395
Anal. Chem. 1992, 6 4 , 395-404
Empirical Procedure That Uses Molecular Structure To Predict Enantioselectivity of Chiral Stationary Phases Alain Berthed,+ San-Chun Chang, and Daniel W. Armstrong* Department of Chemistry, University of Missouri-Rolla,
Rolla, Missouri 65401
A total of 121 racemlc compounds were separated In the normal-phase mode on a (S)-(l-naphthylethyl)carbamoylated @cyclodextrin(S-NEC-&CD) bonded phase and 74 on the R equlvaient (R-NEC) chlrai stationary phase (CSP). All compounds are of the type that have four substituents on a stereogenic center, rather than an “axis of chirality”. I t is shown that the binary solvent pair used as the mobile phase has a slgnlfhnt influence on chiral recognitlon. However, the proportions of the components of a specHic palr have little effect. From the results, the lndhrldual contrlbutions to chlral recognition by these CSPs were estimated for 8 1 different substituents of the stereogenk center. Varying the arrangement of these 8 1 substituents could produce over 1.6 million compounds. Hydrogen was chosen as the reference substituent and was asslgned a 0 caVmoi free energy. The chlrai recognition increased when sp2-hybridlzed carbons were connected to the stereogenic center. Conversely, sp3-hybrldired carbons decreased the enantioseiectivlty. Amldo groups increased the chiral recognitlon, especially when assoclated with ?r-acld (3,5-dlnitrobenzoyl) or ?r-basic (naphthyl) groups. This approach does not allow one to know which enantiomer elutes first. However, the “substituent energy” list for chiral compounds can be used to obtaln an estimated value for the enantloselectivity of a compound by adding the energy contributions of the four substituents connected to the stereogenk center. I n thk way one can predlct a priori whether or not a compound wlll separate on a CSP and estlmate Its separation factor (a).Theoretically, thls approach can be used for most CSPs, provlded a sufficient data base is generated on them.
INTRODUCTION Each optical isomer of a racemic drug may have different therapeutic as well as toxicologic effects, in vivo.’ The U.S. Food and Drug administration may soon require biotoxicity and bioefficacy studies on not only the racemate but also on each enantiomer before granting approval for marketing a new active chiral molecule.2 High-performance liquid chromatography (HPLC) using commercially available chiral stationary phases (CSP) is now a recognized technique for stereochemical a n a l y ~ i s . ~ ~ ~ Chiral recognition, in chromatographic terms, means preferential interaction of one enantiomer with the CSP. To date, the trend is to design new CSPs capable of resolving different classes of compound^.^ The problem is that the number and types of available CSPs has grown tremendously in the past few years. The user may be confused by a choice of more than 50 CSPs? The enantioselectivity of many CSPs overlap, making some redundant. On the other hand there is a significant number of racemates that cannot be resolved by LC despite the large number of available CSPs. t On leave from Laboratoire des Sciences Analytiques, Universite de Lyon I, UA CNRS 435,69622 Villeurbanne, France.
We recently designed a naphthylethyl isocyanate derivatized j3-cyclodextrin (8-CD) stationary phase.7 The naphthylethyl isocyanate derivative is chiral and can be used in normal-phase or reversed-phase conditions to resolve different types of enantiomer^.^,^ Two CSPs were synthesized: the (R)-(-)1-(1-naphthylethy1)carbamoylated(3-CD (R-NEC-@-CD) and the corresponding S(+) phase (S-NEC-P-CD). The goal of this work is to try to answer the question a priori: Does a solute have a chance to be resolved by a NEC-8-CD CSP? By separating a significant number of enantiomers, we have obtained a data base that was used to extract the enantioselectivity contribution of each substituent from the stereogenic center. The two NEC-P-CD stationary phases (R and S) were studied in the normal-phase mode with apolar hexane-isopropyl alcohol mobile phases and more polar acetonitrileethanol mobile phases.
EXPERIMENTAL SECTION Chemicals. The structures of the solutes are given in the tables. The derivatization procedure was extensively described elsewhere.’f’ The derivatizing agents were obtained from various sources, Sigma (St. Louis, MO), Aldrich (Milwaukee, WI), Alfa (Ward Ha,MA), Fisher (St. Louis, MO), and Kodak (Rochester, NY). They consisted of carboxylic acid chlorides and thionyl chloride. Various amines and alcohols were also used! The dansyl amino acids and dinitropyridinyl amino acid derivatives were obtained from Sigma. Columns. The preparation of the 8-CD bonded stationary phase and the (naphthylethy1)carbamoylation have been described.7V8 The cyclodextrin silica coverage was approximately 0.2 pmol/m2. The R-NEC derivatization added an average 6.7 (R)-(naphthylethy1)cbamoylunits/CD ring leading to a R-NEC coverage of 1.34 bmol/m2. The S-NEC derivatization added only about 3.5 S-NEC units/CD ring which was a 0.7 pmol/m2 coverage.8 The R-NEC and S-NEC stationary phases (5 rm) were slurry packed in 25 cm, 4.6 mm i.d., columns. These columns can be obtained from Advanced Separation Technologies Inc. (Whippany, NJ). Chromatographic Apparatus. A Shimadzu pump, Model LC-6A, a Shimadzu SPD-6A UV detector, and a Waters R401 differential refractometer detector were used with a Shimadzu CR2AX integrator. All experiments were done in the normalphase mode with hexaneisopropylalcohol or ethanol-acetonitrile mobile phases. THEORY Enantiomers are nonsuperimposible mirror images of one another. Although they differ in configuration in an isotropic environment, their properties that govern classical chromatographic retention, i.e., vapor pressure, solubility, density or ionization constants, are identical. In the presence of a chiral stationary phase (CSP) some interactions must occur differently between the two enantiomers and the stationary phase in order to obtain stereose ive resolution.l* The three-point interaction model (Dalg esh’l) requires three points of attachment between the solute and some part of the stationary phase. Only one of the two enantiomers can better fulfill the three-point attachment, and it becomes more retained than the other.12 This model is oversimplifiedsince the interactions are not all necessarily attractive: steric hindrance, a repulsive
J@
0003-2700/92/0364-0395$03.00/00 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
Table I. Substituent Free Energy Contributions (cal/mol) for Enantiomeric Recognition on (R)and (S)-(Naphthylethy1)carbamate-SubstitutedCyclodextrin Chiral Stationary Phasesa no. 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
empirical formula
substituents structure of R -Br
-c1
reference substituent -COOH -NH-CHO methyl -NH-CO-NHp -CHZ-OH -0-CH3 -COO-CH3 -NH-CO-CHs ethyl -CHZ-O-CHB -CHOH-CH3 -CH2-S-CH, cyclopropyl -COO-CHp-CH, n-propyl isopropyl n-butyl -CHz-CH-(CH3)2 -CH(CH&CH&H3 tert-butyl -NH-DNBPy -COO-(CH2)3-CH3 solketal -CO-NH-t-Bu - -NH-CO-O-t-Bu n-pentyl phenyl ( 4)) 4-OH
-0-a, -cyclohexyl +hexyl -0-CO-DNB -COO-Q)-NOz -NH-CO-DNB -CO-NH-DNB -0-CO-NH-DNB -NH-CO-@ 40-NH-@ -NH-CO-O-@ -CH&-Cl -CH& -CH&OH -CH&OH(meta) -CH-NH-cyclohexyl -(CHz)&H3 -CH-N(CH3)-cyclohexyl XH2-0-CO-DNB -CH,-NH-CO-DNB -N(eH,)-CO-DNB -CH,-O-CO-NH-DNB -1-benzocyclobutene -CH-NH-CHZ-@ -CH-N(CH&@ -CH2-CH2-@ -CH&-O-CHB -CHz-(3-indole) -CH2-O-(7-indole) - (1-indanyl) -CH-NH-CHZ-CH& -CO-N(CH3)-CH& -CO-O-(CHz)7-CH3 - (1-naphthyl) -(l-tetrahydronaphthyl) -(2-tetrahvdrona~hthvl) -CH,-OLQ)-~H~-%H=CH~ -CHZ-O+D-O-CH~-CH=CHZ -CHz-O-@-CHz-CH20CH3 -CH-0-( 1-naphthyl) -NH-CO- (1-naphthyl) -CH-NH-( 1-naphthyl) -CH2-(1-naphthyl)
S-NEC-CSP Hex-IPA ACN-EtOH -27 -40 0
R-NEC-CSP Hex-IPA ACN-EtOH -27
0 7
0
0
-34 27*3b
-8 1
-7 -54 20;
7
-128; -94; 27 14 -81 40* -142; -14
-7
-27
94 -88 14; -47 -68
-27 -20 -27 -27
0
0
-74 -122; -94; 14 14
-ai
-115; -27 -108; 54 -81 -20; -40 -47
-27 -34 -27 -34 -47 -14;
-81;
0; 0;
47 142; -20@*1C 68 0;
-81 81 68; 54; 81 -81 142 14 230 263 182 27 148 27; 14 0 94; -27* 155 -54; 148 94 20; 243 68; -122a* 122 94 27 14* 142 94 230; 108 -2O@* -108@* 20; 14; 34; 0 54 162
47 135;
-27 81;
-54 74 68;
61;
94 -68 142 68
196
54
14
162 27
34
-27 0 27;
7 -81 115; 47;
0 -34;
-54; 68 20; 243 47;
-7; 0; 14;
-202
120; -54; -20
189; 108 54
34 7;
34
20 -34;
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
397
Table I (Continued) substituents
empirical formula
no.
CllH9 CllH90 ClZH9 ClZH13N202S C12H14N306 C13Hg0
76 77 78 79 80 81
structure of R -CH2-(2-naphthyl) -CH2-O-(1-napthyl) -(1-acenaphthenyl) -NH-DNS CH~-N(~-BU)-CO-DNB C H(0)-0-DNB
S-NEC-CSP Hex-IPA ACN-DOH -7*
20* -34 @ *
-230
14*
20*
61
61 101
88 Synergistic and Antagonistic Substituent Interactions no asymmetry NHCO-DNB and phenyl, add 175 CONH-DNB and phenyl, add 175 486* NHCO-DNB and naphthyl, add -142* NHCO-DNB and cyclohexyl
two identical Substituents 38 and 30 39 and 30 38 and 66 38 and 34 two different DNB derivatives decrease the energy by
R-NEC-CSP ACNHex-IPA EtOH
74 88
cancel all value 148 27 432*
O* -209
Relative free energy values were determined for two different mobile-phase solvent systems, hexane and isopropyl alcohol (Hex-PA) and acetonitrile and ethanol (AcN-EtOH). In each case hydrogen is taken as the reference substituent and is arbitrarily assigned a free energy value of zero. The energy values listed in the table depend on the reference energy chosen for the hydrogen atom, no. 3. Abbreviations: = 1-(3,5-dinitrobenzene);DNBPy = C5H2N301= 2-(3,5-dinitropyridine);DNS = ClzHl2NO2S= dansyl derivative. All DNB = C6H3N204 are para substituted, unless otherwise indicated. bValuesmarked with an asterisk refer to a nonredundant substituent, benzene rings (0) i.e. a substituent appearing in less than two compounds. @I means the asymmetric carbon is part of the ring structure. interaction, is often involved in chiral recognition mechanisms. The capacity factor, k', of a solute is the chromatographic parameter directly related to all solute-stationary phase interactions. Expressing each solute-stationary phase interaction with its corresponding molecular free energy of adsorption, AGi, yields
quantum chemistry, the models used remain semiempirical and they sometimes lead to differing conclusions.13J8 In this work, we propose a different approach based only on experimental results. To estimate the A(AG,) of a given compound, we assumed that the free energy difference, A(AG,), is the sum of four terms, each term being related to one of the four different substituents attached to the stereogenic center:
in which 4 is the volume ratio of stationary phase over mobile phase. The enantioselectivity, a,is expressed by the ratio of the capacity factors of the two enantiomers:
A(AG,) = (AGcll - AG,iz) + (AGCzi - AGCzz) + (AGc3i - A G c d + (AGc4i - AGc42) ( 5 )
a = k\/ktl
with
k', > k',
(2)
Using eq 1 a = exp[Cjl(AGi/RT) - Ciz(AGi/RT)I
(3)
In the case of enantiomers, all solute-stationary phase interactions except the chiral interactions, ZAG,, are identical and cancel. a = expC[(AGC1- AG,&/RTI = exp[(l/RT)A(AG,)] (4)
In eq 4, A(AG,) represents the difference in molecular free energy of the chiral interaction for the two enantiomer^.^ The A(AG,) energy difference is solely responsible for enantioselectivity (eq 4). A(AG,) is a measure of the degree of chiral recognition between two enantiomers and a given CSP. Without additional experiments to determine which enantiomer is selectively retained, only the absolute value of A(AGJ can be determined. The IA(AG,)l energy difference does not give any indication as to which enantiomer is most retained, i.e., the elution order. Computational studies of the interactions of chiral molecules which chiral stationary phases were done by several authors.13-" For example, the A(AG,) energy difference of weakly bound diastereoisomericcomplexes as transient species in chiral chromatographywas fully computed for the retention of trifluoro-l-(9-anthryl)ethanolon a ((3,5-dinitrobenzoyl)pheny1)glycine CSP.16 These studies were done with CSPs and solutes able to form 7mr interactions and hydrogen bonds. Although computational studies use molecular mechanics and
Although this is a simple model, it is not based on unreasonable assumptions. Somewhat analogous statistical mechanical approaches have been used in which a multitude of different additive interactions occur between a solute and the chiral stationary phase.lg The second assumption is that the chiral interaction contribution of a given substituent is independent of the others. Obviously, this assumption should be qualified. If two substituents are identical, the asymmetry disappears, which implies A(AGJ = 0. For some compounds we found a synergistic effect on the chiral recognition energy due to two particular substituents. Such effects are noted in the tables and are discussed in the following section. RESULTS AND DISCUSSION Assessment of the Substituent Free Energies. Table I lists the substituents considered in this study. The empirical formulas are in alphabetical order according to the letters of the atomic symbols. Also, moieties with fewer atoms (within a group) are listed before those with more atoms. Table I1 lists the chromatographic results that were used to obtain the free energy contributionslisted in Table I. A total of 126 chiral compounds were analyzed on the two CSPs. This was 126 X 4 = 504 contributions on the 126 stereogenic centers. There were only 81 different substituents because of redundancy. For example, hydrogen was one of the four substituents in 124 of the 126 compounds. For this reason, the hydrogen atom substituent was chosen as the reference substituent with the arbitrary value of 0 cal/mol for chiral free energy contribution. If a substituent has a positive energy value in Table I, it means that it increases somewhat the chiral recognition by the NEC-p-CD phase compared to hydrogen. A negative energy
398
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
Table 11. Separation Data for a Variety of Derivatized Enantiomeric Solutes on R-NEC and S-NEC-B-CD Chiral Stationary Phases Rsubstituent
compd
70 error
% V/V
modifer
CSP
k 'a
Mobile Phase, Hexane-Isopropyl Alcohol (IPA) Amines 30% IPA 30% IPA 30% IPA 30% IPA 30% IPA 30% IPA 30% IPA 10% IPA 10% IPA 30% IPA 30% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 2% IPA 2% IPA 30% IPA
S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC S-NEC R-NEC
5.82R 3.33s 1.75R 1.67s 1.28R 1.17s 1.00R 7.82R 6.359 4.56R 2.85R 6.90R 7.39s 435R 5.08s 3.83R 4.50s 8.07R 5.72R 3.11R 3.59R 2.20R 2.55R 2.58R 2.68R 8.32 6.14 1.56 10.21 6.47 1.20
14 13.4 1.4 1.4 1.3
3.59 3.05 1.14 1.14 1.11 1.10 1.08 1.08 1.07 2.02 1.77 1.11 1.05 1.06 1.03 1.03 1.01 1.17 1.43 1.11 1.08 1.06 1.07 1.04 1.05 1.2 1.12 0.89--' 0.890.851.80
0
0.5 0.7 0.8 6.4 5.8 1.9 0.6 1.0 0 0.6 0.6 2.7 4.5 1.2 1.0 1.0 1.0 0.7 0.7 2.6 2.0 0 0 0 4.2
3.59 3.05 1.14 1.14 1.11 1.09 1.04 1.04 1.06 2.03 1.77 1.13 1.02 1.06 1.00 1.03 1.03 1.18 1.43 1.09 1.08 1.08 1.08 1.06 1.06 1.18 1.12 1.00 1.00 1.00 1.83
(1,2,3,4-tetrahydro-l-naphthyl)amine*
38*9b 38* 73 73 41 41 11 11 11 38 38 73 73 41 41 11 11 38 38 73 73 41 41 11 11 38 38 73 73 41 38
1-aminoindan*
38
30% IPA
R-NEC
1.53
4.7
1.85
1.84
0.6
38
20% IPA
R-NEC
4.76
3.5
1.31
1.3
0.8
38 38 73 73 73 41 41 11 11 43 28
Amino Esters 40% IPA 40% IPA 30% IPA 10% IPA 30% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA
S-NEC R-NEC S-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC
5.03D 3.39~ 1.75D 6.33D 6.67D 5.36D 5.49L 5.75 6.30 4.42D 1.68
6.3 4.3 0.6 1.0 0.6 0.7 0.6 0 0 0.7 0
1.94 1.55 1.06 1.06 1.03 1.04 l.03 1.00 1.00 1.05 1.00
1.45 1.43 1.07 1.07 1.08 1.02 1.07 1.00 1.05 1.07 1.02
25 7.7 0.9 0.9 4.8 1.9 3.8 0 5 1.8 2
38
30% IPA
R-NEC
2.40
4.6
1.42
1.33
6.3
38
40% IPA
R-NEC
4.00
1.6
1.15
1.47
27.8
38
30% IPA
R-NEC
3.72
2.6
1.70
1.55
8.8
(14-naphthy1)ethyl)amine S
H
R
(methylbenzy1)amine CH3-CH-NHR
I
(1-(cyclohexy1)ethyl)amine CHI-CH-NHR
3-aminoheptane CH~-(CH~)~-CH-CH~CHJ
I
NHR
dR
2-amino-l-methoxypropane*
1.1
0 0 0 0 0.9 3.8 3.8 0.9 0.5 0 1.8 2.9 0 3.0 0 1.9 0.9 0 1.8 0 1.8 0.9 1.9 0.9 1.7 0 0 0 0 1.6
CH3-O-CH+X-CH3
I
NHR
DL-methionine methyl ester CH&-CH&H&H-CO&H3
I
NHR
DL-phenylalanine p-nitrophenyl ester
DL-norleucine methyl ester CH~-(CH&-CH-COZCH~
I
NHR
p-chlorophenylalanine methyl ester
NHR
a-methyl-p-tyrosine methyl ester CH,
I -
NHR
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
399
Table I1 (Continued) Rsubstituent
modifer
CSP
38
30% IPA
Amino Alcohols 36 10% IPA 36 10% IPA
compd a-methyl-m-tyrosine methyl ester
0
% V/V
% error
k'"
R,
aelp
R-NEC
5.44
2.0
1.30
1.38
6.1
S-NEC R-NEC
11.36R 12.499
0.8 0.7
1.06 1.05
1.05 1.05
0 0
adc
CH.
-c:H,-;;CO,CH, I "
HO'
NHR
propranolol*
alprenolol*
36
10% IPA
S-NEC
5.29R
0.8
1.06
1.06
0
metoprolol*
36
10% IPA
S-NEC
13.98
1.0
1.08
1.08
0
36
10% IPA
S-NEC
7.98
0.7
1.05
1.05
0
36
20% IPA
S-NEC
11.32
0.6
1.04
1.04
0
53 53 53 53
10% IPA 10% IPA 10% IPA 10% IPA
S-NEC R-NEC S-NEC R-NEC
15.38 16.28 11.65 11.67
0.6 1.1 0 1.1
1.03 1.07 1.00 1.07
1.05 1.07 1.05 1.07
1.8 0 5 0
norphenylephrine*
36 36
30% IPA 30% IPA
S-NEC R-NEC
11.22 12.78
0 0.6
1.00 1.04
1.00 1.04
0 0
phenylalaninol
38 38
10% IPA 10% IPA
S-NEC R-NEC
7.865 9.96
2.0 0
1.16 1.00
1.17 0.96-
0.8 0
2-phenylgly cinol
38 38
20% IPA 20% IPA
S-NEC S-NEC
10.53s 12.42R
2.5 1.7
1.27 1.21
1.34 1.24
5.5 2.5
p-chlorophenylalaninol
38 38
20% IPA 20% IPA
S-NEC R-NEC
8.93 9.32
1.5 1.8
1.15 1.18
1.20 1.11
4.3 3.8
methionol
38 38
20% IPA 20% IPA
S-NEC R-NEC
9.23 9.78
0.7 0.8
1.05 1.06
1.12 1.07
6.7 0.9
38 38
10% IPA 10% IPA
S-NEC R-NEC
8.16s 9.05R
1.2 0.8
1.08 1.06
1.08 0.98-
0 6.0
38 38
10% IPA 10% IPA
S-NEC R-NEC
8.66B 9.263
1.9 1.7
1.14 1.12
1.14 1.12
0 0
38 38
10% IPA 10% IPA
S-NEC R-NEC
7.81 8.58
1.5 1.1
1.09 1.07
1.10 1.01
0.9 5.6
CH3-0-(CH2),-
-
+ 2 H 2 ~ H - C H ~ N -I C & D N B
OR
HC(CH&
oxprenolol* OR
pindolol*
(SP andR,S)
ephedrine
(R,Rm d S , S )
CH3-SCH~-CH~H-CH+CO-DNB
I
NHR
leucinol (CH3)&M-CH&H-CHWO-DNB
I
NHR
2-amino-3-methyl-l-butanol* (CH3)2CH-CH-C:H&U2O-DNB
I
NHR
2-amino-1-hexanol CH~-(CHZ)~-CH-CH$~O-DNB
I
NHR
400
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
Table I1 (Continued) compd 2-amino-1-pentanol CHr(CH&-CH-CHWO-DNB
Rsubstituent
modifer
CSP
k
R,
aexp
38 38
10% IPA 10% IPA
S-NEC R-NEC
3.20 11.11
0 0.7
1.00 1.05
1.02 0.95
2 5.0
38 38
10% IPA 10% IPA
S-NEC R-NEC
10.56R 14.0
0.7 0
1.05 1.00
1.02 0.96-
2.8 0
38 38
10% IPA 10% IPA
S-NEC R-NEC
15.0 15.43
0.6 0.6
1.02 1.03
1.03 0.96-
1.0 3.0
36 36
10% IPA 10% IPA
S-NEC R-NEC
15.46 17.30
0 0.6
1.00 1.02
0.790.81-
0 2.0
36
Alcohols 10% IPA
S-NEC
10.21
1.9
1.12
1.12
0
36
10% IPA
S-NEC
8.24
3.2
1.28
1.28
0
54
10% IPA
S-NEC
11.54
1.4
1.09
1.09
0
40
10% IPA
S-NEC
6.92
2.1
1.22
1.22
0
40
10% IPA
S-NEC
6.55
4.0
1.32
1.27
3.8
Carboxylic Acids 39 20% IPA 74 2% IPA 42 2% IPA 57 2% IPA 56 2% IPA 64 2% IPA 63 2% IPA 48 2% IPA 50 2% IPA 72 2% IPA 74 2% IPA 42 2% IPA 57 2% IPA 56 2% IPA 64 2% IPA 63 2% IPA 48 2% IPA 50 2% IPA 72 2% IPA 27 2% IPA 74 2% IPA 42 2% IPA 57 2% IPA 56 2% IPA 64 2 % IPA 63 2% IPA 48 2% IPA 50 2% IPA 72 2% IPA 27 2% IPA
S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC s-NEC s-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC S-NEC
3.78 12.39 6.86 2.61 7.67 4.01 6.11 2.58 2.44 10.16 21.64 11.6 12.36 11.96 4.60 8.6 4.76 2.53 10.44
2.7 1.6 1.2 0 0 0 0.8 1.0 1.1 0 1.9 1.7 0 0.6 0
1.28 1.08 1.07 1.00 1.00 1.00 1.04 1.07 1.08 1.00 1.11 1.10 1.00 1.02
1.30 1.08 1.06 0.971.01 0.971.05 1.07 1.06 0.821.11 1.08
1.2
1.06 1.10 1.08 1.00 1.00 1.09 1.08 1.00 1.02 1.00 1.06 1.07 1.06 1.00 1.00
1.6 0 0.9 0 1.0 0 0.9 0 1.8 0 0 1.8 0 0.9 0 0.9 0
% V/V
% aca~c
error
I
NHR
2-amin0-1-butanol CH&H&H-CH2060-DNB
I
NHR
2-amino-1-propanol CH3-C:KCH&CO-DNB
I
NHR
1-amino-2-propanol CH3-CH-CHfiH-CO-DNB
I
OR
1-acenaphthenol*
1-indanol*
9R solketal*
r
r
O s cyclopropylbenzyl alcohol*
QS”
1-phenyl-1-propanol
CH3-CH&H-OR
I
2-chloropropionic acid 0
II
CHdH-GR
I
CI
2-bromopropionic acid 0
II
CH&H-C-R
I
0r
2-bromobutyric acid 0
II
CH3-CH&H-GR
I
Br
1.56
16.00 9.00 2.04 9.03 2.50 6.50 3.42 1.96 9.92 1.45
1.5 1.2 0 0 1.6 1.8 0 0.6 0 1.0 1.1
1.0 0 0
1.00
0.99-
1.03 0.991.07 1.10 1.08 0.840.941.09 1.07 0.981.02 0.981.06 1.08 1.07 0.830.93-
0
0 0 0 0.9 0 0 0 0 0.9 0.9 0 0
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
401
Table I1 (Continued) compd
1,2,3,4-tetrahydro-2-naphthamide
R substituent
modifer
CSP
k’”
Ra
aexp
39
20% IPA
S-NEC
4.17
1.1
1.10
1.10
0
39
20% IPA
S-NEC
6.60
0.7
1.04
1.04
0
39
20% IPA
S-NEC
6.18
5.9
1.52
1.53
0.7
2.6 2.0 1.8 1.2 1.3 1.2 0.5 0 0 0 0.6 0 1.0 0.6 1.8 0.6 2.5 2.7 3.0 0.6 2.0 0 1.2 1.7 0.6 0 0 0 0 0 0.6 0 0.6 1.2 1.2 1.4 2.0 2.0 0.6
0.7
1.04
0.7
1.05 1.10 1.00 1.00 1.00 1.03 1.04 1.03 1.03
1.14 1.10 1.07 1.04 1.04 1.06 1.02 0.990.970.971.01 0.991.06 1.03 1.09 1.08 1.12 1.11 1.16 1.06 1.10 1.00 1.06 1.02 1.05 0.950.990.990.990.991.04 0.951.12 1.05 1.15 1.07 1.08 1.05 1.02 0.990.990.981.05 1.03 1.07 1.06 1.05 1.05 0.990.990.961.01 1.04 1.03 1.06
0 0 1.8 1.8 2.8 0 0.9 0 0 0 5.6 0 0 0 0 5.9 0.9 3.5 1.7 2.9 0 0 0 6.4 0.9 0 0 0
0 1.0 0.6 1.0
1.14 1.10 1.09 1.06 1.07 1.06 1.03 1.00 1.00 1.00 1.07 1.00 1.06 1.03 1.09 1.02 1.13 1.15 1.18 1.03 1.10 1.00 1.06 1.09 1.04 1.00 1.00 1.00 1.00 1.00 1.04 1.00 1.02 1.06 1.06 1.07 1.10 1.10 1.02 1.00 1.00 1.00 1.05 1.03 1.05
%
% v/v acdc
error
0
1-benzocyclobutenecaboxamide* 0 @-LH-R
2-phenoxypropionamide* 0
Mobile Phase, Acetonitrile (ACN)-Ethanol (1% Acetic Acid v/v) Amino Acids 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 11 50% ACN 11 50% ACN 43 50% ACN 50% ACN 28 50% ACN 5 5 50% ACN 7* 50% ACN 50% ACN 7 24 50% ACN 24 75% ACN 79 50% ACN 79 50% ACN 38 50% ACN 50% ACN 38 73 50% ACN 50% ACN 73 41 50% ACN 41 50% ACN 11 0% ACN 11 0% ACN 43 50% ACN 43 0% ACN 28 50% ACN 0% ACN 28 0% ACN 5 0% ACN 5 24 50% ACN 75% ACN 24 50% ACN 79 50% ACN 79 38 50% ACN 50% ACN 38 50% ACN 73 50% ACN 73 41 0% ACN 11 0% ACN 24 50% ACN 24 75% ACN 79 50% ACN 50% ACN 79 38 50% ACN 50% ACN 38 50% ACN 73 50% ACN 73 41 50% ACN 24 50% ACN 24 50% ACN 50% ACN 79 50% ACN 79
38 38 73 73 41 41
DL-phenylalanine
DL-tryptophan
DL-methionine CH~S-CH~~HTCKC~ZH
I
NHR
DL-leucine (CH~)2CH-CH&WO+l
I NHR
S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC S-NEC S-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC R-NEC S-NEC R-NEC
5.12L 7.98~ 3.83~ 4.89~ 3.00~ 4.051, 2.39~ 2.78~ 2.30 0.99 4.05L 4.33L 2.79 2.85 10.28 14.42 4.60~ 6.01~ 4.161. 6.22~ 2.91~ 4.07 2.321. 3.25~ 8.581, 10.74 0.98 9.18 0.76 4.62 8.47 10.65 6.74 14.01 3.16~ 4.30~ 3.941. 5.63~ 3.00~ 3.84 9.71 5.96 10.95 19.40 4.371. 5.86~ 2.12L 2.55~ 1.32 1.61 1.03 4.58 5.20 1.68~ 2.12L
0 0
1.2 0 0 0
0.6 0.7 0.6 0.6
0
0 0 0 9.8 0.9 8.5 0 1.8 4.5 0 0 0 0 0 0
1.8 1.8
0 4.5 0 0 0
1.9 0 0 2.9
402
ANALYTICAL CHEMISTRY, VOL. 64,
NO.4, FEBRUARY 15, 1992
Table I1 (Continued) compd DL-norleucine CH~-(CHZ)&+CW
I
NHR
DL-norvaline CH~-(CHZ)&H-COZH
I
NHR
DL-serine H&CH&H-COzH
I
NHR
DL-alanine CH,-CH-COzH
I
NHR
DL-valine (CH3)zCH-CH-H
I
NHR
R substituent
% v/v modifer
24 24
50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 50% ACN 10% ACN 50% ACN
79 79 24 24
79 79 24 24
79 79 41 24 24 11
79 79
50% ACN 0% ACN 50% ACN 50% ACN
%
CSP S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC R-NEC S-NEC S-NEC R-NEC S-NEC S-NEC R-NEC
k'" 5.63 7.60 2.38 3.12 5.65 8.20 2.40 2.99 10.05 17.00 8.13 10.89 3.32 8.00 16.00 2.02 2.00~ 2.57~
Re
aexp
0.7 0.7 1.0 0.7 0.7 0.7 0.7 1.0 0 0.7 1.0 1.0 0.6 0.6 0.7 0 1.7 1.5
1.04 1.04 1.05 1.04 1.04 1.04 1.04 1.05 1.00 1.04 1.05 1.05 1.03 1.03 1.04 1.00 1.09 1.08
acelc
1.03 1.04 1.06 1.06 1.04 1.04 1.06 1.06 1.01 1.04 1.04 1.06 0.981.03 0.990.981.07 1.07
error 0.9 0 0.9 1.8 0 0 1.8 0.9 1.0 0 0.9 0.9 3.0 0 4.0 0 1.8 0.9
-
Capacity factor of the first eluted enantiomer; configuration indicated as R, S, L, or D, when known. *Entries marked with an asterisk refer to a compound containing a nonredundant substituent (see text). Values marked with a mean the calculated a was lower than 1; for the error estimation adcwas set to 1.00. 'The 48 compounds, whose chromatographic data have been listed in ref 8, were also used in this studv.
value in Table I means that the substituent somewhat decreases the chiral recognition relative to hydrogen. We stress that the hydrogen reference choice is completely arbitrary. Only the sum for the four substituents is known (eqs 4 and 5) and related to a. For example, choosing 60 cal/mol for the hydrogen atom substituent energy would have decreased by 20 cal/mol the free energy of all other substituents listed in Table I. Clearly, changing the arbitrary reference energy for hydrogen could also change some of the substituent values in Table I from positive to negative values. AE the chiral data base increases to include more compounds without hydrogen substituents on the stereogenic center, the free energy value assigned to hydrogen can be refined along with all other energy values. The results listed in Table I1 were put into a computer spreadsheet producing one equation (eq 4) per compound studied, with three unknown values corresponding to the free energy of the three substituents. As stated previously, the fourth substituent, H, has the reference value AG, = 0 cal/mol. The equations can be combined assuming that a substituent provides the same energy contribution regardless of the nature of the other three substituents (assumption 2). The equation system was solved while the following criterion was minimized:
The error, E, is the s u m of the absolute value of the difference between the calculated and experimental enantioselectivity factor for the set of compounds. When the calculated a value was lower than unity, acdcwas set to unity (no resolution). However, the a c t d y computed adc values, lower than 1,are listed in Table 11with a y-n symbol in order to provide more information on the precision of the method. Table I11 lists the number of compounds that were used on each CSP and with each mobile phase. Some recently published results were included to enlarge the data base.8 The average error between the experimental and calculated a values was lower than 3%. It should be noted that this error is lowered by the number of nonredundant substituents. A nonredundant substituent is one that exists only on one compound in the study. The computer fitting affects a free energy to that particular substituent which makes the calculated value exactly equal to the experimental a with a zero
Table 111. Number of Compounds, Nonredundant Substituents, Error, and Maximum Error on the Calculated Enantioselectivity no. of nonredund subst
% av a erior
% max a error
2.9 3.1
30 25
ACN-Ethanol Mobile Phases S-NEC-8-CD CSP 54 9 1.5 R-NEC-8-CD CSP 45 a 1.8
12 9
no. of
stationary phase
compds
Hexane-IPA Mobile Phases S-NEC-B-CD CSP 112 27 R-NEC-8-CD CSP 74 16
error. Free energy values obtained with nonredundant substituents are pointed out by an asterisk in Table I. Table I11 shows that there were approximately 20% nonredundant substituents. Taking into account the error decrease due to nonredundant substituents, the average error on calculated a values is in the 4% range. How Tables I and I1 Are Related. Consider, for example, the first compound of Table I1 which is the dinitrobenzoyl derivative of (1-(1-naphthy1)ethyl)amine. Its stereogenic center bears a hydrogen atom, a methyl group, a naphthyl group, and a (3,5-dinitrobenzoyl)amide group. These four substituents are listed in Table I as 3, 6,66, and 38, respectively. Only substituent 38 is indicated in Table I1 because the three others are shown with the molecule. With the S-NEC-@-CD chiral stationary phase, the energy listed in Table I for the four substituents are 0, -74, 108, and 230 cal/mol, respectively. The sum of the four energies is 264 cal/mol. A 486 cal/mol "bonus" energy should be added because of the synergistic effect of substituents 66 and 38 when they are present in the same molecule (see end of Table I). An explanation of these additional effects is given in the subsequent section on T-T interactions. Therefore, the total energy is 750 cal/mol for the compound. This energy should be divided by the product RT (586 cal/mol at 295 K), and eq 4 gives the calculated a value as a,,lc = exp(750/586) = 3.59. This value is listed in Table 11. With the R-NEC CSP, the energies of the four substituents are 0, -81,108, and 196, respectively. The bonus is only 432 cal/mol, giving a total energy of 655 cal/mol. adc is exp(655/586) = 3.05. Note that in these cases the calculated aCalc values correspond exactly
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
43
. p 3
........... 1 %
...........................
o=T
I1
q8
cH%
CHIRALITY .~ ~
}OPPOSITE
......~.
=O
.... SAME
S-NEC
R-NEC
Ftgure 1. SlmpHfii representation of the two stationary phases used: (left) (SHnaphthylethyl)carbamoylatsdj3syclodextrin stationary phase: (right)(RHnaphthylethyl)carbamoyJatedj3-cyclodextrin stationary phase. The chlralities of the naphthylethyl moieties are opposite (I). The chiralties of the secondary hydroxyl (I I I) and the &CD cavity (IV) are Mentical. The carbamoyl groups (11) are not chiral. SG is silica gel.
to the experimental a values because substituents 66 and 38 appeared together only in this particular compound (see the nonredundant substituent discussion, vide supra). Mobile-Phase Effects. We observed that moderate changes in the proportions of the components of a specific binary mobile phase could have very significant effects on solute retention times and capacity factors and little or no effect on enantioselectivity. Comparable observations were found in the literature.20 The results in Table I were obtained for two binary mixtures: isopropyl alcohol (1PA)-hexane varying from 2% v/v to 50% v/v of IPA in hexane and acetonitrile (ACN)-ethanol. The ethanol percentage varied from 50% v/v to 100% v/v. Acetic acid (1%) was added to the acetonitrile-ethanol mobile phases. Table I shows that the absolute chiral free energy for a given substituent tends to be lower with the more polar acetonitrile-ethanol phases than with less polar hexane-IPA phases independent of the substituent polarity. Stationary-Phase Effects. It has been shown that the chiral recognition process by NEC-derivatized j3-CD involves two different processes: the so-called Pirkle-type recognition process (?minteraction, hydrogen bonding, dipole stacking, and steric hindrance) and the CD inclusion capability.8~~ Both processes could act synergistically or antagonistically or independently.* As shown by Figure 1,the NEC moieties are bonded to the chiral cyclodextrin molecule. The S-NEC CSP was less substituted (3.5 S-NEC/CD ring) than the R-NEC CSP (6.7 S-NEC/CD). However, the cyclodextrin seems to have a greater synergistic effect with the S-NEC moiety. Therefore more compounds were analyzed on this CSP (Table 111). Alkyl groups (6 (methyl), 18 (propyl), 20 (butyl), 21 (isobutyl)) seem to decrease the chiral recognition of the R-NEC CSP more than that of the S-NEC CSP. When a carbonyl group is attached to the stereogenic center, the chiral recognition seems to be enhanced on the R-NEC CSP (substituents 10 (-COO-CH3), 17 (-COO-CH2-CH3), 65 (-COO-Oct)). Most substituents produced comparable energy values on both CSPs. The largest difference between the two CSPs is observed for substituent 60 (-CH2-(3-indole)) which is the substituent in tryptophan. The positive 27 cal/mol on the S-NEC phase became a negative -202 cal/mol on the R-NEC phase. Substituent Effects. The important point of this study is the relative free energy value of each substituent compared to the others since the absolute values depend on the reference chosen. sp3 versus sp2. The highest free energy values (39, 263 cal/mol; 48,155 cal/mol; 63,142 cal/mol) were obtained when an sp2-hybridized carbon was attached to the stereogenic center. The lowest free energy values (6, -74 cal/mol; 12, -81 cal/mol; 18, -81 cal/mol; 23, -81 cal/mol) were obtained with
403
alkyl substituents or when an sp3-hybridized carbon was attached to the stereogenic carbon. It was noted previously that chiral recognition on various CSPs was enhanced when sp2 carbons were connected to the stereogenic carbon.3*4-21*22 The insertion of a methylene group between an sp2-hybridized carbon and the stereogenic center decreased the free energy by about 80 cal/mol (compare substituents 30 and 45, or 66 and 75). An oxygen insertion seems also to decrease the free energy by more than 30 cal/mol (30 and 32; 39 and 40). 1 r ? r Interactions. The effect of 7~ interactions in chiral recognition by Pirkle-type CSPs was extensively studied.45J0J3J8The derivatized NEC-j3-CD phases bear a s-basic naphthyl group (Figure 1). 71-7~ interactions with ?r-acidgroups (nitro or dinitro groups) are possible. All substituents of Table I with a s-acid 3,fi-dinitrobenzoylgroup (DNB) have a highly positive free energy. It seems that acid-base 7r-r interactions increase the chiral recognition. However, when two different a-acid DNB derivatives were present in a compound, the enantioselectivity decreased significantly. Apparently, a compound having two different s-acidic groups can form diastereomeric complexes in which the enantioselectivity of one complex is opposite to that of the other. Therefore they tend to partially or totally cancel one another (antagonistic effect), resulting in a poorer enantiomeric separation. In a few cases, two substituents were found to have synergistic effects (e.g., -NHCO-DNB and naphthyl or phenyl). Only in theses cases (five in all) were the contributions of the substituents not additive. Hence a fifth contribution was included at the end of Table I under the heading Synergistic and Antagonistic Substituent Interactions for these special cases. For example, when two different competing ?r-acidic substituents are present in the same compound, the A(AG,) sum must be decreased by 230 &/mol (Table I). Conversely, when certain ?r-acidic and ?r-basicgroups are present in the same compound, vide supra, the A(AG,) sum is increased (Table I). Hydrogen Bonding and Dipole Stacking. The amido group -CO-NH- is most often responsible for hydrogen bonding and/or dipole stacking. NEC amido groups and secondary hydroxyls at the mouth of the j3-CD cavity can be involved in hydrogen bonding (Figure 1). Wainer showed that the solute-amido stationary phase-amido hydrogen bonds were most important.23 Reversing the linkage between a solute and its ?r-acidic or *-basic moiety from “NH-CO-” to “-CO-NH-” reversed the elution order.24 All substituents containing the amido sequence have a positive free energy. As pointed out earlier, the energy is higher if the carbonyl group, with its sp2-hybridizedcarbon, is connected to the stereogenic center. The energy is lower if the NH group is first. For example, the -NH-CO-a> substituent 41 has 27 cal/mol, its isomer -CO-NH-@ 42 has 148cal/mol. The phenyl group alone (30) has an intermediate value of 81 cal/mol. Similar results were obtained with the naphthyl substituents 73 (-NH-COnaphthyl), 66 (naphthyl), and 74 (-CO-NH-naphthyl), with energies of 54, 108, and 162 cal/mol, respectively. However, when hydrogen bonds and *-A interactions were associated, the stereogenic center was less sensitive to the hybridization. Substituents 38 (-NH-CO-DNB) and 40 (-040-NH-DNB) have 230 and 182 cal/mol energy values, respectively; 39 (CO-NH-DNB) has 263 cal/mol. The decreasing effect of a methylene insertion is obvious with substituents 52 (-CH2NH-CO-DNB), only 20 cal/mol, and 54 (-CH2-O-CO-NHDNB), 68 cal/mol. The replacement of the amido hydrogen by a methyl group does not significantly change the chiral recognition (substituents 48 and 50,38 and 53, and also 56 and 64). Steric effects may compensate for the loss of one hydrogen bond. Steric effects are likely responsible for the large energy increase
404
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
between substituent 52, 20 cal/mol, and the tert-butyl-nitrogen-substituted equivalent 80, with 101 cal/mol. Ring Effects. The cyclohexyl energy (34, 81 cal/mol) is much higher than the hexyl homologue energy (35, -81 cal/mol). Although an sp3-hybridized carbon is attached to the stereogenic center, steric effects and compactness of the cyclohexyl ring allow it to be recognized by the CSPs. When the asymmetric carbon is part of a ring (substituent indicated by @ in Table I), the method presented here did not allow distinction of one side of the ring from the other. The same energy value was assigned to both sides, although one side was often an sp2-hybridized carbon while the other side was sp3-hybridized. All ring substituent energies were negative (a,Table I). Several Chiral Centers. The last substituent, 81 (-CH(@)-O-DNB), contains a stereogenic center. It is derived from ephedrine which has two stereogenic centers and can be resolved into four optical isomers. In this case, two substituents should be studied, the R-configuration and the S-configuration, with different free energies.
CONCLUSION The purely empirical approach for chiral recognition presented in this work is not intended to replace fundamental studies of mechanism. However, it is useful for the end-user of enantiomeric separations in that the list of substituent energies can be used to predict the feasibility of other separation on the NEC CSPs. The 81 substituents listed in this study could make more than 1.6 million different chiral compounds. We studied only 121 of these. The DNB derivative of octyl ester (1-naphthy1)glycinehas an asymmetric carbon with substituents 65 (+230 cal/mol), 66 (+lo8 cal/ mol), and 39 (+230 cal/mol), with an additional 486 cal/mol bonus for associating 39 and 66. We did not analyze a racemic mixture of this compound that would have the maximum theoretical free energy for enantiomeric recognition (1.05 kcal/mol) by the S-NEC-P-CD CSP. It should produce an enantioselectivity value a,as high as 6. This example shows how this work could be used. It is likely that this compound would be fully resolved on this CSP. If the addition of the energy contribution for the four substituents of a given racemate produces a positive energy value, the NEC-8-CD phase is likely appropriate and will resolve the racemate. If the calculation produces a close to zero or negative energy value, another CSP should be tried. This same type of em-
pirical approach could be used for most other CSPs provided a sufficient data base is obtained.
ACKNOWLEDGMENT Support of this work by the National Institute of Health (Grant NIH BMT 2R01 GM3692-05A2) is gratefully acknowledged. Also, we would like to acknowledge Gary Bertrand, University of Missouri-Rolla, for helpful discussions and manuscript proofreading. REFERENCES (1) Coutts, R. T.; Baker, 0. B. Chlrallty 1080, 7 , 99. (2) De Camp, W. H. Chlrallty 1080, 7 , 2. (3) Krstulovlc, A. M., Ed. Chkal Separatbns by HPLC, Appllcatbns to Pharmaceutical Compounds; E. Horwood Ltd., Wlley: Chlchester, U. K., 198% (4) Zief, M., Crane, L. J., Eds. Chromatogrephic Chiral Separations; Chomatographic Science Series, Vol. 40; M. Dekker: New York, 1988. (5) Plrkle, W. H.; Pochapsky, T. C. Chem. Rev. 1080, 8 9 , 347. (8) Armstrong, D. W.; Hllton, M. H.; Coffin, L. LC-GC,1001, 9(Sep),646. (7) Armstrong. D. W.; Stalcup, A. M.; Hllton, M. H.; Duncan, J. D.; Fauikner, J. R.; Chang, S. C. Anal. Chem. 1000, 62, 1610. (8) Stalcup, A. M.; Chang, S. C.; Armstrong, D. W. J . Chromatogr. 1001, 540, 113. (9) Armstrong, D. W.;Chang. S. C.; Lee, S. H. J . Chromatogr. 1001, 539, 83. (IO) Plrkle, W. H.; Maiher, 0.;Hyun, M. H. J . Llq. Chromatogr. 1986, 9 , 443. (11) Dalgllesh, C. E. J . Chem. SOC.1052, 137. (12) Lochmuller. C. H.; Souter, R. W. J . Chromatogr. 1875, 773, 283. (13) Toplol, S.; Sabio, M.; Moroz, J.; Caldweil. W.B. J . Am. Chem. Soc. 1088, 710, 8367. (14) Llpkowttz, K. B.; Baker, B. Anal. Chem. 1000, 62, 774. (15) Llpkowb. K. B.; Landwer, J. M.; Darden, T. Anal. Chem. 1088, 5 8 , 1611. (16) Lipkowltz. K. B.; Demter, D. A.; Zegarra, R.; Larter, R.; Darden, T. J . Am. Chem. SOC. 1088, 770, 3446. (17) Plrkle, W. H.; Pochapsky, T. C.; Malher, G. S.; Field, R. E. J . Chromatogr. 1085, 348, 89. (18) Toplol, S.; Sablo, M. J . Chromatogr. 1080, 467, 129. (19) Boehm, R. E.; Meltire, D. E.; Armstrong, D. W. Anal. Chem. 1088, 60. 522. (20) Zlei, M. I n Chromatographic Chlral Separations; Z l e f , M., Crane, L. J., Eds.; Chromatographic Science Series, Vol. 40; M. Dekker: New York, 1988; pp 315-335. (21) Berthod, A.; Jin, H. L.; Stalcup, A. M.; Armstrong, D. W.Chlral/iy 1000, 2.38. (22) Wainer, I. W.; Alembik, M. C. I n Chromato@-aphlc Chlral Separations; Zief, M., Crane, L. J., Eds.; Chromatographic, Sclence Series, Vol 40; M. Dekker: New York, 1988. pp 355-384. (23) Wainer, I . W. In Chlral Separatbns by HPLC, Appllcetlons to Pharmaceutical Compounds; Krstuiovlc, A. M., Ed.; E. Horwood Ltd., Why: Chlchester. U.K., 1989; pp 194-207. (24) Walner, I. W.; Alembik. M. C. J . Chromatogr. 1088, 367, 59.
RECEIVED for review August 16,1991. Accepted November 13, 1991.
