Anal. Chem. 1995, 67,1580-1587
Mechanistic Aspects of the Stereospecific Interaction for Aminoindanol with a Crown Ether Column Richard A. Thompson, Zhihong Ge, Nelu Grinberg,* Dean Ellison, and Patrkia W a y
Merck Research Laboratotbs, R80Y- 115, P.O. Box 2000,Rahway, New Jersey 07065-0914
Investigations into the mechanistic aspects of the stereospecific interaction of the four optical isomers of aminoindanol on a silica based crown ether column were performed. The nature and concentration of the mobile phase's counteranion&ected the hydrophobic interaction but had little effect on the inclusion interaction. Minimal changes in the separation factor of the enantiomers were observed in the pH range of 1-5.2, but a minimum in the capacity factor was observed at pH 3.75. Van't Hoff plots indicated a high entropy and a positive enthalpy at pH 5.2, while a lower entropy and a negative enthalpy were observed at and below pH 3.75. Hill plots indicated that there were more active binding sites at pH 3.0 as compared to pH 1.0 and that the binding ratio of aminoindanol to active sites was also greater. Apparently at higher pH values, as the silica becomes deprotonated, there is an additional electrostatic interaction between the protonated aminoindanol and the deprotonated silica sites. In the last two decades, particularly in the pharmaceutical industry, a special emphasis has been placed on the organic synthesis of enantiomerically pure compounds. This emphasis can be attributed in large part to a heightened awareness that pharmacological and toxicological differences can exist between enantiomers.'-9 Consequently, chromatographic enantioseparations have become a priority and are still a challenge despite the exponential development in chiral technologies. Currently there are two major methods for achieving a chiral separation. One method is indirect and is accomplished through derivatizationwith an enantiomerically pure agent to form a diastereomer, followed by separation on an achiral stationary phase. Alternatively, separation can be attained through chiral additives in the mobile phase or with chiral stationary phases. Separations performed using chiral additives in the mobile phase are less predictable, in mechanistic terms, than those using chiral stationary phases. (1) Blaschke, G.; Kraft, H. P.; Fickentscher, IC; Kohler, F. Anneim. Forsch., 1979,29,1640. (2) Blaschke, G.; Kraft,H. P.; Markgraf, H. Chem. Bey. 1980,113, 2318. (3) Albert, A. Selective Toxicity, 5th ed.; Chapman, Hall, London, 1973. (4) Sternbach, L. H. J. Med. Chem. 1979,22,1. (5) Yost, Y.;Holtzmann, J. J. Phorm. Sci. 1979,68 (9), 1181. (6) Jaffe, J. A. Arthitis Rheum. 1970,13,436. (7) Wacker, A; Heyl, E.; Chandra, P. Anneim. Forsch. 1975,30,395. (8) Nelson, W. L.; Burke, T. R, Jr. J. Om. Chem. 1978,43,3641. (9) I€ M.; Giacomini, Nelson, W. L.; Pershe, R A; Valdivieso, L.; TurnerTamiyasu, JS.; Blaskche, T. F. /. Pharmocokinet. Biopharm. 1986,335356.
1580 Analytical Chemistry, Vol. 67, No. 9, May 7, 1995
Separations performed with chiral stationary phases can be accomplished through a number of different mechanisms. The major mechanisms are ligand exchange, n-JCinteraction, and inclusion. Chiral separations based on inclusion are achieved through a mechanism by which the guest molecule is included into the cavity of a host molecule. The exterior of the host molecule generally possesses functional groups that act as steric barriers or interact with the guest molecule in a fashion that induces enantioselectivity. The interaction within the cavity of the host molecule may be hydrophobic (e.g., cyclodextrins) or hydrophilic (e.g., crown ethers). This paper focuses on interactions with crown ethers. Crown ethers can be described as heteroatomic macrocycles with repeating units of (-X-C2H4-) where X, the heteroatom, is commonly oxygen but may also be sulfur or nitrogen atoms. These cyclic polyethers were first synthesized by Pedersen.lo Crown ethers possess hydrophobic exteriors and hydrophilic cavities.The hydrophilic character of the cavity results from the presence of the heteroatoms. Consequently, the cavity has a strong affinity for cations,1°-13 predominantly through electrostatic interactions between the cation and the heteroatoms. The strength of this affinity is dependent upon the size and the charge of the guest cation and the size of the cavity of the host crown ether. The largest stability constants are obtained for those cations whose ionic diameters are a little less than the diameter of the crown ether cavity, thus providing for a tight fit.14 Crown ethers, especially lkrown-6 ethers, can complex not only inorganic cations but also alkylammonium compounds.15The inclusion interaction is based primarily bn hydrogen bonding between the hydrogens of the ammonium group and the heteroatom of the crown ether. Additional electrostatic interaction occurs between the nitrogen and the crown ether's oxygen lone pair electrons. Cram took advantage of the ability of crown ethers to bind alkylammonium compounds to perform enanti~separations.~~-~ (10) Pedersen, C. J. J. Am. Chem. SOC.1967,89,2495. (11) Pedersen, C. J. J. Am. Chem. SOC.1967,89,7017. (12) Pedersen, C. J. 1.Am. Chem. SOC. 1970,92,386. (13) Pedersen, C. J. J. Am. Chem. SOC. 1970,92,391. (14) Frensdorff, H. IC]. Am. Chem. SOC.1971,93,600. (15) Tiiko, J. M.; Hegelson, R; Newcomb, M.; Gokel, G.; Cram, D. J. J. Am. Chem. SOC.1974,96,7097. (16) Hegelson, R C.; 'Ilmko, J. M.; Cram, D. J. J. Am Chem. SOC. 1974,96, 7380. (17) Cram, D. J.; Cram, J. M. Science 1974,183,801. (18) Hegelson, R C.; Koga, JS.; " k o , J. M.; Cram, D. J.J. Am. Chem. SOC.1974, 95,302. (19) Gokel, G. W.; Xmko, J. M.; Cram, D. J. J. Chem. Soc., Chem. Commun. 1975,394. (20) Gokel, G. W.; Timko, J. M.; Cram, D. J. J. Chem. SOC.,Chem. Commun.
1975,444. 0003-2700/95/0367-1580$9.00/0 Q 1995 American Chemical Society
OH
1-Aminoindan-2-01
1-Aminoindan
danol (Figure l), and the crown ether used in the Crownpak CR column. Aminoindanol possesses two chiral centers, resulting in four stereoisomers (two cis and two trans enantiomers). Determination of thermodynamic parameters through van't Hoff plots for all four isomers were successfully undertaken. We have investigated the role of hydrophobic interactions between the aromatic portions of the crown ether and the aminoindanol. The role of the counterion and its effect on the interaction between aminoindanol and the crown ether were examined through variation of the counterion. We have compared interaction of the crown ether with 1-aminoindan(Figure 1) to gain insight into the effect of the hydroxy group during interaction. Furthermore, we have performed binding studies between the cis isomers of aminoindanol and the crown ether as an independent means of determining the binding constants at various pH values. EXPERIMENTAL SECTION
Chromatographic Equipment. Chromatograms were processed using PE Nelson Access Chrom version 1.7 software (PE Nelson, Cupertino, CA). The columns used were Crownpak CR(+) and CR(-), 150 x 4.0 mm (Chiral Technologies Inc., Exton, PA), which consist of either the R or the S form of the optically active crown ether 2,3:4,5bis[1,2-(3phenylnaphtho)I-1,6,9,12,15,Crown Ether [CR(-)I l~hexaoxaacycloeicosa-2,4diene attached to a 5 pm silica gel substrate. The column temperature was controlled by a Jones Figure 1. Structures of aminoindanol, aminoindan, and the crown ether. Chromatography Model 7950 temperature controller (Lakewood, CO). Chromatographic Conditions. The mobile phase was preStructural features that enhance the ability of the host molecule pared by addition of the acid of the desired counteranion, to to bind alkylammonium compounds were in~estigated.'~It was deionized water in quantities to give the desired pH. (1S,2R)discovered that the introduction of bulky groups, such as binapthyl cis-1-aminoindan-2-01(&I), (lR,2S)-cis-l-aminoindan-2-01 (cis-II), groups, onto the exterior of the crown ether could provide steric (lS,2S)-truns-l-aminoindan-2-01 (trans-I),(lR,2R)-truns-l-aminoinbarriers and induce enantioselective interactions with the guest dan-2-01 (trans-II) (Merck Research Labs), and 1-aminoindan molecule.21 (Aldrich Chemicals, Milwaukee, WI) were used to investigate The use of crown ethers for enantioseparations was later interactions with the crown ether. applied to liquid chromatography by Cram and c ~ - w o r k e r s . ~ ~ - ~ ~ Samples were prepared by dissolution into the mobile phase Crown ethers were covalently bound to a silica gel or polystyrene and were introduced into the chromatographic system through a matrix. Chromatographic enantioseparationswere found possible 10 pL loop. All separations were performed at a flow rate of 0.6 for a number of amino esters and amino acids. Shinbo et al. found mL/min with UV detection at 210 nm. Capacity factors were that chiral separations could also be performed on a reverseddetermined as defined by phase column that was dynamically coated with a crown ether.26 The crown ether they used is shown in Figure 1. This crown k' = (t, - tato ether has been utilized in a commercially available column (Crownpak CR) and has been used to separate a large number of amines, amino alcohols, amino acids, and amino e s t e r ~ . ~ ~ - ~ l where tr is the retention time of the analyte peak and to is the first perturbation in the base line observed after injection. In this paper we present the results of our investigation into Determination of Binding Constants by Hill Plots. Two the stereospecific interaction between an amino alcohol, aminoinCrownpak CR(+) columns were emptied, and the packmg material was washed with deionized water and dried under vacuum at 40 (21) Cram, D. J.; Cram, J. M.; Hegelson, R C.; Sousa, L. R; Timko, J. M.; Newcomb, M.; Moreau, P.; DeJong, F.; Gokel, G. W.; Hoffman, D. H.; "C. Packing material was weighed (40.00 f 0.40 mg) and placed Domier, L A; Peacock, S. C.; Madam, IC;Kaplan,L. Pure Appl. Chem. 1975, in a 4 mL amber HPLC vial. Stock solutions containing various 43, 327. (22) Sousa, L. R; Sogah, G. D. Y.; Hoffman, D. H.; Cram, D. J.J. Am. Chem. SOC. concentrations of cis4 or cis-I1 were prepared in perchloric acid 1978,100, 4569. at pH 1 and 3. Two milliliters of each stock solution was placed (23) Sogah, G. D. Y.; Cram, D. J. J. Am. Chem. SOC.1979,101, 3035. in individualvials containing the packing material. The mixtures (24) Newcomb, M.; Toner, J.; Hegelson, R J. Am. Chem. SOC.1979,101,4941. (25) Ligenfelter, D.; Hegelson, R; Cram, D. J.J Org. Chem. 1981,46, 393. were then equilibrated by placement on a rotator (Roto-Torque (26) Shinbo, T.; Yamaguchi, T.; Nishmura, IC; Suguira, M.J Chromatogr 1987, 7367, Cole Parmer, Chicago, IL) for a period of 24 h. M e r 24 h, 405, 145. the vials were removed from the rotator and filtered with a 0.45 (27) Hilton, M.; Armstrong, D. W. J. Liq. Chromatogr. 1991,14, 9. (28) Castelnovo, P. Chirality 1993,5, 181. pm polypropylene filter (Whatman Puradisc 22PP, Arbor Tech(29) Vaccher, C.; Berthelot, P.; Debaert, M. J Chromatogr. 1993,645, 95. nologies, Ann Arbor, MI). The concentration of cis-aminoindanol (30) Application Guidefor Chiral Column Selection, 2nd ed.; Chiral Technologies in the filtered solutions was determined by HPLC using a Inc.: Exton, PA, 1993. Crownpak CR(-) column with perchloric acid, pH 3, as the mobile (31) Kersten, B. J. Liq. Chromatogr. 1994,17,33.
w
Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
1581
Table I.Capacity Factors and Separation Factors as a Function of the Counteranion
k' trans-I k' trans41 a
k' cis4 k' cis-n a
H3P04
HN03
TFA
HC104
1.45 1.86 1.28 1.60 1.90 1.19
2.00 2.59 1.29
3.26 4.15 1.27
4.14 5.36 1.30
2.21 2.64 1.20
3.54 4.20 1.19
4.51 5.42 1.20
phase. The original stock solution for each sample was used as a reference to calculate the concentration of the sample after equilibration. The amount of cis-aminoindanoladsorbed onto the packing material was determined as the difference between the original concentration of the solution and its concentration following equilibration with the packing material. pKa Determination. The pKa of aminoindanol was determined through potentiometric titrations with hydrochloric acid using a Metrohm 665 dosimat and a 670 titroprocessor (Brinkmann, Westbury, NY). RESULTS AND DISCUSSION
Role of the Counterion. Previous studies on the effect of counterions on a chiral separation on a cellulose phase had indicated that the counterion plays a role in the retention and resolution of enantiomer^.^^ It was found that counteranionsthat were more polarizable increased both the retention and the resolution between the enantiomers of basic compounds. This observation is compatible with the theory of cha~tropicity.~~ An anion with a high chaotropicity is characterized by high polarazibility with a consequent low degree of hydration. Such ions undergo relatively small changes in enthalpic energy during transfer from an aqueous phase to an organic phase as compared to less chaotropic anions.32 Chaotropic counteranions,when ionpaired to the analyte, facilitate the approach of the analyte to the stationary phase and thus increase their interaction. The effect of various anions on the chiral separation of the four optical isomers of aminoindanol was investigated. The capacity factors of the four isomers were measured for mobile phases containing nitric acid, phosphoric acid, tritluoroacetic acid, and perchloric acid. The pH of all mobile phases was 2.0. Table 1 shows the capacity and separation factors for the four isomers of aminoindanol under the above conditions. The results indicate that the capacity factor decreases as follows:
portions of the aminoindanol ion pair and the crown ether. This hydrophobic interaction is affected by the chaotropicity of the counteranion and consequently, a change in the capacity factor is observed with variation of the counteranion. However, no such change is observed with the separation factor, which indicates that chaotropicity does not affect the inclusion mechanism. Effect of the Concentrationof the Counterion. It is known that the capacity factor of a solute is related to the change in partial molar free energy incurred during the transfer of solute between the mobile phase and the stationary phase.% This relationship is represented by the equation
In k'
= -(AG"/RT)
+ In
where Cp represents the phase ratio. The free energy can be broken down into enthalpic and entropic terms to give the van't Hoff equation
In k'
= -(AH"/RT)
+ AS"/R + In rD
(32) Ishikawa, A; Shibata, T./. Liq. Chromutogr. 1993, 16, 859. (33) Hatefi, Y.; Hanstein, W. G . PYOC.Nutl. Acad. Sci. U.S.A. 1969, 62, 1129.
1582 Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
(2)
Consequently, a plot of In k' vs (1/3 should be linear, with a slope of -(m/R) and an intercept of (AS"IR In Cp). Additionally, for a pair of enantiomers
+
In a = -AAG"/RT = -(AAHo/RT)
+ AASo/R
(3)
From eq 3, one can calculate the difference in free energy, and from plots of In a vs (1/7J, the difference in enthalpy and entropy between the two enantiomers can be calculated. Van't Hoff plots were generated for the four isomers of aminoindanol as a function of perchlorate concentration. The pKa of aminoindanol was determined to be 8.3. Since in the pH range of 1-4 the aminoindanol is fully protonated and perchlorate is fully deprotonated, the interaction is expected to be independent of the proton concentration. In going from pH 1 to 4, however, there is a large change in the concentration of the counteranion that may affect the interaction between the aminoindanol and crown ether. The capacity factors of the four isomers were measured from 5 to 45 "C at five intervals on a Crownpak CR(-) column. Aqueous solutions of perchloric acid were used as the mobile phase. The perchloric acid solutions were adjusted to pH's of 1.0, 2.0, 2.4, 3.0, and 3.8 All 20 plots of In k' vs (l/T) were linear indicating no change in the retention mechanism at that specific pH as a function of temperature (Table 2). The entropic term (AS In @) decreased with decreasing concentration of perchlorate ion (Figure 2). This decrease reflects an increase in the order of the mobile phase as there are progressively fewer ions to disrupt the hydrogen bonding between the water molecules. Consequently, the hydration shield around the aminoindanol is more structured, and the hydrophobic interaction is decreased. This decrease is reflected in the decrease in capacity factor with decreasing concentration of perchlorate. Diminution in the hydrophobic interaction is also reflected in the observation that the separation factor between pairs of diastereomers decreases with decreasing perchlorate concentration. The separation between the diastereomericpairs (cis-1trum-Iand cis1I:trum-II) is based primarily on hydrophobic interactions since
+
The separation factors for both pairs of enantiomers were unchanged and thus independent of the nature of the anion. Under acidic conditions, the amine group of aminoindanol is protonated. Enantiomeric resolution is determined by the strength of the inclusion of the protonated amine group into the crown ether. Enantioselectivity is provided by interaction of the guest molecule with the bulky aromatic groups, attached to the crown ether, which act as chiral barriers. In addition to the inclusion phenomena, there is also a nonspecific interaction which occurs between the hydrophobic
(1)
(34) Martin, A J. P.Biochem. SOC.Symp. 1949,3, 4.
Table 2. PH*
AH Values for Aminoindanol as a Function of
PH
trum-I1
trans-I
1
-708.1 (0.997) -642.2 (0.995) -637.6 (0.998) -709.6 (0.997) -803.7 (0.998)
2 2.4 3.0 3.75
-892.8 (0.997) -784.1 (0.996) -775.0 (0.998) -825.9 (0.997) -911.9 (0.998)
cis-I -632.1 (0.996) -605.4 (0.998) -609.5 (0.997) -670.4 (0.991) -674.9 (0.998)
-m-
cis-I1 -750.9 (0.992) -703.1 (0.998) -703.6 (0.997) -748.9 (0.998) -750.9 (0.998)
Terms in parentheses represent the correlation coefficients for the plots. (I
Cis I
--t CIS I1
-400
t
- 1000 -l2O0
-2.0
1'
1.b
'
115
210
'
215
I
310
I
'
3f5
J.0
- log [clo,-]
-2.5
t
-3.0
0
+
5 a
-m-
Trans I ---c Trans I1
-3.5
-4.0 -4.5
-5.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
- log [cl04-1 -l2Oo
2.0
2.5
3.5
4.0
Table 3. Retention of the Cis Isomers as a Function of pH at 25 "C
-3.5 -4.0
+
K
1.5
Figure 3. Influence of perchlorate ion concentration on A H for the cis enantiomers (top) and the trans enantiomers (bottom).
-3.0
33 a
1.0
- log [c104-]
-2.5
0
1 3.0
-4.5 -5.0
-5.5
PH
k' cis-I
k' c~s-II
a
1.0 2.0 2.4 3.0 3.75 4.3 5.2
8.8 5.95 4.55 2.69 0.96 2.47 3.44
10.75 6.95 5.26 3.13 1.13 3.02 4.19
1.22 1.17 1.16 1.16 1.18 1.22 1.22
-5.0
t
4
-6.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
- log (cI04-1 Figure 2. Influence of perchlorate ion concentration on the entropic term for the cis enantiomers (top) and the trans enantiomers (bottom).
each diastereomeric pair will be included in an identical manner. No significant trend was observed with the enthalpy as a function of the concentration of perchlorate ion (Figure 3). Additional separations were performed at 25 "C,for the cis isomers at pH 4.3 and 5.2. A significant increase in the capacity
factor was observed in going from pH 3.75 to 4.3 to 5.2 Vable 3). The increase in capacity factor can be attributed to an increase in electrostatic interaction between the positively charged aminoindanol and the negatively charged silanol sites as the silica becomes deprotonated. Irregular retention behavior in reversed-phase systems has been previously investigated and attributed to this type of silanophilic effect.35-s8 (35) Nahum, A; Horvath, C. J. Chromatop. 1981,203, 53. (36) Klaas, E. B.; Horvath, C.; Melander, W.; Nahum, A.J. Chromatogr. 1981, 203,65. (37) Melander, W.; Stoveken, J.; Horvath, C.J. Chromatop. 1980,199, 35.
Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
1583
1.55
L
1.50
/
1.45 1.40 lu
-
1.35
C
1.30 1.25 1.20
-a-o-
Trans Cis
3.0
3.5
1.15 0.0031
0.0032
0.0033
0.0034
1.0
1.5
1.0
1.5
2.0 2.5 log (c104-1
-
1K"K I
I
I
r
I
I
I
4.0
1
0.20 8
-
0.18
C
0.16 0.14 0.12
-
-
-0.451
r V.
*"
0.0031
0.0032
0.0033
0.0034
1/T, K
Figure 4. Influence of temperature on the K (top) and a (bottom) values for the cis enantiomers at pH 5.2. Other chromatographic conditions are as described in the text.
To further investigate the silanophilic effect, van't Hoff plots were performed at pH 5.2 for the two cis isomers. The plots indicated a deviation from linearity which was more pronounced for &-I (R = 0.98, Figure 4). Much more significant was the fact that the van? Hoff plot indicated a positive value for the enthalpy (negative slope), and the entropic term was much more positive than that at the lower pH values. These results indicate that the mechanism occurring at pH 5.2 is predominantly entropically driven. An entropically driven enantiomeric separation has been previously reported.% In that case, the enthalpy was negative and the separation factor increased with increasing temperature. However, in our case, the separation factor decreased with increasing temperature for the cis enantiomers of aminoindanol (Figure 4). These results indicate that the inclusion mechanism, which controls the enantioseparation, is still enthalpically driven but that there also exists another mechanism that is entropically (38) Sokolowski, A; Wahlund, K G.1.Chmmatugr. 1980,189, 299. (39) Cabrera, K; Lubda, D. J. Chromatogr. 1994, 666,433.
1584 Analytical Chemistry, Vol. 67,No. 9, May 7, 7995
,
-0.50
I
2.0
,
, 2.5
- log [C104']
,
E::, 1
3.0
3.5
4.0
Figure 5. Influence of perchlorate ion concentration on AAH (top) and AAS (bottom) for the cis and trans enantiomers.
driven. This mechanism would be the electrostatic interaction that is occurring between the positively charged aminoindanol and the negatively charged silanol sites. Examination of the separation factors of the cis enantiomers and the trans enantiomers as a function of the perchlorate concentration showed a slight decrease in separation factor with decreasing perchlorate concentration up to pH 3, and then a very slight increase up to pH 3.75. A breakdown of the data into A A l P and AAS" terms revealed that while AAH' was increasing with decreasing perchlorate concentration, the AAS" term was also increasing (Figure 5). The changes in these two factors were virtually canceling each other out, resulting in only a small change in AAG" and consequently a small change in separation factor with respect to temperature. In fact, plots of A M " vs AAS" for the cis pair and the trans pair of enantiomers from pH 1to 3 are linear, with correlation coefficients of 0.997 and 0.996 and slopes of 0.0030 and 0.0027, respectively. The observed linearity is associated with a phenomenon called enthalpy-entropy compen-
-
Racemic CISRrans AI 74000
s Trans I1
Y
1.4
>
s
Trans I
54222
5
0
10
15 Time (min)
20
25
30
Figure 6. Chromatogram of a synthetic mixture of the four stereoisomers of aminoindanol at 25 "C using an aqueous perchloric acid (pH 1.0) solution as the mobile phase. Other chromatographic conditions are as described in text.
*
1.3
1.2
26 1.0
1.5
2.0 2.5 log [C104']
-
24 22
3.0
3.5
4.0
Figure 8. Influence of perchlorate ion concentrationon the a values of the two enantiomers of aminoindan at 25 "C. Chromatographic conditions are as described in the text.
20 18 y
16 14 12 10 8 1.0
1.5
2.0
2.5
3.0
3.5
4.0
- log (clO4-J Figure 7. Influenceof perchlorate ion concentrationon the K values of the two enantiomers of aminoindan at 25 "C. Chromatographic conditions are as described in the text.
sation,40 and it indicates that the inclusion mechanism, which causes enantioselectivity,is practically indifferent to the concentration of perchlorate in the eluent up to pH 3.0. Furthermore, the slopes represent the reciprocal of the temperature at which perfect compensation would occur. The observed slopes correspond to temperatures of 331 and 363 K and represent the temperatures at which the enantioseparationwould be totally independent of mobile phase composition from pH 1 to 3. Contribution of the Hydroxy Group to the Interaction with the Crown Ether. The hydroxy group on aminoindanol would be expected to affect the interaction with the crown ether. The observation that there is resolution between each pair of diastereomers (cis4 and trans-I and cis-I1 and truns-II) between pH 1 and 3.75 (Figure 6) supports this expectation, since they differ only in the positioning of the hydroxy group. The hydroxy group could provide steric hindrance to inclusion and reduce hydrophobic interactions. Consequently, the interaction between 1-aminoindan and the crown ether was examined to determine what effect the absence of the hydroxy group had on the interaction. Experiments were performed on the CR(-) column with (40) Melander, W.; Campbell, D. E.; Horvath, C.J Chromatogr. 1978,158,215225.
perchloric acid, pH 2, as the mobile phase. Amiioindan is more strongly retained than any of the isomers of aminoindanol. The capacity factor for the most retained enantiomer of aminoindan at 25 "C (16.0) is more than twice that of cis-I1 (6.95), which is the most strongly retained isomer of aminoindanol. The separation factor for aminoindan (1.23) was approximately equal to that for the trans isomers (1.25) and greater than that for the cis isomers of aminoindanol(l.17). Apparently, for cis-aminoindanol, where the hydroxy group is on the same side of the molecule as the amino group, the inclusion into the crown ether is reduced due to steric hindrance. The combination of both observations indicates that the absence of the hydroxy group enhances the hydrophobic interaction and its presence on the same side as the amine group hinders inclusion. The capacity factor and separation factor were measured as a function of perchlorate ion concentration and consequently pH (Figures 7 and 8). The capacity factor was observed to decrease going from pH 1to 3.2, and it then started to increase again. The separation factor was observed to decrease slowly up to pH 2.6, and it then started to rise again. The observed changes in trends can be attributed to the influence of the silanophilic effect. Binding Studies. The binding that describes the interaction between aminoindanol and the crown ether can be represented bY
A
+ CSAC
(4)
where A is the aminoindanol, C is the crown ether, and AC is the complex formed. An equation is obtained where
This equation shows that the ratio of bound or complexed sites to unbound or uncomplexed sites is proportional to the product of the equilibrium constant and the concentration of free ligand. Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
1585
0.0
-
-1.0-0.5
-g,
'
-2.5 -5.5
I
-5.0
t
I
-4.5
t
I
-4.0
-1.5-2.0
-
-2.5
-3.0
1
-3.5
-3.6
-3.4 -3.2
-3.0
-2.8
-2.6
-2.4
log [Ccis free1 [MI
log PCIS free1 [MI
Figure 10. Hill plot for cis-l at pH 3.0.
-5.5
-5.0
-4.5
-4.0
-3.5
-3.0
log V c i s free1 [MI
Figure 9. Hill plots for cis-I (top) and cis-ll (bottom) at pH 1.O.
A plot of log([AC]/[C]) vs [A] should give a straight line with a slope of 1and an intercept of K1. Such plots are known as a Hill plots.41-43 The total number of sites available for binding is determined by equilibrating a known mass of CR(+) stationary phase with increasing concentrations of one isomer of cis-aminoindanol. When the amount of adsorbed aminoindanol, as determined by HPLC, has reached a plateau, the sites are judged to be saturated. This concentrationof adsorbed aminoindanol is equated with the total concentration of available sites. Binding studies were performed for both cis enantiomers of aminoindanol at pH 1.0 using perchloric acid. Data were collected for binding between 0.5%and 80%of the available sites, and Hill plots were generated. The difference between the total available sites and the amount of adsorbed aminoindanol ([AC]) was defined as [ C] . (41)Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers. Volume 2: Protein Adsorption; J. D., Andrade, Ed.; Plenum Press: New York, 1985. (42) Wyman, J.; Gill, S. J. Binding and Linkage: Functional Chemistry of Biological Macromolecules; University Science Books: Mill Valley, CA, 1990. (43) Cantor, C. R; Schimmel, P. R Biophysical Chemistry Part III: n i e Behavior of Biological Macromolecules; W. H. Freeman and Co.: New York, 1980.
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Both Hill plots were linear with slopes of 1 (R = 0.996 for both plots; Figure 9). KI was determined to be 537 and 692 mol-' for &-I and cis-11, respectively. These values give a AGO of 3722 and 3872 cal/mol for the respective isomers and a AAG" of 150 cal/ mol. Calculation of AAG" on the basis of the separation factor obtained on a CR(+) column, with perchloric acid (PH 1) as the mobile phase, gives a value of 166.7 cal/mol. These results obtained for AAG" based on two independent methods are in good agreement. A Hill plot was also generated for cis-I at pH 3 (Figure 10). This plot was also linear but had a slope of 2.25 and a log KI of 6.15 (R = 0.998). Additionally, the number of available sites was 8.8 times greater than at pH 1. nYo possibilities can be forwarded for these findings. One possibility is that the mechanism for interaction has changed such that the aminoindanol is interacting with the crown ether in an approximate ratio of 2:1, aminoindanol to crown ether. Two to one ratios of amino compounds to crown ether in crystal states have been previously reportedu and were found to be energetically feasible by molecular m0deling.4~A more likely postulation, however, is that the silanophilicinteraction is coming inb effect, leading to nonspecific electrostatic interaction between the silanol sites of the packing material and the protonated aminoindanol. Binding would be based on inclusion as well as additional interaction at the silanol sites. This heterogeneous binding would explain why the number of available binding sites increased much more than 2-fold. CONCLUSION
We have presented here our findings about the interaction of aminoindanol with a crown ether. This interaction was found to be dependent upon the nature of the counteranion. The counte rion affected the capacity factor, whereby more chaotropic anions increased the capacity factor. The counteranion had no or minimal effect on the separation factor. An increase in the capacity (44)Colquhoun, H.; Stoddart, J. F.; Williams, D. J.j.Chem. Soc., Chem. Commun. 1981,847. (45) Leunuink, F. T. H.; Harkema, S.; Briels, W. J.; Feil, D. /. Comput. Chem. 1993,14, 899.
factor was noted with increasing concentration of the perchlorate anion. A significant increase in capacity factor was observed with perchloric acid at pH 5.2, with only a slight change in separation factor. A van’t Hoff plot at pH 5.2 indicated a dramatic increase in entropy together with a positive enthalpy, which is the converse of the van’t Hoff plots generated at lower pH values. Through the use of Hill plots, it was determined that the interactions at pH 1 and 3 were different. There were more available sites, a higher stability constant, and a greater ratio of ligands to binding sites at pH 3.0. These factors can be attributed to increased nonspecik electrostatic interaction as silanol sites become deprotonated.
ACKNOWLEWMENT We are indebted to Chiral Technologies Inc. for supplying us with the crown ether and the Crownpak columns which made this study possible and also for providing us with fruitful discussion. We would also like to express our gratitude toward Dr. Ken Ryan of Merck and Co. for providing us with the four optical isomers of aminoindanol. Received for review November 9, February 22, 1995.@
1994.
Accepted
AC941099T @
Abstract published in Aduance ACS Abstracts, April 1, 1995.
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