Relationship between Retention and Effective Selector Concentration

(4) Rogan, M. M.; Altria, K. D.; Goodall, D. M. Chirality 1994, 6, 25-40. (5) Lloyd, D. K.; Li, S.; Ryan, P. J. Chromatogr. A 1995, 694, 285-296. (6) ...
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Correspondence Anal. Chem. 1996, 68, 3270-3273

Relationship between Retention and Effective Selector Concentration in Affinity Capillary Electrophoresis and High-Performance Liquid Chromatography Abdelwahab Ahmed, Houda Ibrahim,† Florent Pastore´,‡ and David K. Lloyd*

Meakins-Christie Laboratories, McGill University, 3626 St. Urbain, Montreal, PQ, H2X 2P2, Canada

Many chiral selectors are used in both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). One would expect their behavior in both techniques to be similar, since the underlying molecular interactions should be the same, and to a great extent this is the case. However, our work with human serum albumin as a buffer additive in CE has shown that compounds separated by this technique cannot always be separated by HPLC with a human serum albumin chiral stationary phase, and vice versa. In this paper, results are presented which suggest that these discrepancies can be understood if one considers the concentration of protein present in the column in both separation systems. Human serum albumin is usually used at tens of micromolar concentrations in CE, while the effective concentration of the immobilized protein in a HPLC column may approach millimolar levels. This disparity in protein concentration is shown to be reflected in the capacity factors for a test solute (benzoin) in a variety of mobile phases/background electrolytes. Although this effect of selector concentration is demonstrated for a protein chiral selector it should be generally observed when the same phase (not necessarily chiral) is used in HPLC or CE. Human serum albumin (HSA) is an acidic, water-soluble protein, MW ≈ 66 000, which acts as a transport protein in vivo.1 It has been used to develop chiral stationary phases in highperformance liquid chromatography (HPLC).2 Capillary electrophoresis (CE) has recently become popular for performing chiral separations,3,4 using a variety of different chiral selectors. Proteins such as albumin (human and bovine) have been used as chiral selectors in what has been called affinity capillary electrophoresis, with the protein dissolved in solution in the CE background † Current address: Advanced Separation Technologies, Inc., 37 Leslie Court, P.O. Box 297, Whippany, NJ 07981. ‡ On leave from Ecole Nationale Supe ´ rieure de Chimie de Paris, 11, rue Pierre et Marie Curie, F-75231, Paris Cedex 05, France. (1) Peters, T. Adv. Protein Chem. 1985, 37, 161-245. (2) Domenici, E.; Bertucci, C.; Salvadori, P.; Fe´lix, G.; Cahagne, S.; Motellier, S.; Wainer, I. W. Chromatographia 1989, 29, 170-176. (3) Terabe, S.; Otsuka, K.; Nishi, H. J. Chromatogr. A 1994, 666, 295-319. (4) Rogan, M. M.; Altria, K. D.; Goodall, D. M. Chirality 1994, 6, 25-40.

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electrolyte (BGE). Such separations have recently been reviewed.5 It has been shown that as well as performing chiral separations, one can also determine binding constants between proteins and nonchiral6 or chiral7 analytes and investigate the effect of displacers.7 Our earlier studies were carried out with benzoin (a neutral test analyte) and promethazine and propiomazine (basic solutes). Despite good enantioresolution in CE, we could not achieve separations of all of these compounds using a commercial HPLC HSA-chiral stationary phase (CSP). Furthermore, attempts to separate a variety of compounds separable with a HSA-CSP by CE using HSA as a BGE additive were rarely successful. Similarly, Valtcheva et al.8 have reported separations using cellobiohydrolase I in CE which they could not repeat using the immobilized protein in HPLC. This is somewhat surprising, since retention on proteins in CE and HPLC is expected to be via relatively well-defined binding mechanisms which, given certain constraints of allowable operating parameters, should be the same in both separation techniques. Indeed, similarities have been demonstrated for other systems, such as chiral resolution using cyclodextrins in CE and HPLC.9,10 There could be a variety of reasons for the observed differences between our CE and HPLC results with HSA. The properties of HSA used in the CE and HPLC systems may not be the same due to changes induced during immobilization, although some commercially prepared columns have been shown in general to conserve the protein’s native binding properties.11 In CE, the analyte and protein mobilities may in some cases be too similar to achieve a separation, but this does not always explain why separations achieved by CE could not be repeated in HPLC (although the great improvement in efficiency in CE is the (5) Lloyd, D. K.; Li, S.; Ryan, P. J. Chromatogr. A 1995, 694, 285-296. (6) Gomez, F. A.; Avila, L. Z.; Chu, Y. H.; Whitesides, G. M. Anal. Chem. 1994, 66, 1785-1791. (7) Lloyd, D. K.; Li, S.; Ryan, P. Chirality 1994, 6, 230-238. (8) Valtcheva, L.; Mohammed, J.; Pettersson, G.; Hjerten, S. J. Chromatogr. 1993, 638, 263-267. (9) Penn, S. G.; Liu, G.; Bergstro¨m, E. T.; Goodall, D. M.; Loran, J. S. J. Chromatogr. 1994, 680, 147-155. (10) Piperaki, S.; Penn, S. G.; Goodall, D. M. J. Chromatogr. A 1995, 700, 5967. (11) Domenici, E.; Bertucci, C.; Salvadori, P.; Wainer, I. W. J. Pharm. Sci. 1991, 80, 164-166. S0003-2700(96)00373-3 CCC: $12.00

© 1996 American Chemical Society

explanation in some cases). Another possibility is that the preparation of the HSA was not the same; this has a significant effect on the protein’s properties1 and may complicate comparisons between CE and HPLC results, and this is difficult to control if one is using commercial columns. Consequently, we decided to make our own HSA-CSP to perform chiral HPLC separations and to use the HSA left over from the column preparation to perform CE measurements. In this way the source and preparation of the protein would be eliminated as a variable, and we would be able to directly compare the results obtained by each technique. In this study, benzoin was used as a test analyte, and its retention was measured in terms of capacity factors in both CE and HPLC. Differences in retention are explained in terms of the effective protein concentration in each system. EXPERIMENTAL SECTION Capillary Electrophoresis. Separations of benzoin enantiomers were performed using CE with 1-propanol as an additive to the background electrolyte. These results have been presented elsewhere, as part of a study using a variety of organic modifiers,12 so only brief experimental details are given here. An Applied Biosystems (Foster City, CA) Model 270A-HT capillary electrophoresis system was used, with UV absorbance detection at 247 nm and a 0.5 s rise time. Fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) were 72 cm long × 50 µm internal diameter, 365 µm external diameter, 50 cm long to the detector. Between runs the capillary was rinsed with 0.1 M NaOH, water, and run buffer for 2 min each, using a vacuum of ∼70 kPa applied to the capillary outlet. Separations were performed at +30 kV (current 50-60 µA), at an oven temperature of 30 °C. Samples were introduced hydrodynamically for a time of 1.0 s using a vacuum of 17 kPa. Immediately before the sample, an equivalent volume of water was injected to give a more distinctive electroosmotic flow marker peak. Phosphate buffer was prepared by mixing 100 mM aqueous solutions of analytical grade dibasic sodium hydrogen phosphate and monobasic sodium hydrogen phosphate (BDH, Toronto, Canada) to give pH 7.0. An appropriate amount of HSA was dissolved in the 100 mM sodium phosphate buffer to obtain a 70 µM solution of HSA. Running buffer was prepared by dilution of the above solution with appropriate amounts of water and organic modifier to give a BGE containing 50 mM phosphate buffer, 35 µM HSA, and the desired volume percentage of organic modifiers. Racemic benzoin analyte (100 µM) was dissolved in water/ methanol (99:1). Liquid Chromatography. The stationary phase was prepared using Excil silica (3 µm particles, 8 nm pore size), obtained from CSC (St. Laurent, PQ, Canada). Immobilization of HSA followed the in situ procedure of Fe´lix and Liu.13 In brief, epoxy silica was prepared by refluxing activated silica with (glicidoxypropyl)trimethoxysilane in xylene for 36 h under anhydrous conditions. After washing, the epoxy silica was packed into a 50 × 4.6 mm stainless steel column using a slurry packer operating at 5000 psi with hexane as the packing solvent. The column was then dried, and HSA was immobilized by recirculating a 10 mg mL-1 solution of HSA in 50 mM pH 7.5 potassium phosphate buffer through the column. By measuring photometrically the starting and finishing concentrations of HSA in the immobilization buffer, the (12) Ahmed, A.; Lloyd, D. K., submitted to J. Chromatogr. (13) Fe´lix, G.; Liu, M. Bio-Sciences 1989, 8, 2-6.

amount of protein immobilized on the column was found to be 47 mg of HSA/1 g of silica. Unreacted epoxy groups were blocked with glycine, and the column was then rinsed with mobile phase before use. The column was tested with compounds known to be resolved (enantiomers of temazepam and ketorolac were successfully separated) to check that the column was active. Some packing was then removed for making packed capillaries, and the remainder was then packed into a 30 × 4.6 mm column using a pressure of ∼2000 psi with a phosphate buffer/ethanol (90:10) packing solvent. This was the column used for the k′ measurements described here. Chromatography was performed using an ABI Spectroflow 400 analytical pump with a flow rate of 0.8 mL min-1 and a SpectraPhysics Model 100 UV absorbance detector operated at 240 nm with a 1 s time constant. Injections were 20 µL, and data were collected using a Spectra-Physics DataJet integrator and stored on a personal computer running Spectra-Physics Winner system software. Mobile phases were made up using 50 mM sodium phosphate buffer, pH 7, with varying volume percentages of 1-propanol. The column was held at a temperature of 30 °C by use of a water jacket. RESULTS AND DISCUSSION Provided that the underlying interactions between the protein and analyte remain the same in both CE and HPLC, the amount of free and protein-bound analyte in the column should be related by the equilibrium binding constant,

K ) [AP]/[A][P]

(1)

where [A] and [AP] are the free and protein-bound analyte concentrations and [P] is the free protein concentration. The binding constant can be related to the capacity factor, k′, using the expression well-known in affinity chromatography14

k′ ) KmP/V

(2)

where mP is the number of moles of protein binding sites available in the column and V is the column void volume. mP/V is, of course, simply the effective molar concentration of available binding sites within the column. In HPLC, k′ may be calculated from the elution time of the analyte and the column dead time. In CE, the capacity factor is defined in terms of the motions of the free analyte and the analyte-selector complex15 and can be calculated from their effective mobilities, using the expression16

k′ ) (µeff - µ0)/(µcomp - µeff)

(3)

where µ0 is the effective mobility of the free analyte (no protein present in the BGE), µcomp is the effective mobility of the analyteprotein complex, and µeff is the effective mobility of the analyte measured in the presence of the protein in the BGE. k′ can be a little more difficult to calculate in CE than in HPLC, since µcomp may not be equal to the effective mobility of the free protein, except when small, neutral analytes are bound, and all mobilities will vary when the composition of the BGE is changed.3,12 In the present case, benzoin is a small, neutral molecule and so one can safely assume that µcomp is equal to the mobility of the free protein. (14) Walters, R. R. In Analytical Affinity Chromatography; Chaiken, I. M., Ed.; CRC Press: Boca Raton, FL, 1981; Chapter 3. (15) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 113-116. (16) Khaledi, M. G.; Smith, S. C.; Strasters, J. K. Anal. Chem. 1991, 63, 18201830.

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Table 1. k′ as a Function of Percent (v/v) 1-Propanol Measured in HPLC and CEa

a

Figure 1. Analysis of benzoin using (A) CE, applied voltage, +30 kV, capillary 72 cm long, 50 cm to detector (50 µm I.D.) and (B) HPLC, column 50 × 4.6 mm HSA, flow rate, 0.8 mL min-1. Mobile phase/ background electrolyte, 50 mM phosphate, pH 7/1-propanol (99.5: 0.5). Other conditions as in text.

Calculation of k′ is further simplified in this case, since µ0 ) 0. Another complicating factor in CE could be binding of the analyte to protein adhered to the capillary surface; we have previously shown this to be a minor effect at the concentrations of protein additive used here.7 If the underlying interactions are the same in both HPLC and CE, one would expect K to be equal in both cases when the separation conditions are the same. The capacity factors in both techniques should be related by eq 2, where mP/V is replaced in CE simply by the concentration of protein added to the BGE. Thus if k′ is measured under a variety of identical conditions in CE and HPLC, a plot of k′HPLC vs k′CE should be a straight line, with a slope equal to the ratio of the concentrations of the protein in each system. We have recently demonstrated the effect of a variety of organic modifiers on the retention of benzoin enantiomers in CE, using HSA as a chiral selector.12 The data presented for benzoin with 0, 0.5, 1, 1.5, and 2% (v/v) 1-propanol as an organic modifier were used for comparison with HPLC measurements made under the same conditions. An electropherogram and a chromatogram of benzoin with 0.5% 1-propanol are shown in Figure 1. Clearly there is excellent resolution of the enantiomers by CE, while in HPLC there is just a slight indication of some separation. Capacity factors for benzoin in both systems are shown in Table 1, as a function of added 1-propanol (the CE values are taken as the average of the k′ for the two enantiomers). In HPLC, k′ was measured at various analyte concentrations and flow rates with consistant results, indicating that overloading was not occurring. In Figure 2, a plot of k′HPLC vs k′CE is shown. Clearly the variation 3272 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996

% (v/v) 1-propanol

k′(HPLC)

k′(CE)

0 0.5 1 1.5 2

5.58 4.42 3.65 3.31 3.24

0.196 0.124 0.106 0.089 0.086

Experimental conditions in text.

Figure 2. Plot of k′ determined by HPLC vs k′ as determined by CE. The error bars represent the standard deviation of six measurements (HPLC) or triplicate measurements (CE). The line is a linear regression fit to the data.

of k′ as a function of added 1-propanol is nonlinear in both CE and HPLC (Table 1), so the linear relationship between k′ measured in each system as illustrated in Figure 1 is unlikely to be a chance occurrence. The slope is 21 ( 2 and the intercept on the k′HPLC axis is 1.5 ( 0.3, with r2 ) 0.970. A complicating factor in HPLC is nonspecific retention of the analyte on the support. To see whether this was likely to be very significant, k′ for benzoin was measured by packed-capillary chromatography using the epoxy silica support. It was found that there was some retention for benzoin under a variety of mobile phase conditions with k′ generally ∼0.5 and that k′ was little affected by the proportion of organic modifier in the mobile phase. Thus it seems that retention on the support may be in part responsible for the nonzero intercept in Figure 2. From the slope of the line the relative concentration of the protein in the two systems can be determined to be 21:1; i.e., since the concentration of HSA in the CE experiments was 35 µM, this suggests that the effective HSA concentration in the HPLC experiments is 0.73 mM. This can be compared to the 17.4 mg of HSA known to be immobilized in the column (calculated from the known amount of packing in the column, and the measured amount of HSA immobilized on the packing, as described in the Experimental Section). The void volume is 0.41 mL; and thus the effective protein concentration in the HPLC column can be calculated to be 0.64 mM, in reasonable agreement with the value calculated from the k′ data. Calculation of K from the above CE measurements gives values similar to those previously determined.7 These results allow us to explain why somewhat different operating conditions are generally used in HPLC and CE with

protein selectors. HPLC measurements with HSA and other protein CSPs are typically performed with several percent of organic modifier in the mobile phase; k′ would be unacceptably high for typical solutes with K ) 103-105 in plain buffers and concentrations of immobilized protein approaching 1 mM. The modifier is needed to reduce K and thus k′ to an acceptable range. Conversely, in CE, the use of unmodified buffers is common, and this can be understood when one realizes that, with the low concentration of protein typically used, k′ will be low and its further diminution with organic modifiers is not necessary. One should also note that with protein chiral selectors there may be more than one binding process by which the analyte is retained and that, for example, on addition of organic modifier enantioselectivity will be lost long before all retention is lost because the modifier affects the two processes differently.12 This may explain why selectivity observed in CE may not always be reproduced in HPLC, since obtention of optimum k′ values will require different amounts of modifier. Of course, other factors are also important, particularly the efficiency. In Figure 1, N for the CE separation is >105, while for HPLC, N < 400. If one calculates the minimum efficiency required, Nreq, to achieve the separation under these

conditions, using the expression17

(17) Riley, C. M. In High Performance Liquid Chromatography: Fundamental Principles and Practice; Lough, W. J., Wainer, I. W., Eds.; Blackie: Glasgow, 1996; Chapter 2. (18) Ferguson, P. D.; Goodall, D. M.; Loran, J. S. 8th Int. Symp. High Perf. Capillary Electrophoresis, Orlando, FL, Jan 1996; Abstr. P121. Ferguson, P. D.; Goodall, D. M.; Loran, J. S. J. Chromatogr., in press.

Received for review April 17, 1996. Accepted June 24, 1996.X

Nreq ) {6[R/(R - 1)][(k′ + 1)/k′]}2

(4)

with R ) 1.23 (enantioselectivity measured by CE) and k′ ) 5.05 (HPLC retention measured with that column), one finds that Nreq ≈ 1500. Therefore it is hardly surprising that no enantioresolution is observed for benzoin in HPLC, given the low efficiency characteristic of the HSA phase. These measurements illustrate that, although one should in principle be able to extrapolate between measurements made in CE and HPLC, practical limitations on the operating parameters may in practice present difficulties in doing this when using protein phases. Selectors such as cyclodextrins with less complex binding mechanisms may allow for more general comparisons between HPLC and CE, and a quantitative description of retention using cyclodextrins similar to that described here has recently been reported.18 ACKNOWLEDGMENT This work was funded by the Natural Sciences and Engineering Research Council of Canada. D.K.L. is recipient of a chercheurboursier award from Fonds de la Recherche en Sante´ du Que´bec.

AC960373B X

Abstract published in Advance ACS Abstracts, August 1, 1996.

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