Anal. Chem. 1996, 68, 2379-2385
Unusual Maxima in HPLC Retention of an Homologous Series Consisting of Longer Alkyl Chain Substitutions Steven M. Fields,* Edward W. Huber, Roy J. Vaz, and Nicholas W. Brake†
Analytical and Structural Sciences, Hoechst Marion Roussel, 2110 East Galbraith Road, Cincinnati, Ohio 45215
A series of compounds were studied which had increasing alkyl chain length between an aromatic group and a tertiary amine. In this homologous series, a maximum in retention versus alkyl chain length was found for a wide range of mobile phases using either a silica gel column or a cyanopropyl-bonded silica gel column. One nonaqueous mobile phase using an octadecyl-bonded silica gel column also showed this maximum. The selectivity between E and Z geometric isomers found with the shorter chain analogues decreased with increasing chain length greater than about pentyl, with eventual complete loss of resolution. A few normal-phase solvents produced a reversal of the usual E-Z elution order. 1H NMR assignments are described for the individual geometric isomers. NMR studies and ab initio calculations indicate that a folded conformation in solution is possible for the longer chain analogues. The unusual retention patterns are most likely due to a combination of localized adsorption and analyte solution conformation effects. The monotonic retention changes of members of an homologous series in gas or liquid chromatography are well understood and even exploited in the development of retention indexes. In partition chromatography, the relationship is often described by the Martin equation, where adjusted retention volume or time is a logarithmic function of the number of homologue units. Adsorption chromatography also yields predictable retention patterns, but the relationship frequently deviates from the Martin equation. In either mode of chromatography with a liquid mobile phase, if the homologous series consists of an increase in the length of a linear alkyl chain, then the expected retention patterns can be predicted on the basis of the change in polarity and hydrophobicity.1 Retention should increase with the chain length in reversed-phase HPLC. Conversely, retention should decrease in normal-phase HPLC (liquid-solid adsorption chromatography), but the magnitude of the retention changes should be much smaller than in the partition-based retention of reversed-phase HPLC. In this study, we considered a series of compounds which consisted of lengthening the alkyl chain of clomiphene (n ) 2 in Figure 1) from ethyl up to dodecyl (n ) 12). Due to the synthetic route, each of the analogues was produced as a mixture of the E and Z geometric isomers. The individual isomers needed to be * Present address: Alza Corp., Palo Alto, CA 94303. † Present address: Duramed Pharmaceuticals, Inc., Cincinnati, OH 45213. (1) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; Wiley-Interscience: New York, 1979. S0003-2700(96)00054-6 CCC: $12.00
© 1996 American Chemical Society
Figure 1. General structure of clomiphene analogues, shown as the E geometric isomer. Analogues studied ranged from n ) 2 to 12; n ) 2 is clomiphene.
isolated from the reaction product mixtures. The method of choice for these separations was normal-phase HPLC, due to the ease of solvent removal after fractionation and the high-resolution separation of the geometric isomers using a silica gel column.2,3 Previously, a number of related triphenylethylenes containing alkyl chains of n ) 2-4 and various other chemical substitutions were readily fractionated using slight modifications to the USP test method4 for clomiphene. It was expected, based on these results, that the fractionations of this homologous series with longer alkyl chain lengths would pose no complications in method development. However, modification of these methods for separation of the longer chain analogue isomers was not feasible due to unusual retention patterns and selectivity changes. A significant effort was required to develop HPLC separation conditions for the entire analogue series. This report describes the development of HPLC separations, along with spectroscopic and molecular modeling data which provide some insights into the retention mechanisms. EXPERIMENTAL SECTION The mobile phase delivery system was a Waters 600E pump and controller (Waters Corp., Milford, MA). Detection was by UV absorbance at 280 nm using a Waters Model 484 detector. The injector was a Waters 712 WISP. Injection volumes were 10100 µL. Samples were taken up in mobile phase or mobile phase/ CHCl3 mixtures (up to 50% CHCl3). The HPLC columns were µPorasil, silica gel, 3.9 mm i.d. × 300 mm, irregular 10 µm particle, obtained from Waters; Spherisorb S5 CN, cyanopropyl, 4.6 mm i.d. × 250 mm, spherical 5 µm particle, obtained from Phenomenex (Torrance, CA); µBondapak C18, octadecyl, 3.9 mm i.d. × 300 mm, irregular 10 µm particle, obtained from Waters; and PVA(2) Engelhardt, H.; Elgass, H. Liquid Chromatography on Silica and Alumina as Stationary Phases. In High-Performance Liquid Chromatography, Advances and Perspectives, Vol. 2; Horvath, C., Ed.; Academic Press: New York, 1980. (3) Snyder, L. R. Mobile Phase Effects in Liquid-solid Chromatography. In HighPerformance Liquid Chromatography: Advances and Perspectives, Vol. 3; Horvath, C., Ed.; Academic Press: New York, 1983. (4) Clomiphene citrate. U.S. Pharmacopeia XXII; USP Convention, Inc.: Rockville, MD, 1990.
Analytical Chemistry, Vol. 68, No. 14, July 15, 1996 2379
Table 1. Summary of Retention Patterns and Isomer Resolution E-Z isomer resolutionb for n column type silica gel (µPorasil)
PVA-Sil cyanopropyl
C18
mobile phase
(v/v)a
tr max versus n?
CHCl3/hexane/TEA 20-100/80-0/0.1-0.2 0/100/0.5-2 EA/hexane/TEA 5-20/95-80/0.1 50/50/0.05-0.1 ACN/CHCl3/hexane/TEA 5/15/80/0.1 IPA/hexane/TEA 10-20/90-80/0.05 MTBE/hexane/TEA 40/60/0.1 CHCl3/hexane/TEA 50-100/50-0/0.1-0.2 CHCl3/hexane/TEA 30-50/70-50/0.1-0.2 IPA/hexane/TEA 5/95/0.02-0.1 EA/hexane/TEA 20/80/0.1 ACN/CHCl3/hexane/TEA 5/15/80/0.1 CHCl3/hexane/TEA 20-100/80-0/0.05 CHCl3/ACN/TEA 10-50/90-50/0.05 0/100/0.05 EA/ACN/TEA 50/50/0.05 MeOH/ACN/TEA 10-100/90-0/0.05 H2O/ACN/TEA 5/95/0.1
2
5
7
10
12
isomer order
∼ ∼
∼ ∼
++ +
++ ∼
+
-
-
-
EZ
∼
-
-
-
-
EZ
yes
-
∼
∼
-
-
ZE
yes
++
++
+
EZ
yes
++
++
++
+
EZ
yes
+
+
+
-
EZ
yes
+
∼
-
-
EZ
yes
+
++
+
-
EZ
yes
-
∼
-
no no
∼
∼ +
+ ∼
no
∼
+
∼
no
-
∼
no
∼
+
yes yes
+ -
yes yes
+ -
yes
+
yes
+
-
EZ ZE ZE ZE
-
EZ
+ -
+ -
EZ EZ
∼
∼
EZ
∼
+
EZ
+
++
EZ
a Mobile phase abbreviations: TEA, triethylamine; EA, ethyl acetate; ACN, acetonitrile; IPA, isopropyl alcohol; MTBE, methyl tert-butyl ether; MeOH, methanol. b Resolution symbols: -, coelution; ∼, slight separation with 0.6 < Rs < 1.0; +, good separation with 1.0 < Rs < 1.4; ++, greater than baseline resolution.
Sil, cross-linked poly(vinyl alcohol), 4.6 mm i.d. × 250 mm, spherical 5 µm particle, obtained from YMC, Inc. (Wilmington, NC). The mobile phases used are listed in Table 1. The compositions are shown in volume percent. Column flow rates were 1 or 2 mL/min in all separations. 1H NMR spectra were obtained using a Varian Unity-300 NMR spectrometer (Palo Alto, CA). Spectra were obtained in the solvents noted at ambient temperature (∼25 °C). All 1H spectra were referenced to TMS (δ ) 0.00). To determine the rotation of the phenyl rings relative to the plane of the chloroethylene bond, an analogue without the alkoxyamine side chain was subjected to a systematic search.5 The minimum energy structure was then optimized using an ab initio quantum program (Spartan 3.0, available from Wavefunction, Inc., Irvine, CA), using the split valence basis set 3-21G(*). Analogues with n ) 5 and 9 were then constructed from the optimal geometry of the minimum energy structure. These molecules were then optimized with the constraints described by F(x) ) 0 for 4 < x < 5 and F(x) ) kx2 for x < 4 and x > 5, where x is the distance between the phenyl ring centroid and a methyl carbon (Figure 2) and k ) 200 kcal/Å2. After removal of the constraint, each analogue was optimized again and then placed at the center of a preoptimized box of CHCl3 (as provided by SYBYL6.04, courtesy of W. Jorgenson); all solvent molecules that
had van der Waals overlap with the solute molecule were eliminated. The minimal periodic boxes for the two structures were cubic and contained 204 and 273 solvent molecules for the n ) 5 and 9 analogues, respectively. The structures in the periodic box were optimized using the Tripos 52 force field6 using minimal periodic boundary conditions. The two systems were then
(5) Learch, A. R. In Reviews in Computational Chemistry, Vol. 2; Lipkowitz, K. B., Boyd, D. B., Eds.; VCH Publishers: New York, 1991.
(6) Clark, M.; Cramer, R. D.; Van Opdenbosh, N. J. Comput. Chem. 1989, 10, 982-1012.
2380 Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
Figure 2. Distances monitored over the molecular dynamics simulation, shown in the starting geometry of the n ) 9 analogue. Also shown are the distances between the ortho protons of the phenyl trans to the Cl and the centers of the two other phenyl rings.
subjected to a constant temperature7 molecular dynamics simulation,8 as implemented in SYBYL6.04 (Tripos Associates, Inc., St. Louis, MO) for a period of 200 ps, with an integration step size of 1 fs. The first 100 ps was an equilibration process, and the structure was recorded every picosecond. The distances between the C2 carbon of the N-ethyl and the centroid of the phenyl ring shown in Figure 2 were monitored during the dynamics. The “distance 1” in the simulations started at 5.34 Å for the n ) 5 analogue and at 4.37 Å for the n ) 9 analogue. RESULTS AND DISCUSSION The separations developed during this study used mobile phases ranging from hexane to methanol and columns ranging from silica gel to octadecyl-bonded silica gel (C18). A major consideration in the separation method development was the ease with which a mobile phase could be removed from a fractionated analyte. Volatile organic solvents were preferred over aqueous solvent systems. Since the clomiphene analogues contain a tertiary aliphatic amine, the addition of triethylamine (TEA) to all of the mobile phases, usually at about 0.1%-0.2%, was necessary to prevent irreversible adsorption of the analytes onto the acidic silanol groups of the silica gel. Retention Maxima and Isomer Resolution. Initially, the retention characteristics of the clomiphene analogues were examined under conditions used for the fractionation of the n ) 5 analogue, which were very similar to those of the USP method for analysis of clomiphene.4 A very unusual retention pattern was found which complicated the development of separation methods for the longer chain analogues. A maximum in retention versus alkyl chain length was found at about n ) 4-5 on the silica gel and the cyano columns (Figures 3 and 4) and with one set of conditions on the C18 column (Figure 5). The nonaqueous mobile phase of CHCl3/hexane/TEA showed the only retention maximum with the C18 column. The entire set of conditions tested is listed in Table 1. Clomiphene (n ) 2) was the least retained of all the analogues with most of the mobile phases on the silica gel and CN columns, often with substantially faster elution compared to that of the n ) 12 analogue (Figure 4). In systems where the retention of all analogues was low (e.g., IPA/hexane/TEA), clomiphene retention was closer to the n ) 12 retention. A notable exception to this pattern was the hexane/TEA system, where the clomiphene retention was longer than that of the n ) 7 analogue. On the C18 column, only the ACN and ACN/H2O mobile phases produced the expected logarithmic dependence of retention on alkyl chain length (Figure 5). The order of elution was as predicted from hydrophobic interaction principles, where the more hydrophobic components were retained longer. The other mobile phases, including methanol/TEA, showed a nearly linear dependence of retention on chain length. For nearly all of the HPLC systems which produced a maximum in retention, there was little or no isomer separation for analogues with chain lengths greater than n ) 5. It is rare that geometric isomers cannot be separated by NPLC2,3 due to the specificity of localized adsorption phenomena. The ethyl acetate/hexane/TEA (20/80/0.1 v/v) mobile phase with the µPorasil column produced high resolution for the n ) 7-10 (7) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Dinola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684-3690. (8) McCammon, J. A.; Harvey, S. C. Dynamics of Proteins and Nucleic Acids; Cambridge University Press: Cambridge, U.K., 1987.
Figure 3. Retention maximum versus alkyl chain length on silica gel column. Conditions: µPorasil, CHCl3/hexane/TEA (80/20/0.1 v/v); n ) alkyl chain length.
Figure 4. Retention data on µPorasil and Spherisorb S5 CN columns. Solvent abbreviations: C, CHCl3; H, hexane; T, TEA; Me, methyl tert-butyl ether; I, 2-propanol; E, ethyl acetate. Where varied compositions were tested, the data shown are representative of the composition range.
analogues, but its usefulness for fractionations was limited by low sample solubility. The E and Z isomers eluted in the same order (E-Z) on the PVA, CN, and C18 columns regardless of the mobile phase used. However, on the µPorasil column (silica gel), the retention order of the isomers was found to be sensitive to the mobile phase used. For example, the elution order was E-Z in the CHCl3/hexane/ Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
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Figure 5. Retention data on µBondapak C18 column. Solvent abbreviations: C, CHCl3; H, hexane; T, TEA; M, MeOH; E, ethyl acetate; A, CH3CN; W, water. Where varied compositions were tested, the data shown are representative of the composition range.
TEA system, but the order was Z-E when the CHCl3 was not present and TEA was 1-2% (Table 1). Two solvents, ethyl acetate and methyl tert-butyl ether, in hexane/TEA mixtures, also gave elution orders of Z-E on the µPorasil column. In contrast, the same ethyl acetate mixture gave an elution order of E-Z on the CN column. Low levels of water in HPLC solvents can greatly influence the adsorption of polar analytes such as amines and acids on silica gel columns. Water is present in nearly all solvents at some low level, and it is difficult to exclude completely from a chromatographic experiment. The solvents used were HPLC grade, with lot analyses of 0.001% in hexane and 0.004% in CHCl3. Determination of water in both these solvents by Karl-Fischer titration confirmed these levels. A mobile phase of 80/20/0.1 (v/v) CHCl3/ hexane/TEA spiked with additional water was used to test the effect of a higher concentration. The solubilities of water in CHCl3 and hexane were used to calculate the solubility in the mixture, which was done using the volumetric ratio of the solvents. After vigorous stirring, no water droplets were visible. The total water concentration was then about 0.05%. No significant difference in the isomer separation was observed, and the maximum in retention existed and was still at about n ) 4-5. Identification of Peaks. The isomer elution order for any particular analogue was determined using the isomer peak area ratio and NMR analysis of isolated fractions. Several of the analogues were synthesized with an enriched amount of the E isomer, at approximately a 2:1 ratio over the Z isomer. The clomiphene reference standard conformed to USP specifications and contained about 65% of the E isomer. If there was a separation of the isomers in a chromatogram, then the peak area ratio was used to tentatively assign the elution order of the isomers. Geometric conformation was made by NMR analysis of fractionated samples, as discussed below. The individual isomer samples were then chromatographed to confirm the peak assignments. In all cases, the NMR data supported the assignments made using the peak area ratio technique. 2382 Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
Figure 6.
1H
NMR spectra of representative aromatic protons.
The NMR assignments were based on the observed chemical shift of the ortho protons (relative to the ethylene group, H-1 in Figure 6) of the aromatic ring trans to the chlorine. Analysis of a large series of triphenylethylenes has indicated that in DMSOd6, these protons are shifted upfield, consistent with a conformation in which the trans (relative to the chlorine) aromatic ring is rotated relative to the remaining two rings, resulting in the ortho protons being placed in the shielding cone above the adjacent rings. The distances between the protons and the centroids of the benzene rings are around 3.5 Å (Figure 2). In addition, none of the phenyl rings are coplanar with the ethylene bond. The angle between the planes defined by the ethylene-chlorine bonds (-CdC-Cl) and the phenyl rings was -119.8° for the phenyl cis to the Cl, 128° for the phenyl trans to the Cl, and -123.3° for the phenyl R to the Cl. Determination of olefin geometry thus became a matter of assignments. For the E isomer, a typical 1H NMR spectrum is shown in the top of Figure 6. Two sets of two protons (H-1 and H-2) are shifted upfield relative to the remaining 10 aromatic protons. NOE (to the ether methylene) and decoupling experiments show these to be the protons of the phenoxy ring, consistent with the discussion above. By contrast, for the Z isomers, the pattern shown in Figure 6 (bottom spectrum) is observed. The phenoxy protons ortho to the oxygen (H-3) are shifted upfield, but the meta protons (relative to the oxygen, H-4), no longer in the shielding cone of the adjacent aromatic rings, are observed downfield. By contrast, the ortho protons (H-1) of the aromatic ring trans to the chlorine are now shifted upfield. Solution Conformations. 1H NMR spectra of the n ) 2, 5, 9, and 12 analogues were obtained in an attempt to determine if a folded solution conformation was possible, which could aid in explaining the observed HPLC data. Spectra were obtained in CDCl3, CD3CN, and CD3CN/D2O (95/5 v/v). The chemical shifts of the methylene groups R to the oxygen and nitrogen as well as those of the methyl groups are listed in Table 2. For comparison, the observed chemical shifts in CDCl3 of the n ) 9 and 12 diethylamine alkyl alcohols are also listed in Table 2. For the triphenylethylenes, no significant differences were observed for the protons R to the oxygen in the n ) 2, 5, 9, or 12 analogues (the n ) 2 shifts are about 0.1 ppm downfield due to
Table 2. Proton NMR Chemical Shifts (ppm) in Various Solvents solvent analogue na 2 5 9 12 9 OH 12 OH 2 5 9 12 9 OH 12 OH 2 5 9 12 9 OH 12 OH 2 5 9 12
CDCl3
CD3CN
CD3CN/D2O
-CH2- of the Ethylamine 2.60, 2.65 2.52, 2.58 2.55, 2.52 2.46 3.11 3.02,3.03 3.11 3.02 2.50 2.53 -CH2- of Alkyl Chain R to Nitrogen 2.81, 2.90 2.72, 2.81 2.4-2.5 2.37 2.85-3.04 2.9 2.97 2.9 2.40 2.38 Terminal Methyl (-CH3) 1.04, 1.08 0.95, 1.00 1.01, 1.04 0.94, 0.96 1.38, 1.39 1.26, 1.27 1.40 1.27 1.02 1.02 -CH2- of Alkyl Chain R to Oxygen 3.94, 4.07 3.90, 4.04 3.85, 3.98 3.85, 3.99 3.83, 3.97 3.83, 3.97 3.84, 3.97 3.83, 3.97
2.52, 2.58 2.48, 2.46 3.08 3.08
2.73, 2.82 2.38, 2.41 2.95 2.95
0.95, 1.00 0.94, 0.96 1.21, 1.22 1.24
3.91, 4.05 3.84, 3.98 3.82, 3.96 3.81, 3.95
Figure 7. Distances monitored over the dynamics simulation period for the n ) 5 analogue, as described in Figure 2.
a OH denotes the corresponding diethylamine alkyl alcohols (HO(CH2)nNEt2).
the β amine functionality). By contrast, a large (0.4-0.5 ppm) downfield shift of the protons near the amine was observed in the n ) 9 and 12 analogues compared to the n ) 2 or 5 compounds or the n ) 9 or 12 alcohols. This was observed in all the solvents examined. This observation suggests that these analogues can assume a conformation in which the amine tail is in a deshielding region of the triphenylethylene group that is not possible for the n ) 2 or 5 analogues or the alcohols. The time-averaged conformation observed by NMR is such that a downfield shift is therefore observed. It should also be noted that no changes in chemical shift were observed upon dilution, indicating that intramolecular and not intermolecular interactions are responsible. These NMR observations indicate that a conformation is possible in solution for the longer chain analogues that is not possible for the shorter chain analogues. Molecular dynamics simulations were also conducted on the n ) 5 and 9 analogues. For the n ) 5 analogue, monitoring of the intramolecular distances showed that the distance between the phenyl and the amine tail continues to increase over the simulation period (Figure 7). However, for the n ) 9 analogue (Figure 8), the distances equilibrate and are much shorter than those for the n ) 5 analogue. Also in the n ) 9 analogue simulation, there is rotation around the last bond between the C9 alkyl carbon and the amine nitrogen during the time simulation period from 10 to 80 ps. This dynamics simulation is in agreement with the proposed solution folding suggested by the NMR data, which is that, as the alkyl chain length increases, the amine group end lies in the vicinity of the phenyl ring. Since there are no strong intramolecular interactions that could cause the folding, such as hydrogen bonding, either hydrophobic interactions
Figure 8. Distances monitored over the dynamics simulation period for the n ) 9 analogue, as described in Figure 2.
between the phenyl and the ethyl groups of the amine or hydrodynamic forces induce the molecule to adopt the folded conformation. HPLC Retention Mechanisms. It was not possible to conclusively identify the cause of the retention maxima and associated losses in E-Z isomer resolution, nor the cause of the Analytical Chemistry, Vol. 68, No. 14, July 15, 1996
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isomer elution order reversals. However, interpreting the data in terms of a displacement model for adsorption chromatography3 suggested some possible explanations. It was beyond the scope of this report to attempt an in-depth analysis of the data in terms of the model. Rather, the analogue retention maxima and isomer selectivity are discussed below in terms of the interaction concepts of the model in an attempt to reconcile the observed chromatographic data with the suggested solution conformations. The displacement model has, as a premise, that retention can be viewed as a competitive adsorption equilibrium between analyte (A) and mobile phase (M) molecules for column active sites:
A + nMad T Aad + nM
where the subscript ad indicates adsorption onto the column and n is the number of solvent molecules displaced by the adsorption of the analyte molecule. The distribution coefficient, K, proportional to retention, is related to column, mobile phase, and analyte properties by the following expression:
log K ) R(S° - Ai°)
where R is the adsorbent activity parameter, S° is the adsorption energy of solute i onto a standard adsorbent (defined as R ) 1), Ai is the molecular area of the solute i which adsorbs onto the surface, and ° is the solvent strength parameter. For a particular column and mobile phase, the parameters R and ° are constants. For small molecules with low adsorption energies, S° and Ai can be calculated from the group contributions of the individual molecular functional groups. However, for larger molecules with multiple, polar adsorptive groups, the additivity can be affected by the molecular conformation and steric factors. Using the group contribution parameters for solutes on an alumina surface,9 the effect of increasing the alkyl chain length from n ) 2 to 12 would be to increase S° by about 2%, taking into account the rotation of the phenyl rings about the ethylene bond plane, which reduces the number of interactions. In contrast, the effect on Ai is much larger in magnitude, on the order of 25100%, depending upon how the solution folding conformation is taken into account. The solvent strength parameter ° for the mobile phases used with the silica gel, PVA, and cyanopropyl columns would range from about 0.1 to 0.5. Assuming a value of ° of 0.1 or greater, Ai° would increase faster than S°, and thus the term (S° - Ai°) would decrease and retention should decrease as the alkyl chain length increases. The maxima in retention can then be considered as a function of the term (S° Ai°). One or both of the individual terms must be a nonlinear function of the alkyl chain length, as the difference of two linear functions would not produce a retention maximum. The apparent anomaly in the retention maxima is the low retention of the shortest chain analogues, since the retention pattern from about n ) 5-12 follows the basic mechanistic prediction. Intramolecular delocalization3 describes the situation where one functional group of a molecule is strongly adsorbed (localized) such that geometric constraints prevent or reduce the magnitude of adsorption of other functional groups. In the case of clomiphene (n ) 2), this could arise by localized adsorption of either (9) Karger, B. L.; Snyder, L. R.; Horvath, C. An Introduction to Separation Science; Wiley-Interscience: New York, 1973.
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the amine or the triphenylethylene groups. Although the hydrogen bonding potential of the amine with a silanol would be the strongest single adsorption, it is not known if that adsorption energy is greater than the combined energy of the triphenylethylene group. Since a necessity of geometric isomer separation is the adsorption of the triphenylethylene portion of the molecule, the intramolecular delocalization may arise from the aromatic function. Regardless of which part of the molecule is localized, it is then plausible that the intramolecular delocalization decreases as the alkyl chain length increases, eventually becoming insignificant. Thus, S° might increase rapidly as the alkyl chain increases from n ) 2 to 4, and thereafter increase at a slower rate. A steady, although not necessarily linear, increase in Ai° at the same time would then yield an observed difference term (S° - Ai°) with a maximum. An inconsistency in the previous argument relates to the lack of isomer separation with longer alkyl chain lengths. If the intramolecular delocalization is the cause of the low clomiphene retention and longer alkyl chains reduce the effect, then the isomers should be resolved with longer alkyl chains because the molecule is then large enough and flexible enough to permit adsorption of both the amine and the aromatic groups. This inconsistency can be addressed by considering the ramifications of the folded solution conformation of the longer chain analogues. If the amine and the chlorophenyl end of the ethylene adsorb, then other isomer-specific adsorptions could be delocalized, yielding an adsorption conformation independent of the isomeric configuration. This possibility could then explain the loss of isomer selectivity with the longer chain analogues, where the isomer selectivity is presumed to be dependent upon the differential adsorption of triphenylethylene part of the molecule. A rationale for the maximum using CHCl3/hexane/TEA with the C18 column is more difficult to establish, due to the partitioning interactions and low number of adsorption sites. The monotonic increases in retention with more traditional mobile phases suggest that the mechanism is related to the hydrophobic effect. Likewise, the isomer resolution must also be a function of the analogue hydrophobicity. The isomer selectivity changes and elution order reversals observed with solvent changes are related to the nature of the analyte-surface interactions. Changes in solvent affect the competitive equilibrium in two ways:2-4 by potentially changing the number of solvent molecules displaced by the adsorbing analyte and by changing the solvent strength parameter °. There was no strong relationship between the strong solvent classification of localizing character10 and the isomer elution order. The basic localizing solvents MTBE and TEA (the latter used without other strong solvents) produced the Z-E elution order, while IPA did not. The nonbasic localizing solvent ethyl acetate also produced the Z-E order. CONCLUSIONS A maxima in retention versus alkyl chain length for an homologous series was found for a wide range of mobile phases using either a silica gel column or a cyanopropyl-bonded silica gel column. One nonaqeous mobile phase (chloroform/hexane) using an octadecyl-bonded silica gel column also exhibited the (10) Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. Practical HPLC Method Development; Wiley-Interscience: New York, 1988.
maximum. The E and Z isomer resolution and elution order were dependent upon the alkyl chain length, the type of solvent, and the column. NMR and molecular dynamics simulations indicate that a folded conformation in solution is possible for the longer chain analogues. Interpretation of the data in terms of the displacement model of adsorption chromatography suggests that the observed phenomena may be due to a combination of localized adsorption and analyte solution conformation. The uniqueness of these data is reinforced by the fact that a literature search
revealed no other instances of a retention maximum within an homologous series in HPLC.
Received for review January 18, 1996. Accepted April 30, 1996.X AC960054K X
Abstract published in Advance ACS Abstracts, June 1, 1996.
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