids because of the effective steric bulk of these R groups. In 5 and 9 we find two states populated at room temperature. In 5 the syn form exists in -57% excess, while in 9 the syn form exists in -68% excess. The rotational barrier in 5 is now over 6 kcal mol-' and almost 14 kcal mol-' for the tertbutyl derivative, 9. The height of these barriers is of little consequence because the syn and anti forms can be interconverted by rotation over a small 4 kcal mol-' barrier between w = 200' and w = 350'. In summary, the free carboxylicacids evaluated by several computational methods are conformationally more flexible than one would a priori predict. Generally two major forms are accessible to these compounds: syn and anti. In only one instance (7) does the anti form predominate; all other acids are predicted to exist mainly in the syn form. Furthermore,
' 0
plane as well as below the plane. These time-consuming searches preclude MNDO calculations. Hence all calculations were performed with MM2C. As before, the NOz groups were replaced with CHO groups, which, along with the amide, were fixed coplanar with the aromatic ring to which they are attached. Beginning with the syn conformer the C2-C3 bond was twisted in 20' increments for a full 360' rotation. The direction of twisting was such that the amide C=O first encounters the R group and then the carboxylate. The results for 2,4,6, and 8 are presented in Figures 7-10, respectively. Beginning with the alanine derivative, 2, we find three minima in Figure 7 of which two are important. The lowenergy minima at w = 40' and w = 20O0 correspond to the syn and anti conformers, respectively. The syn form is populated in excess of 85% at room temperature. In contrast to the corresponding free carboxylic acid, we note that the structure at w = 320' in 1 is absent in 2. Nonetheless, similar conclusions can be formulated about both molecules; two forms, syn and anti, exist with the syn form more heavily populated than the anti form. It is also evident that the barrier separating syn from anti has been reduced from -6 kcal mol-' in 1 to - 4 kcal mol-' in 2. The phenylglycine derivative, 4, is presented in Figure 8. Here we find four minimum-energy conformations, with s y n forms at w = 40' and w = 280' being most stable. The anti
ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
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7 6
-
5
n 0 E
\ 4 0 0
Y
- 3 0
L
W
2 I
u50,
100
150
200
250
300
350
3
0
0 w (degrees)
w (degrees)
Flgure 7. MM2C minimum-energy reaction pathway for clockwise rotation around C2-N3 in ammonium carboxylate 2.
Figure 9. MM2C minimum-energy reaction pathway for clockwise rotation around C2-N3 in ammonium carboxylate 6.
7 1
n
" 0
50
100
150
250
v ,
300
350
I0
Figure 8. MM2C minimumenergy reaction pathway for clockwise rotation around C2-N3 in ammonium carboxylate 4.
Figure 10. MM2C minimumenergy reaction pathway for clockwise rotation around C2-N3 in ammonium carboxylate 8.
form is effectively not populated, but, if a strong intermolecular association with a suitable eluent occurs, the system could adapt the anti form for templating purposes. (Yet to be resolved is the issue of the importance these high-energy conformers play in enantiomer recognition.) The flexibility of the phenyl group to rotate in 4 has also been addressed. Using the most stable structure at w = 280°, we twisted about the C2-C24 bond in 20" increments beginning with w' = H21-C2-C34-C25 = 0" to w' = 180". The most stable orientation has w' = 3O, and the barrier for phenyl group twisting is only 3.58 kcal mol-'. Hence the twisting of this group for interaction with adsorbed solutes is feasible. Finally, we note that the interaction of the carboxylatewith the amide C = O is less destabilizing in 4 than in 3; to rotate from one of the syn forms to the anti form now costs less than 3 kcal mol-I. In summary, it appears that the ionic phenylglycine derivative prefers to exist in one of two syn forms, but it could
attain the anti conformation for possible complexing with adsorbates. The valine derivative, 6, was next studied. In Figure 9 we fiid three minima with the syn form at w = 40" and anti form at w = 200" most stable and equally populated. Again the interaction of the polar carboxylate with the polar amide C=O is less destabilizing than in the free carboxylic acid, 7; the rotational barrier for syn s anti interconversion is reduced to -3 kcal mol-'. We conclude that a rapid syn F? anti interconversion exists with both forms populated at room temperature. Finally, the leucine derivative, 8, is presented in Figure 10. Again three minima are found. Here the energy minimum is the anti conformer at w = 200°, but the syn form at w = 20" and a skewed syn form at w = 280" are also low-energy, stable structures. As with the other ammonium carboxylates the association of the pendant ammonium ion with the amide
w
200
(degrees)
~i
(degrees)
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 8, JULY 1986
r------
i
_ I -
Flgwe 11. Stereoview of the anti form of ammonium carboxylate 4, computed by MMPC.
I Flgure 12. Stereoview of the syn form of ammonium carboxylate 8, computed by MMPC.
C=O lowers the repulsive interaction of the COz group with the amide. The conformational behavior of these ionically bound dinitrobenzoylderivatives of amino acids is quite different than that of their parent acids; indeed we anticipate they will be different than their covalently bound analogues. Like the free carboxylic acids, the fiist encounter of the amide C = O with R is repulsive and destabilizing. However, unlike the carboxylic acids the ionized carboxylate group generally tends to be stabilized by the amide in the range w = 200-30O0. There are two counterbalancing effects at play here: one is the inherent repulsion of the negative carboxylateby the polar amide C=O and the other is the electrostatic attraction of the amide C=O to the pendant ammonium ion. In all instances the barrier to rotation has been reduced because of this attraction, and several new minima could be found. Generally these ionic CSP’s have flatter and more complex multidimensional potential energy surfaces than do their corresponding acids. The phenylglycine and leucine derivatives are commercially available and are commonly used in many laboratories, so in Figures 11and 12 we provide three-dimensionalviews of these structures in their most stable form.
Several additional points need to be made about these ionic CSP’s. First, the reaction coordinates are minimum-energy reaction paths (MERP) that are composites of many H21C2-N3-C4 rotations, each with a unique orientation of O’C
‘ 0
and R groups. During our explorations on the multidimensional potential energy surfaces of these molecules it was common to find other minimum-energy structures nearly degenerate in energy with the lowest energy structures presented in the figures. Consequentlythese CSP’s can adapt other, higher energy structures not described here. In as much as we attempted to search as much conformational space as possible, we cannot be assured that the global minima were always located. This is not a problem unique to the work presented here, but rather is endemic to all optimizations. The second point to be made is that the C2-N3 MERP involves a correlated, dynamic motion of the carboxylate group, the R group, and the position of the ammonium ion. For example, in valine derivative 6 the isopropyl group orientation defined by w’= H 2 5 4 2 4 - C l - H 2 1 is in the range of -170O for w = Oo and 20° but rotates to avoid nonbonded
Anal. Chem. lQ86, 58,1617-1625
interactions to w ’ - 4 5 ’ when w is in the range 40-120’ and then it rotates back to w‘ -180’ in the range w = 140-340’. The carboxylate also rotates along the Cl-C2 axis, and the ammonium slides from one face of the planar carboxylate to the other face. The motion of the isobutyl group in 9 is even more complex. The third and final point is that the calculations employed an effective dielectric constant o f t = 1.50. This is reasonable for ionic CSP’sin hydrocarbon solvent, but the results become less tenable for more polar solvents. Additionally, even though solvent molecules were not included in these calculations, explicitly including nonpolar organic solvents would not change the qualitative trends presented above. Our results are consistent with chemical intuition and highlight the fact that these ionic CSP’s are even more conformationally flexible that one would a priori predict. Both syn and anti conformations exist, and the barrier to interconversion is small. Furthermore the pendant ammonium group plays a key role by coordinating with both the carboxylate and with the amide C=O. The conformational flexibility of the phenyl group in the phenylglycine CSP and the isobutyl group in the lycine CSP is great enough to allow for a wide range of orientations for templating purposes; perhaps it is this inherent flexibility that serves to make these CSP’sso effective. We are currently investigating the conformational and dynamic behavior of the covalently bonded analogues and will report this at a later date. Registry No. 1, 102208-23-1;2, 102208-24-2;3, 74927-72-3; 4, 102073-88-1; 5, 98243-65-3; 6, 102073-89-2; 7, 98243-66-4; 8, 102073-90-5;9, 102110-09-8.
LITERATURE CITED (1) Asymmetric Synfhesis; Morrison, J. D., Ed.; Academic Press: New York, 1983.
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(2) March, J. Advanced Organic Chemistry, 3rd ed.; Wiley: New York, 1981; Chapter 2. (3) Willstatter. R. Ber. Msch. Chem. Ges. 1904, 3 7 , 3758. (4) Prelog, V.; Wieland, P. He&. Chlm. Acta 1944, 2 7 , 1227. (5) GICAv. E.; Feibush, 6.; Charles-Sigler, R. Tetrahedron Lett. 1966, 1009. (6) Blaschke, G. Angew. Cbem., Inf. Ed. Engl. 1980, t 9 , 13. (7) Schuria V. Angew. Chem., Int. Ed. Engl. 1984, 2 3 , 747. (8) Armstrong, D. W. J . Liq. Chromafog. 1084, 7(S-2), 353. (9) Plrkle, W. H.; Finn, J. M. In Asymmetric Synthesis; Morrison, J. D., Ed.; Academic Press: New York, 1983; pp 87-124. (IO) Alienmark, S. J . Biochem. Biophys. Methods 1984, 9 , 1-2$. (11) Davankov. V. A.; Kurganov. A. A.; Bochkov, A. S. Adv. Chromatogr. 1084, 2 7 , 71-116. (12) Pirkle, W. H.: Finn, .I. M.; Hamper, B. C. J . Am. Chem. Soc. 1981, 703, 3964. (13) Pirkle, W. H.; Finn, J. M. J . Org. Chem. 1981, 46, 2935. (14) Pirkle, W. H.; Schrelner, J. L. J . Org. Chem. 1081, 46, 4988. (15) Pirkia, W. H.; Finn, J. M. J . Org. Chem. i982, 47, 4037. (16) Pirkle, W. H.; Hyun, M. H. J . OIg. Chem. 1984, 4 9 , 3043. (17) Plrkie, W. H.; Hyun, M. H.; Bank, B. J . Chromatogr. 1884, 376, 585. (18) Pirkle, W.H.; Hyun, M. H.; Tsipouras, A.; Hamper, B. C.; Banks, 6. J . Pharm. Biomed. Anal. 1084, 2 , 173. (19) Wainer, I . W.; Doyle, T. D. LC Mag. 1084, 2 , 68. (20) Boyd, D. 8.; Lipkowltz, K. B. J . Chem. Educ. 1982, 59. 269. (21) Osawa, E.; Musso, H. Angew. Chem., Int. Ed. Engl. 1983, 2 2 , 1. (22) Burkert, U.;Aillnger, N. L. Molecular Mechanics; American Chemical Society: Washington, DC, 1982; ACS Monograph 177. (23) Clark, T. A Handbook of Computetional Chemlstty: A Practical OUMe to Chemical Structure and Energy Calcu&tions ; Wiley-Interscience: New York. 1985. (24) Dewar, M. J. S.; Thiei, W.J . Am. Chem. Soc.1977, 99, 4899. (25) Stewart, J. P. QCpEBuH. 1083, 3(2), 455. (26) Allinger, N. L.; Yuh, Y. H. Ouantum Chem. hogram Exch. 1081, 73, 395. (27) Meyer, A. Y. In The Chemistry of Functional Groups, Supplement D ; Patai, S., Rappoport, Z., E&.; Wiley: New York, 1983; Chapter 1.
RECEIVED for review December 20,1985. Accepted March 4, 1986.
Measurement of Reaction Rate Constants in the Liquid Chromatographic Reactor: Mass Transfer Effects Alexander H. T. Chu and Stanley H. Langer* Department of Chemical Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706 The adaptation of llquid chromatographk c o h n s as chemkal reactors for r e a c t h klnetk studies Is examlned. The overall for pyrldlne and 4-plcollne catalyzed rate constants, k-, esteriflcatlon reactlons of tetrachloroterephthaloyl chlorlde were determined for a llquld chromatographk reactor (LCR). I t b shown that wHh a rate constant for reactbn In the moMle phase, a valid rate constant for thls system can be obtalned for reactbn In the statlonary phase. Thus conversbns In each phase could then be evaluated. PosSiMlttks for complkatlons from mass transfer effects in the LCR system were examlned, and reglmes where they should be consldered are determined. Longitudinal dtffuslon, micropore diffusion, and interfacial sorptlon and desorption were specifically considered. However, analysls shows that none of these processes slgnlllcantly affected the klnetk measurements carried out in this study where chemlcal reactlan rates are skw. Experimental llmltatlons on rate constant measurements for flrst-order and pseudo-flrst-order reactlons are lndlcated and dlscussed.
The extension of chromatographic reaction rate measurements to reactions occurring in liquid chromatographic col0003-2700/86/0358-1617$01.50/0
umns not only expands the application of these systems but also has promise of providing special information about the stationary phase to complement information obtained from physical measurements and other chemical methods (I, 2). Such information initially would come from the application of chromatographictheory with the use of appropriate kinetic systems obeying simple rate laws. However, this application is predicated on the column operating as an ideal chromatographic reactor, a behavior which could be more limited in liquid chromatographythan with gas chromatographybecause of mass transport processes. Therefore, the effects of these processes are examined here. Earlier we showed that with a reversed-phase column and a reactive solute solvolysis reactions in the stationary phase had to be considered and that an octadecylsilyl stationaryphase composition could be investigated with rate constant measurements (I, 2). As such, on-column chemical rate measurements can be subject to mass transfer effects from transport (physical) rate processes in the packed chromatographic bed; these effects must be examined systematically so that they can be eliminated or minimized and the limits of reaction rate measurements in liquid chromatographic columns identified. The criteria for “ideal chromatographic 0 1986 American Chemical Society
