Anal. Chem. 1999, 71, 928-938
Influence of Thermal Annealing on the Thermodynamic and Mass-Transfer Kinetic Properties of D- and L-Phenylalanine Anilide on Imprinted Polymeric Stationary Phases Yibai Chen,†,‡ Marianna Kele,†,‡ Peter Sajonz,§ Bo 1 rje Sellergren,*,| and Georges Guiochon*,†,‡
Department of Chemistry, The University of Tennessee, Knoxville, Tennessee 37996-1600, Division of Chemical and Analytical Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, Merck Research Laboratories, P.O. Box 2000, R 80Y-335, Rahway, New Jersey 07065, and Department of Inorganic Chemistry and Analytical Chemistry, Johannes Gutenberg University, Mainz, Joh.-Joachim-Becherweg 24, D-55099 Mainz, Germany
An investigation of the material, chromatographic, thermodynamic, and kinetic properties of thermally treated (i.e., annealed) polymeric stationary phases imprinted with L-phenylalanine anilide (L-PA) was carried out. The imprinting procedure of the solid phase used in this study was the same as for the untreated imprinted stationary phase studied previously. However, after polymerization, these new stationary phases were treated at elevated temperatures (50, 120, 140, and 160 °C) for 24 h. The treatment at 120 and 140 °C led to a larger decrease in the retention of L-PA than that of D-PA. The polymer treated at 160 °C could no longer resolve the D,L-PA racemate. The heat treatments were accompanied by a decrease in swelling and an increase in density causing an increase in the density of the remaining active sites. The polymer treated at 120 °C was chosen for classical frontal analysis. The adsorption isotherms and the masstransfer rate coefficients of D- and L-PA were derived from the experimental breakthrough curves. This study was carried out in the same temperature and concentration ranges as the previous one. A comparative discussion of the properties of the two polymeric molecular imprinted stationary phases is presented. The thermal treatment increases the saturation capacity of the packing material by one-third to one-half, reduces markedly the separation factor of the two enantiomers, and slightly accelerates their mass-transfer kinetics. There seems to be no interactions on the annealed polymer between the selective L-PA imprinted sites and the D-PA molecules.
Imprinted chiral stationary phases were developed in the early 1950s.1 These new materials are rapidly attracting interest for the separation of enantiomers and the preparation of optically pure compounds because of alleged advantages in simplicity, flexibility, †
The University of Tennessee. Oak Ridge National Laboratory. § Merck Research Laboratories. | Johannes Gutenberg University. (1) Curti, R.; Colombo, U. J. Am. Chem. Soc. 1952, 74, 3961. ‡
928 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
and economy. A better understanding of the physicochemical nature of the equilibrium involved in the separation, of the chromatographic behavior of the stationary phases made with these polymeric materials, and of their characteristics would allow significant improvements of the performance of imprinted stationary phases. One of the problems arising from the use of imprinted stationary phases is the peak broadening observed in many cases.2 It is generally believed that this broadening arises from a slow step in the mass-transfer kinetics of the enantiomers, probably a slow kinetics of binding to and dissociating from the selective sites3,4 and/or to overloading of the high-energy binding sites.2 In a previous study, the separation of D- and L-phenylalanine anilide (PA) enantiomers was chosen to evaluate an L-phenylalanine anilide (L-PA) imprinted stationary phase.5 The adsorption isotherms were determined using classical staircase frontal analysis.6 The experimental data fitted well to both the Freundlich and the bilangmuir isotherm models. The best values of the numerical coefficients derived for the bilangmuir model indicated that the enantioselective sites of the imprinted stationary phase had a much lower saturation capacity for the nonimprinted enantiomer, D-PA, than the imprinted one (L-PA). This confirmed the high selectivity for D-PA and L-PA achieved by the L-PA imprinted stationary phase used. In the kinetic study, the masstransfer rate coefficient (kf) was calculated by fitting the breakthrough curve to the transport model of chromatography.6 The mass-transfer rate coefficients were small, indicating a slow masstransfer kinetics. The mass-transfer coefficient kf for L-PA exhibited a strong dependence on the sample concentration while for D-PA this coefficient was higher and less affected by the sample concentration.5 For use of imprinted materials in applications demanding a high selectivity, e.g., in separation assays or sensors, it is necessary, on one hand, to reduce the number of nonselective (2) (3) (4) (5)
Sellergren, B.; Shea, K. J. J. Chromatogr. 1995, 690, 29. Wulff, G.; Oberkobusch, D.; Minarik, M. React. Polym. 1985, 3, 261. Sellergren, B. Chirality 1989, 1, 63. Sajonz, P.; Kele, M.; Zhong, G.; Sellergren, B.; Guiochon, G. J. Chromatogr. A. 1998, 810, 1-17. (6) Guiochon, G.; Goishan-Shirazi, S.; Katti, A. Fundamentals of Preparative and Nonlinear Chromatography; Academic Press: New York, 1994. 10.1021/ac981154o CCC: $18.00
© 1999 American Chemical Society Published on Web 01/30/1999
binding sites and to increase the number of enantioselective binding sites and, on the other hand, to enhance the mass-transfer kinetics. The selectivity of the selective sites (i.e., the binding energies of these sites with the two enantiomers) and their distribution are influenced by factors related to the preparation of the material, e.g., the choice and the relative concentrations of the monomers, the solvent, and the temperature, but they can also be influenced by posttreatments, such as chemical modifications or thermal annealing.7 A recent study showed that the selectivity, the kinetic properties, and the sample loading capacity of imprinted stationary phases can be affected by thermal posttreatments.2,7 The response to the heat treatment of a polymeric material depends on its drystate morphology. A material classified as gellike, with a low porosity and a small internal surface area but with a high swelling factor, is more stable than a less swellable material with a mesoporous morphology.7 This statement is valid with respect to both the onset temperature for mass loss and the enantioselectivity. Compared to untreated materials, a heat-treated gellike material exhibits a lower selectivity at low concentrations and a somewhat better efficiency, whereas a mesoporous material exhibits a significant loss in enantioselectivity. To understand the cause of these differences, imprinted stationary phases similar to the one previously studied but treated at different temperatures were prepared. They were characterized in terms of chromatographic performance and structural properties. The material treated at 120 °C was subjected to a thermodynamic and kinetic study, and the results were compared with the results obtained in our previous work.5 Frontal analysis is the ideal technique for a quantitative study of the interactions between solutes and a stationary phase.6,8,9 The adsorption equilibrium isotherms were determined directly from the frontal analysis experiments.6 The bilangmuir isotherm model was chosen to fit these experimental data. It is the simplest model for a heterogeneous surface covered with two different kinds of adsorption sites, nonselective and enantioselective.6 Despite the simplicity of this model, it provided a remarkably good representation of the experimental results. The fitting parameters extracted from the model were used to calculate the saturation capacities of both enantiomers and their separation factor, parameters that can be used to evaluate the quality of the stationary phase. The masstransfer rate coefficients were determined at various temperatures using a lumped kinetic model.5 THEORY Adsorption Equilibrium Isotherm. The bilangmuir isotherm model was chosen, due to the heterogeneous characteristics of the imprinted stationary phase.5,6 In eq 1, q* is the sample
q*(C) )
a1C a2C + 1 + b1C 1 + b2C
(1)
concentration in the stationary phase at equilibrium with a sample concentration C in the mobile phase, a1, b1, a2, and b2 are numerical parameters. The saturation capacities of the two equilibria involved (7) Sellergren, B.; Shea, K. J. J. Chromatogr. 1993, 635, 31. (8) Sajonz, P.; Zhong, G.; Guiochon, G. J. Chromatogr., A 1996, 728, 15. (9) Sajonz, P.; Zhong, G.; Guiochon, G. J. Chromatogr., A 1996, 731, 1.
in the retention for the two enantiomers and the separation parameters can be derived from the values of these numerical parameters, as discussed later. For a pair of compounds, there are two isotherms and this isotherm model has eight parameters. If the two compounds are enantiomers and if the nonselective and the enantioselective interactions can be accounted for by two separate contributions, the term corresponding to the nonselective interactions is the same for both compounds (Pasteur principle) and the model has only six independent parameters. This is the most general case.10 If the two enantiomers interact with the same enantioselective sites (albeit with different free energies), the saturation capacity of the second term in eq 1 is the same for the two compounds and there are only five parameters. Finally, it is possible that one of the two enantiomers does not interact with the selective sites (a plausible assumption in the case of imprinted polymers), in which case there are only four parameters and the isotherm for the less retained compound is a Langmuir one.10 Thus, the analysis of the adsorption isotherms of the two enantiomers can give valuable information regarding the equilibrium responsible for chiral retention and separation.10 Kinetic Model. The solid-film linear driving force model was used for the kinetic study.5,6 In this model, all contributions to band broadening are combined into one constant, called the masstransfer rate coefficient, kf, which is defined as
∂q/∂t ) kf(q* - q)
(2)
where q* and q are the solute concentrations in the stationary phase, at equilibrium with the mobile-phase concentration C and at time t, respectively. Application of Theoretical Models. Frontal analysis was used as described previously.5,6,8,9 The isotherm data points were obtained by integration of the breakthrough curves, which has the advantage of giving results independent from the mass-transfer kinetics.6,8 The profiles of the breakthrough curves were calculated using the transport model of chromatography6 because the low efficiency observed is, in part, due to the slow mass-transfer kinetics.5 This justifies neglecting the axial dispersion whose contribution would be negligible. These profiles were calculated using the isotherm data previously measured and appropriate values of the rate coefficients. The values of these coefficients best fitting the experimental data were derived by parameter identification as previously described.5 EXPERIMENTAL SECTION Equipment. Two HPLC systems were used in this study. The initial evaluations of chromatographic performance were performed on an HP 1050 systems (Hewlett-Packard, Waldbronn, Germany). The thermodynamic and kinetic data were obtained on an HP 1090 Series II system (Hewlett-Packard, Palo Alto, CA). The latter was equipped with a multisolvent delivery system allowing solvent gradient or the delivery of a binary mobile phase of constant composition, an automatic injection system, a diode array UV detector, a column oven, and a data station. The chromatograms acquired were uploaded to one of the computers at the UT Computer Center. (10) Fornsted, T.; Sajonz, P.; Guiochon, G. Chirality 1998, 10, 375.
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Figure 1. Capacity factors (k′) for D- and L-PA and separation factor (R) on columns packed with untreated L-PA imprinted polymer as described in the Experimental Section. Injection: 1 µmol of D,L-PA in 20 µL of MeCN. Mobile phase: MeCN/water/HOAc 92.5:2.5:5 (v:v:v).
The pore volumes and surface areas were derived from measurements of nitrogen sorption carried out using an ASAP 2010 (Micromeritics, Northcross, GA). The bulk density of the polymer was estimated by weighing an amount of the granulated polymer (25-36 µm) corresponding to ∼0.2 mL in graduated NMR tubes. The polymer swelling was determined by equilibrating a sample in acetonitrile overnight and tapping it until no further change in the bed height was observed. Stationary Phases, Thermal Treatment, and Initial Evaluation. The polymeric imprinted material [poly(methacrylic acidco-ethyleneglycoldimethacrylate] was prepared as described earlier.5 The only difference between the polymers used in this work and the one previously studied5 is the thermal treatment applied to the polymer after synthesis. Chromatographic columns (12.5 × 0.4 cm) were first packed with the untreated material, as previously described.5 The initial chromatographic evaluations were carried out at a flow rate of 1 mL/min and a detection wavelength of 254 nm and using acetone as the void volume marker. It was then verified that the material exhibited the same behavior as before5 in the resolution of D- and L-PA in a mobile phase consisting of MeCN/water/HOAc at 92.5:2.5:5, v:v:v (Figure 1). The columns were unpacked and the materials dried from this solvent mixture and heated under atmospheric pressure at 50 °C or under vacuum, at 120, 140, or 160 °C for 24 h. There was no weight loss at these temperatures. Thermogravimetric analysis showed weight loss to start above 230 °C for this type of material.7 The packing material used previously had not undergone this thermal treatment. The materials treated at 50 and 120 °C were repacked in columns of the same size and those treated at 140 and 160 °C in smaller (5 × 0.46 cm) columns. These columns were used to determine the ability of the treated materials to resolve D- and L-PA at different sample loadings in the same mobile phase as above. 930 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
The material treated at 120 °C was subjected to more detailed thermodynamic and kinetic investigations. A 10 × 0.46 cm column was packed, using a slurry packing technique, with an acetonitrile, water, and acetic acid (92.5:2.5:5, v:v:v) solution as both the slurry and the pushing solvent. The untreated and the thermally treated (annealed) columns are referred to as column I and column II, respectively. Mobile Phase and Samples Used in the Thermodynamic and Kinetic Measurements. The mobile phase was a 70:30 (v: v) mixture of pure acetonitrile and an aqueous buffer. The buffer was prepared by dissolving proper amounts of orthophosphoric acid and sodium hydroxide in water in order to obtain a pH of 5.8. The pH was measured using a calibrated pH meter (American pH II). Solutions of each enantiomer were prepared at three different concentrations, 1.0, 0.1 and 0.01 g/L, by dissolving either D- or L-phenylalanine anilide in the mobile-phase solution. These solutions were used as samples. The samples and the mobile phase were filtered through 0.45 µm Nylon filters (Nalgene Filterware, NYL 150-0045, Nalgene, NY) before injecting into the chromatographic system. Experimental Procedure for Frontal Analysis. (a) Recording of Breakthrough Curves. A seven-step staircase frontal analysis was performed for each sample solution, at each temperature. These steps were from 0 to 5, 5 to 10, 10 to 20, 20 to 40, 40 to 60, 60 to 80, and 80 to 100%. All the experiments were made in the order of increasing concentrations. The column was washed and equilibrated for at least 30 min before changing the sample solution (from low to high). The experiments were first performed at 40 °C for each sample solution and then continued at 50, 60, and 70 °C. The flow rate was controlled at 1 mL/min during all the experiments. The signals were detected and recorded at wavelengths of 260 and 280 nm by the UV detector. The extracolumn time, tx, was measured by injecting acetonitrile after
Figure 2. Elutlon profiles of D,L-PA (50 nmol) injected on columns packed with heat-treated, L-PA imprinted stationary phases. Mobile phase: MeCN/water/HOAc, 92.5:2.5:5 (v:v:v). For the materials treated at 50 and 140 °C, in order of increasing retention, the first two peaks are the split peak of D-PA and the third is L-PA. The columns with the materials treated at 140 and 160 °C were only 5 cm long.
having replaced the column by a zero volume connection. It was found to be tx ) 0.65 min. This transit time through the extracolumn volumes of valves and tubings is longer in the FA experiments because the pump is used to mix the two solutions used for the frontal analyses experiments. (b) Column Characteristics. Analytical-size (10 µL of dilute solution) injections of D-PA and L-PA at 40 °C gave retention times of 7.66 and 21.82 min, respectively. The selectivity factor was approximately 3.1. The efficiency, N, of column II for a nonretained tracer was measured from the width at half-height of the acetone peak at 40 °C. N was approximately 1500 theoretical plates at the beginning of the experiments. It decreased gradually to nearly 1100 plates by their end. The holdup time of this column, to, was 0.757 min and the phase ratio, F () (1 - )/, with total porosity) was 1.19. RESULTS AND DISCUSSION Influence of the Heat Treatment on the Material and Chromatographic Properties of the Polymeric Stationary Phases. The chromatographic properties of the L-PA imprinted polymers were determined before and after the thermal treatment (Figures 1-3). The mobile phase was acetonitrile, with acetic acid and water as modifiers. Drying the polymers at 50 °C overnight resulted in a reduced peak-splitting effect (not shown in Figure 2), without significant changes in the retention factors. This contrasted with the results of heat treatments made at higher temperatures. The most significant effects of the treatments at 120 and 140 °C were a reduced retention of L-PA and a flattening out of the plots of the selectivity versus the sample size, which was of comparable importance at both temperatures. Treatment at 120 °C led to a sharpening of the peaks and no peak splitting for D-PA. The peak asymmetry remained significant, however. The peak asymmetry factor for L-PA on a column packed with a
Figure 3. Separation factor (R) and capacity factors (k′) for D- and L-PA versus the amount of D,L-PA injected on columns packed with heat-treated L-PA imprinted stationary phases. Same column dimensions as for the corresponding Figure 2. Mobile phase: MeCN/water/ HOAc 92.5:2.5:5 (v:v:v).
material treated at 120 °C increased from 3 for a 5-nmol sample to more than 5 for a 200-nmol sample in a mobile phase of MeCN/ potassium phosphate buffer 0.05 M, pH 7, 70:30 v:v.2 The material treated at 140 °C gave a column exhibiting poor efficiency. The increase in retention of D-PA with increasing sample load is Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
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probably associated with the use of acetic acid as a mobile-phase modifier. Acetic acid interacts with the solutes and the retention process becomes endothermic.7,11 The peak-splitting effect, on the other hand, is likely related to the polymer porosity and the mobility of its chain segments. This is seen when comparing the peak profiles on materials treated at 50 and 120 °C. The amount of micropores is related to the extent of diffusional mass-transfer limitations in the material. Due to the abnormal chromatographic behavior with the acetic acid modifier, the remaining investigations were carried out with an aqueous-buffered mobile phase. The imprinted polymers used in this work exhibit material properties typical for a cross-linked polymer prepared in the presence of a good solvent.12 This leads to the formation of a relatively homogeneous network, with a large extent of intermolecular cross-links. Phase separation takes place upon coagulation of the solvent-swollen gel particles, to form grains, which, in turn, coagulate to form the pore system. The grains are interconnected by flexible segments that impart the polymer with a relatively large degree of swelling. Thus, in the dry state, these polymers exhibit no or little porosity. In solution, however, the polymers swell to occupy a volume close to that of a corresponding amount of polymer prepared in a poor solvent, giving a permanently porous structure. The materials are thus quite porous in the swollen state. Despite the high degree of swelling, the extent of unreacted double bonds is quite low, showing that the swelling is due to the heterogeneity of the cross-link density. In a previous study of the thermal posttreatment of suspensionpolymerized trimethylolpropane trimethacrylate (TRIM), it was observed that a large decrease of the swelling and a slight decrease of the average pore size took place at 130 °C.13 Since the original swelling observed prior to the treatment could be restored upon heating the material in a swelling solvent, the reduced swelling was attributed to a thermally induced relaxation process rather than to additional chemical cross-linking. Possibly, the temperature of the treatment was above the glass transition temperature of the interconnecting segments. In this context, it should be noted that atactic poly(methyl methacrylate) has a glass transition temperature of 105 °C. Similar observations were made in this study. The thermal treatment was accompanied by a significant reduction in the swelling and an increase in the bulk density of the dry material (Table 1). So, a much larger amount of the annealed polymer was needed for the packing of a column. This led to a higher density of the enantioselective binding sites, although they may be less accessible than prior to the thermal treatment. The nitrogen sorption data showed that all materials, whether before or after the thermal treatment, exhibited no or little porosity and only a small specific surface area (Table 1). The IR spectra of the polymers showed a slight decrease of the intensity of the CdC stretch band at 1638 cm-1 assigned to unreacted double bonds. This indicates that the heat treatment resulted in only a slight increase of the conversion of pendant double bonds of the materials. However, heating to 140 °C led to the apparent (11) Sellergren, B., Lepisto ¨, M.; Mosbach, K. J. Am. Chem. Soc. 1988, 110, 5853. (12) Guyot, A. In Synthesis and Separations Using Functional Polymers; Sherrington, D. C., Hodge, P. Eds.; Wiley-Interscience: New York, 1988; Chapter 1. (13) Reinholdsson, P.; Hargitai, T.; To¨mell, B.; Isaksson, R. Angew. Macromol. Chem. 1991, 192, 113-132.
932 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
Table 1. Results from Swelling and Nitrogen Sorption Measurements on Thermally Treated L-PA Imprinted Polymersa
T (°C)
swellingb (mL/mL)
densityc (g/mL)
surface aread (m2/g)
pore volumee (mL/g)
pore diamf (Å)
untreated 120 140 160
2.1 1.9 1.6 1.5
0.41 0.60 0.51 0.59
4.4 4.6 4.1 7.1
0.021 0.024 0.021 0.022
189 108 202 127
a Physical characteristics of the 25-36-µm particle size fraction of thermally treated polymers imprinted with L-PA. The polymers were heated under vacuum at the indicated temperatures for 24 h, as described in the Experimental Section. In the nitrogen adsorption measurements the polymers were degassed at 40 °C for 12 h. b Swelling in acetonitrile. c Weight of 1 mL of dry polymer (25-38 µm). d BET surface area using a 40-point pressure table. e Total pore volume of pores less than 2600 Å. f Average pore diameter (BJH).
destruction of most high-energy binding sites. Treatment at 160 °C led to the total loss of the enantioselectivity. Apparently, this temperature exceeds the glass transition temperature of the more highly cross-linked regions of the matrix hosting the binding sites. Thermodynamic Study. All the experiments reported here were carried out with column II, packed with the thermally annealed material. When needed, these results are compared with those obtained on the untreated material (column I) and previously published.5 (a) Fitting of the Experimental Data to a Model. The experimental data afforded by frontal analysis with D-PA and L-PA were first fitted to the bilangmuir isotherm model. The results of this nonlinear regression are shown in Figure 4, in which the experimental data (symbols) and the best isotherms (lines) are compared. A logarithmic scale is used in the figure evenly to distribute the experimental data points in the plots. The values of the parameters obtained are listed in Table 2 for column II (thermally treated), with the same data in Table 3 for column I (untreated). The experimental data were fitted to several isotherm models which will be discussed and compared later in this work. Two separate figures are needed to present the data and results for each enantiomer in order to have enough resolution for the clarity of the discussion of the isotherm models (see later in this paper). Figure 4a and b (D-PA) and Figure 4c and d (L-PA) show that, as the column temperature increases from 40 to 70 °C, the sample concentration in the stationary phase in equilibrium with a given mobile-phase concentration decreases. Both D-PA and L-PA show the same isotherm features. A close comparison of Figure 4a,b and 4c,d shows that the isotherms for L-PA are higher than those for D-PA. The isotherms of D-PA and L-PA at 40 °C are also compared in Figure 5. First, we see that, in equilibrium with the packing material imprinted for L-PA, the solid-phase concentration of L-PA is markedly higher than that of D-PA for any given liquidphase concentration, as indicated by the higher L-PA curve. Second, the distance between the two curves decreases with increasing concentration, indicating saturation of the enantioselective sites long before saturation of the nonselective sites. For example, at C ) 0.01 g/L, the relative difference of the amounts of the two enantiomers adsorbed at equilibrium is ∼50%; at C ) 1 g/L, it is only 8%. Finally, the density of the enantioselective
Figure 4. Experimental isotherm data obtained with column II and best curves obtained by fitting the data to the bilangmuir model. Symbols experimental data. Solid lines, best eight-parameter model; dashed lines, best six-parameter model; dotted lines, best four-parameter model: (a) Solute, D-PA, 40 (+) and 60 (]) °C. (b) Solute, D-PA, 50 (+) and 70 (4) °C. (c) Solute, L-PA, 40 (+) and 60 (4) °C. (d) Solute, L-PA, 50 (+) and 70 (4) °C. Table 2. Best Parameters for the Bilangmuir Models and Fitting Residuals (Column IIa) L-PA
T (°C)
a1
b1 (L/g)
D-PA
a2
b2 (L/g)
a1
b1 (L/g)
Eight-Parameter Model 120 ( 132 6.5 ( 0.1 0.37 ( 0.03 48 ( 27 4.41 ( 0.09 0.23 ( 0.03 26 ( 18 2.73 ( 0.03 0.07 ( 0.01 23 ( 14 2.06 ( 0.03 0.05 ( 0.02
40 50 60 70
8.2 ( 0.5 5.1 ( 0.2 3.2 ( 0.3 2.3 ( 0.1
0.59 ( 0.09 0.32 ( 0.06 0.13 ( 0.09 0.07 ( 0.05
7(4 3.7 ( 0.7 2.2 ( 0.5 1.0 ( 0.2
40 50 60 70
6.4 ( 0.2 4.35 ( 0.01 2.71 ( 0.09 2.02 ( 0.04
0.34 ( 0.05 0.20 ( 0.03 0.05 ( 0.04 0.01 ( 0.2
4.6 ( 0.6 3.0 ( 0.4 1.9 ( 0.2 0.92 ( 0.09
Six-Parameter Model 9(2 6.4 ( 0.2 10 ( 2 4.4 ( 0.1 6(1 2.71 ( 0.09 4.5 ( 0.8 2.02 ( 0.04
40 50 60 70
6.5 ( 0.1 4.54 ( 0.09 2.73 ( 0.05 2.02 ( 0.03
0.36 ( 0.03 0.24 ( 0.03 0.05 ( 0.02 0.01 ( 0.02
4.7 ( 0.5 4.4 ( 0.7 1.9 ( 0.2 0.93 ( 0.08
Four-Parameter Model 10 ( 2 6.5 ( 0.1 0.36 ( 0.03 26 ( 7 4.54 ( 0.09 0.24 ( 0.03 6(1 2.73 ( 0.05 0.05 ( 0.02 4.4 ( 0.8 2.02 ( 0.03 0.01 ( 0.02
a
0.34 ( 0.04 0.20 ( 0.03 0.05 ( 0.04 0.01 ( 0.2
fitting residuals a2
b2 (L/g)
L-PA
D-PA
2(5 1(6 1.2 ( 0.8 1(2
511 ( 2110 948 ( 6690 351 ( 386 1063 ( 6260
0.033 0.010 0.017 0.0044
0.013 0.013 0.0019 0.0035
2(4 1(4 0.6 ( 0.9 0.3 ( 0.6
262 ( 1160 542 ( 3210 118 ( 369 161 ( 668
0.047 0.020 0.022 0.0066
0.014 0.014 0.0026 0.0040
0.047 0.027 0.022 0.0067
0.017 0.022 0.0057 0.0044
0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
Column II was thermally treated (annealed) before packing.
sites in the imprinted polymer is much smaller than that of the nonselective ones, as shown by the data in Tables 2 and 4. (b) Interpretation of the Results. The bilangmuir isotherm model (eq 1) for two enantiomers has been discussed earlier in this paper. The experimental data obtained for the adsorption equilibrium isotherms were fitted to the three main models possible in the present case, the eight-, six-, and four-parameter
models. The results are reported in Tables 2 (column II, this work) and 3 (column I, data from a prior work which have been reevaluated using the same three models5). For further illustration, the three terms of the six-parameter model at 40 °C, the nonselective term, and the enantioselective terms for the two enantiomers are plotted in Figure 5. The curve corresponding to the nonselective term (solid line) is almost superimposed with Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
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Table 3. Best Parameters for the Bilangmuir Models and Fitting Residuals (column Ia) L-PA
D-PA
T (°C)
a1
b1 (L/g)
a2
40 50 60 70
5.6 ( 0.2 3.7 ( 0.1 2.63 ( 0.08 1.99 ( 0.04
0.35 ( 0.05 0.23 ( 0.04 0.15 ( 0.03 0.11 ( 0.02
10.9 ( 0.8 5.0 ( 0.4 2.7 ( 0.2 1.50 ( 0.09
40 50 60 70
5.1 ( 0.1 3.28 ( 0.08 2.29 ( 0.06 1.72 ( 0.05
0.27 ( 0.03 0.17 ( 0.03 0.10 ( 0.03 0.04 ( 0.03
40 50 60 70
5.08 ( 0.08 3.28 ( 0.06 2.32 ( 0.04 1.77 ( 0.02
0.28 ( 0.03 0.17 ( 0.02 0.10 ( 0.02 0.06 ( 0.02
a
b2 (L/g)
b1 (L/g)
fitting residuals a2
b2 (L/g)
L-PA
D-PA
Eight-Parameter Model 65 ( 11 4.60 ( 0.05 0.20 ( 0.01 41 ( 8 3.15 ( 0.03 0.16 ( 0.01 32 ( 6 2.33 ( 0.04 0.14 ( 0.02 29 ( 5 1.73 ( 0.06 0.07 ( 0.03
1.50 ( 0.07 0.83 ( 0.07 0.4 ( 0.3 0.16 ( 0.05
23 ( 4 41 ( 10 109 ( 186 10 ( 9
0.0088 0.0054 0.0025 0.0011
0.00044 0.00034 0.0018 0.00052
9.2 ( 0.7 4.0 ( 0.3 2.2 ( 0.2 1.18 ( 0.09
Six-Parameter Model 33 ( 4 5.1 ( 0.1 14 ( 2 3.28 ( 0.08 9(1 2.29 ( 0.06 6.3 ( 0.9 1.72 ( 0.05
3(2 1(1 0.5 ( 0.6 0.3 ( 0.2
427 ( 636 214 ( 443 125 ( 295 62 ( 85
0.020 0.019 0.012 0.0051
0.013 0.0065 0.0036 0.0020
9.3 ( 0.7 4.0 ( 0.3 2.1 ( 0.2 1.15 ( 0.08
Four-Parameter Model 33 ( 4 5.08 ( 0.08 0.28 ( 0.03 14 ( 2 3.28 ( 0.06 0.17 ( 0.02 9(1 2.32 ( 0.04 0.10 ( 0.02 6.4 ( 0.8 1.77 ( 0.02 0.06 ( 0.02
0.00 0.00 0.00 0.00
0.021 0.019 0.012 0.0059
0.018 0.011 0.0061 0.0023
a1
0.27 ( 0.03 0.17 ( 0.03 0.10 ( 0.03 0.04 ( 0.03
0.00 0.00 0.00 0.00
Column I was not thermally treated.
Figure 5. Comparison of the experimental isotherm data obtained with column II (symbols) and those calculated with the six-parameter bilangmuir model (lines) at 40 °C. D-PA (dashed-dotted line and diamonds), L-PA (dash-dot-dot line and cross symbols). The three different terms of the bilangmuir isotherms for the two enantiomers are also shown. Solid line: first term, common to the two enantiomers and corresponding to the contribution of the nonselective adsorption. Dotted line: second term, enantioselective adsorption of L-PA. Dashed line: third term, enantioselective adsorption for D-PA (see numerical coefficients of the isotherms in Table 2).
the bilangmuir isotherm for D-PA (dashed-dotted line), confirming that most of the interactions between D-PA and the imprinted polymer take place at the nonselective sites. The curve corresponding to the enantioselective term for L-PA (dotted line) is well above that for D-PA (dashed line), showing how much more strongly L-PA is absorbed than D-PA on the chiral selective sites of the polymer. The amount of D-PA adsorbed at equilibrium on the enantioselective sites is at least 4 times lower than that of L-PA at the lowest concentration (0.001 g/L) and at least 70 times lower at the highest concentrations (Figure 5). Quasi-saturation is reached at lower concentrations for L-PA than for D-PA because of the much larger value of b2 for this compound (Table 2). As discussed below, there might even be no adsorption of D-PA on the enantioselective sites. The fitting residuals are also given in Tables 2 and 3. Consideration of these residuals allows an answer to the two 934 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
important questions which arise, (1) which model best accounts for the adsorption data, and (2) are the sites imprinted for L-PA molecules accessible to those of D-PA? The first question amounts to asking whether an increase in the number of parameters of the model, which results in a lower total residual (i.e., the sum of the residuals for L-PA and D-PA) because of the increased flexibility, results in a significantly better fit of the data. If the eight-parameter model is the best, our general model of enantiomeric adsorption would not be satisfactory because the two types of sites would both exhibit some degree of enantioselectivity. This issue is easily settled by using proper statistical methods to analyze the fitting residuals obtained with the three different models. Statistics recognizes that increasing the number of parameters, i.e., of degrees of freedom, allows a higher flexibility of the model, hence lower residuals. The gain resulting from the use of a more sophisticated model is measured by the decrease of the residuals brought by increasing the number of parameters from four to six and eight. As seen in Table 2, this gain is small. To estimate whether there is a significant difference between the fittings of the data provided by the three models, statistics provides the F-test, based on the calculation of the ratio of the residuals of two models and its comparison with a threshold. In the case of the isotherms of D-, L-PA on the thermally treated column, the calculated ratios obtained for the different pairs of models are all smaller than the critical F-value from a standard F-distribution table. A comparison between the experimental results (symbols) and the best eight-, six-, and four-parameter models is shown in Figure 4, in which these three theoretical isotherms are represented by the solid, dashed, and dotted lines, respectively. All models fit the data well, but the more flexible eight-parameter model gives slightly better results. In conclusion, there is no significant difference between the quality of the three models or, in other words, that they all fit the data equally well, within the limits of the experimental errors, at the 95% confidence level. This result allows an answer to the second question, whether there should be any enantioselective sites for D-PA on an L-PA imprinted polymer or, rather, whether D-PA can be adsorbed on the L-PA selective sites. This assumption was tested by using the
Table 4. Saturation Capacities of the Two Materials Calculated from the Bilangmuir Isotherm Parameters qs1 ) a1/b1 (g/L) col I
qs2 ) a2/b2 (g/L) col II
T (°C)
L-PA
D-PA
L-PA
40 50 60 70
16.1 16.1 17.5 18.1
23.0 19.6 16.6 24.7
14.0 16.0 24.4 31.5
col I D-PA
col II
L-PA
Eight-Parameter Model 17.5 0.167 19.1 0.124 38.8 0.0857 44.3 0.0524
qs1 ) a1/b1 (g/L)
D-PA
L-PA
D-PA
0.0661 0.0202 0.00366 0.0154
0.0623 0.0773 0.0857 0.0439
0.00421 0.00121 0.00333 0.000482
qs2 ) a2/b2 (g/L) col I
T (°C)
col I
col II
40 50 60 70
18.8 19.3 23.9 47.4
18.8 22.0 55.5 219
Six-Parameter Model 0.280 0.282 0.244 0.189
40 50 60 70
18.2 19.3 22.5 28.3
18.1 22.0 51.6 200
Four-Parameter Model 0.278 0.282 0.237 0.181
col II
L-PA
four-parameter model (i.e., by assuming that a2 ) b2 ) 0 for D-PA, so that its isotherm consists of the nonselective term only). The coefficients of this model are also listed in Table 2. Obviously, the fitting residuals (L-PA plus D-PA) are higher for the fourparameter model than for the six-parameter one, but as explained above, the difference is not large enough to be significant. The F-test gives a ratio that is smaller than the critical F-value, meaning that the four- and the six-parameter models fit the data equally well, within the limits of the experimental errors. This conclusion agrees with the conclusion derived from Figure 5, that the amount of D-PA adsorbed on the enantioselective sites is very small at best. Note that the use of a four-parameter model modifies only the adsorption model for D-PA. As a result, the isotherm of L-PA is barely changed and the fitting residuals of L-PA are almost the same for the six- and the four-parameter models (see Tables 2 and 3). Faced with three different versions of the same model, which fit the data equally well at all temperatures, we ought to chose the simplest. This suggests that the enantioselective adsorption of D-PA on a polymer imprinted with L-PA is probably negligible while the nonselective adsorption of the two enantiomers is the same. There must be few if any enantioselective imprinted sites in this polymer able to adsorb both L- or D-PA, albeit with a different free energy. These sites adsorb only L-PA; the stationary phase behaves as it was intended to. This conclusion is somewhat at variance with the one reached in our previous work,5 in which the experimental data were fitted only to the eight-parameter model. We revisit this issue in the next section. Comparison of the Thermally Treated and the Untreated Column in the Thermodynamic Study. In view of the conclusion reached above regarding the selection of the best isotherm model for the experimental isotherm data on column II, it was worthwhile to reevaluate our earlier isotherm data on column I. We fitted the experimental data on column I at four temperatures to all three models: the eight-parameter, six-parameter, and four-
D-PA
L-PA
D-PA
0.00653 0.00419 0.00390 0.00463
0.521 0.307 0.324 0.206
0.00653 0.00182 0.00481 0.00164
0 0 0 0
0.465 0.307 0.311 0.209
0 0 0 0
parameter models and calculated the best values of the coefficients and the fitting residuals. They are listed in Table 3 and compared to those obtained for column II (Table 2). The F-test results show, that in this case, as in the previous one, the six- and four-parameter models fit the data with no significant difference at the 95% confidence level. As a matter of fact, the residuals are nearly the same for both columns, with each of the two models. However, this is not so for the eight-parameter model, which gives a lower (3-fold) residual. This result can be interpreted in two ways. We may conclude that the raw packing material contains two different types of sites, both with some, albeit different, enantioselectivity and that the enantioselectivity of one of these types of sites disappears during annealing at moderate temperatures (we know that all enantioselectivity is erased by annealing at 160 °C or above). Alternately, we may conclude that the surprisingly low residuals obtained with the eight-parameter model is a fluke. More experimental data are needed to reach a definitive conclusion. Other comparisons between the performance of the two columns are interesting. Tables 4 and 5 compare the saturation capacities and the separation factors obtained with column I (the untreated column studied previously5) and column II (the thermally treated column studied in this work), respectively. The saturation capacities were calculated from the bilangmuir isotherm coefficients (eight-, six-, and four-parameter models) at each temperature, for the two imprinted polymeric phases, qs1 ) (a1/ b1) for the nonselective sites and qs2 ) (a2/b2) for the enantioselective sites. The isotherm parameters of column I are listed in Table 3. From these data, we see first that the values of the saturation capacity obtained with the six- and the four-parameter models are in good agreement and vary regularly with temperature. The saturation capacity of the nonselective sites (qs1) increases with increasing temperature while that of the enantioselective sites (qs2) decreases. Thus, increasing the column temperature is not favorable for the chiral separation at the preparative level (at least Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
935
Table 5. Separation Factors on the Two Materials Calculated from the Bilangmuir Isotherm Parameters R1 ) a1L/a1D
R2 ) a2L/a2D
T (°C)
col I
col II
40 50 60 70
1.22 1.18 1.13 1.15
40 50 60 70
1.00 1.00 1.00 1.00
col I
R ) (a1L + a2L)/(a1D + a2D) col II
col I
col II
1.27 1.15 1.16 1.12
Eight-Parameter Model 7.27 3.48 6.09 3.25 6.77 1.89 9.62 1.97
2.71 2.21 1.95 1.85
1.82 1.59 1.38 1.29
1.00 1.00 1.00 1.00
Six-Parameter Model 3.31 2.71 4.48 3.04 4.43 3.31 4.08 3.48
1.82 1.75 1.60 1.44
1.36 1.38 1.40 1.29
R1 ) a1L/a1D T (°C)
col I
40 50 60 70
1.00 1.00 1.00 1.00
R ) (a1L + a2L)/(a1D + a2D) col II Four-Parameter Model 1.00 1.00 1.00 1.00
col I
col II
2.82 2.22 1.91 1.65
1.73 1.69 1.69 1.46
with this mobile-phase). Second, by contrast, the sets of values of the four saturation capacities obtained for each column with the eight-parameter model are much less consistent, especially for the enantioselective contributions. This is explained by the difficulty of the calculation. Experimental data are always marred by random errors. In the present case, we need to carry out determination of isotherm data in a huge concentration range. Accurate determinations of the four bilangmuir parameters require measurements both at high concentrations, such that b2C is comparable to 1 (for the accurate estimation of b2), and at low concentrations, such that b1C be smaller than 0.01 (for the accurate estimation of a1). Since the ratio b2/b1 is between 100 and 200, this would require a concentration range exceeding 10 000. This is not uncommon in the investigation of chiral stationary phases.14 The concentration ratio achieved here, 2000, was imposed by the rather high detection limit of D-, L-PA and could not be improved much. The number of data points appears to be insufficient under these conditions for the accurate estimate of eight parameters by fitting of the experimental data. This also justifies the preference of the fourparameter model even for the data obtained with column I (Table 3). Third, we observe that all the values of the saturation capacities of L-PA are higher on column II than on column I (Table 4). Thus, column II would give a higher production rate in preparative chromatography than column I. Finally, the saturation capacity for column II, qs1, seems to increase dramatically at 70 °C. However, this abnormal variation is probably explained by the rapid decline in the value of the coefficient b1 (Table 2). The product b1C becomes lower than 0.10 and, with a maximum concentration of 1 g/L in the mobile-phase, the isotherm is acquired in a region in which its deviation from linear behavior is slight. Accordingly, the error made on the determination of the saturation capacity is so important as to make the value obtained meaningless above 55 °C.
Table 5 shows a comparison of the separation factors for the two columns. R1 (a1L/a1D) and R2 (a2L/a2D) are defined as the separation factors for the nonselective and the enantioselective sites, respectively, while R is the apparent separation factor. In compliance with the features of the model, the separation factor for the nonselective sites on both columns should be unity (R1 ) 1). It is different from 1 with the eight-parameter model but the difference remains small, even for column I, and does not appear to be significant. On the other hand, R2 is different on column I and column II. It is also a function of the column temperature. For both columns, it increases when the temperature increases from 40 and 70 °C, except for column I at 70 °C. On the average, R2 is smaller for column II than for column I. Thus, the apparent separation factor is lower for column II than for column I. It seems to decrease slightly with increasing temperature for both columns. Kinetic Study. Peak broadening in chromatographic separations on imprinted polymers was already observed and attributed to slow mass-transfer4 and/or overloading of imprinted binding sites.2 As in a previous study,5 a lumped kinetic model, the solidfilm linear driving force model, was used to model this kinetics.6 This simple model includes the differential mass balance equation6 of the compound studied and a concentration-related kinetic equation (eq 2). We made the simplifying assumption of lumping all the contributions due to nonequilibrium in the column into a single parameter, the mass-transfer rate coefficient. The frontal analysis breakthrough curves were transferred from the data station of the HP1090 to a PC computer and fitted to a calculated profile. Fitting was accomplished by adjusting the rate coefficient kf. The result cannot be precise. Panels a and b of Figures 6 show plots of the rate coefficients of D-PA and L-PA, respectively versus the concentration (logarithmic scale). For both enantiomers, the mass-transfer rate coefficient increases with increasing temperature and with increasing concentration. This effect is most probably explained by surface diffusion.15
(14) Fornstedt, T.; Sajonz, P.; Guiochon, G. J. Am. Chem. Soc. 1997, 119, 1254.
(15) Miyabe, K.; Guiochon, G. Anal. Chem. 1999, 71, 889-896.
936 Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
Table 6. Mass-Transfer Rate Constants kf for D-PA mass-transfer rate constant (min-1) concn step (g/L)
Figure 6. Plot of the mass-transfer rate coefficient kf versus the sample concentration: 40 (2), 50 (b), 60 (1), and 70 °C (9). (a) Solute, D-PA. (b) Solute, L-PA.
The data used to draw parts a and b of Figures 3 were taken from Table 6a and b (column II), although a few of these data are not reported in the figures, because of overlap. For example, kf for the 0-0.005 g/L range was not included in the figure because there is a data point for the range 0.004-0.006 g/L. We note that these two values of kf are quite different, at 35 and 65 min-1, respectively (Table 6a, column II). The size of the concentration step seems to play an important role in the determination of the mass-transfer rate coefficient. In this work, there is a systematic overlap of the frontal analysis data acquired (see Experimental Section). The averaging process involved in the determination of the rate coefficient in the case of steps of large relative importance is not yet fully understood. The values of the mass-transfer rate coefficient explain some of the characteristics of the imprinted stationary phase. As discussed before, the separation factor on column II increases slightly with increasing column temperature (Table 5) although the resolution tends to decrease. At 40 °C, the mass-transfer rate coefficient (kf) is substantially higher for D-PA than for L-PA. As the temperature increases, both rate coefficients increase and the difference between the rate coefficients of D-PA and L-PA decreases. This effect is insufficient, however, to compensate for the decrease of the separation factor on column II and the resolution decreases. A similar effect was found in the case of column I. The drop in the separation factor with increasing
T ) 40 °C
T ) 50 °C
T ) 60 °C
T ) 70 °C
col I col II col I col II col I col II col I col II
Cn
Cn+1
0.0 0.0005 0.001 0.002 0.004 0.006 0.008 0.0 0.005 0.01 0.02 0.04 0.06 0.08 0.0 0.05 0.1 0.2 0.4 0.6 0.8
0.0005 0.001 0.002 0.004 0.006 0.008 0.01 0.005 0.01 0.02 0.04 0.06 0.08 0.1 0.05 0.1 0.2 0.4 0.6 0.8 1.0
35 45 45 45 45 50 50 50 60 60 60 60 60 60 40 80 80 90 100 80 80
0.0 0.0005 0.001 0.002 0.004 0.006 0.008 0.0 0.005 0.01 0.02 0.04 0.06 0.08 0.0 0.05 0.1 0.2 0.4 0.6 0.8
0.0005 0.001 0.002 0.004 0.006 0.008 0.01 0.005 0.01 0.02 0.04 0.06 0.08 0.1 0.05 0.1 0.2 0.4 0.6 0.8 1.0
8 10 13 15 17 19 20 25 30 40 50 60 70 80 20 80 100 110 110 100 70
For D-PA 45 30 45 60 50 60 50 60 50 70 50 70 50 70 55 40 60 85 60 85 60 95 60 95 65 95 65 95 60 45 80 90 90 90 90 110 100 110 80 110 80 110
50 50 50 55 55 55 55 55 60 60 60 60 65 65 60 80 90 90 100 80 80
For L-PA 10 12 75 17 35 30 25 35 30 30 35 30 30 35 30 30 35 40 40 50 40 50 55 40 50 55 50 50 65 55 50 70 60 60 75 65 60 80 15 30 65 68 70 80 70 90 100 68 90 90 68 90 85 70 80 85 85 60 100
15 15 20 30 30 35 40 35 50 50 50 60 70 70 40 80 85 90 90 80 70
25 45 53 58 65 65 65 35 75 75 83 83 80 80 40 80 80 100 100 100 100
35 70 70 70 80 80 80 25 95 95 105 105 105 105 45 110 110 120 120 120 120
50 55 55 55 55 55 55 55 60 60 60 60 60 60 60 80 90 90 100 80 80
45 80 80 80 90 90 90 30 105 105 115 115 115 115 40 120 120 130 125 120 105
53 53 53 53 58 60 70 70 80 80 80 90 75 100 110 110 115 115 120
15 20 30 30 35 35 35 35 50 50 50 55 60 60 50 80 100 90 80 90 80
45 65 65 65 65 65 80 80 90 90 90 100 85 110 120 120 125 125 130
temperature could not be compensated by the acceleration of mass-transfer and the resolution decreases despite an improved column efficiency. Comparison of the Thermally Treated and the Untreated Columns in the Kinetic Study. The mass-transfer rate coefficients on both columns are listed in Table 6a and b for column I (the untreated column) and column II (the thermally treated column). For D-PA (see Table 6a), the rate constants on column II are, on the average, higher than that on column I. As the temperature increases from 40 °C to 70 °C, the rate coefficients of mass transfer on column II increase (Figure 6) and they do it faster than those on column I (Table 6). In fact, the mass-transfer rate coefficients were nearly independent of the temperature for column I and for both enantiomers. When the concentration increased from 0.0005 to 0.8 g/L, kf for D-PA approximately doubled on column I while its extent of variation on column II in the same concentration range decreased with increasing temperature, from 4-fold at 40 °C to 2-fold at 70 °C. For L-PA, the Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
937
corresponding figures are a 5-6-fold increase on column I (with a slight temperature dependence) and, on column II, a negligible variation at 40 °C and 3-fold increase at 70 °C. These results indicate that the column temperature has a rather moderate influence on the mass-transfer kinetics. These results suggest that the thermal treatment markedly accelerates the kinetics of mass-transfer, especially at low concentrations. Not surprisingly, this effect is much more important for the L-enantiomer. Note that, in this work, we have defined and measured a lumped coefficient of mass-transfer resistances that includes the individual contributions of the mass-transfer kinetics on both the enantioselective and the nonselective sites. The case discussed here involves rather an heterogeneous masstransfer kinetics,16 with, most probably, a marked difference for these two kinetics. CONCLUSION Once more, the bilangmuir isotherm model proved to be suitable for the study of a new chiral separation, that of L- and D-PA on a polymeric imprinted stationary phase.6,10 It describes accurately the thermodynamic behavior of the two enantiomers. This agreement allowed a comparative evaluation of the qualities of different imprinted polymers for the separation of enantiomers and, more specifically, a discussion of the suitability of a dedicated thermal treatment of the polymer prior to its use. On the fundamental front, we were able to conclude that the enantioselective sites of the L-PA imprinted polymer do not seem to interact much, if at all, with the molecules of D-PA. The conditions under which this conclusion was reached suggests that, if it may not be general for imprinted chiral phases, this result is not exceptional. On the more specific problem studied, we showed that the heat treatment imparts higher sample load capacities to the (16) Gotmar, G.; Fornstedt, T.; Guiochon, G. J. Chromatogr., A, in press.
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Analytical Chemistry, Vol. 71, No. 5, March 1, 1999
polymer, indicating that this treatment may be beneficial for the preparative applications of these materials. The kinetics of masstransfer was found to be somewhat faster after annealing, especially at low concentrations. The values obtained for the rate coefficients of mass-transfer showed a strong dependence on the solute concentration in the mobile-phase, which confirms a trend observed previously in all cases studied. The slight acceleration of the mass-transfer kinetics was insufficient, however, to compensate for the decrease in the separation factor upon heat treatment. The net result was a decrease in the enantiomeric resolution. Finally, it is worth noting that, if the mass-transfer kinetics was only moderately improved, the thermal treatment markedly improved the long-term thermal stability of the polymer. The performance of column II drifted less than that of column I during the investigations carried out at temperatures above 50 °C. Forthcoming publications will report on the thermodynamics and mass-transfer kinetics of imprinted polymers with different morphology and of such polymers subjected to chemical posttreatments. This work is important for gaining a better understanding of the recognition process available in these polymers and will allow the optimization of their properties for various uses in separations, assay monitoring, or chemical sensors. ACKNOWLEDGMENT This work was supported in part by Grant CHE-9701680 from the National Science Foundation and by the cooperative agreement between the University of Tennessee and Oak Ridge Laboratory. B.S. thanks Hewlett-Packard (Waldbronn, Germany) for providing the HP 1050 system. Received for review December 22, 1998. AC981154O
October
20,
1998.
Accepted
