Anal. Chem. 1992, 84, 2872-2874
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TECHNICAL NOTES
Protein-Based Capillary Affinity Gel Electrophoresis for the Separation of Optical Isomers Staffan Birnbaumt and Staffan Nilsson' Department of Pure and Applied Biochemistry and Department of Technical Analytical Chemistry, P.O.Box 124, University of Lund, Lund S-22100, Sweden
INTRODUCTION Chiral separation and synthesis are currently of utmost concern, particularly for the preparation and analysis of pharmaceutical compounds. Biological receptors, often the targets of these compounds, discriminate between the enantiomers and thus the optical isomers of a racemic drug will often exhibit completely different pharmacological effects. Methods for chiral separation and analysisinclude liquid (LC), gas (GC) as well as supercritical fluid (SFC) chromatography in which the chiral selector has either been used in conjunction withthe mobile or stationary phase. Among the various chiral stationary phases (CSP) investigated, immobilized protein phases such as ovomucoid, a-acid glycoprotein, as well as bovine serum albumin (BSA) and its proteolytic peptide fragment have been used.1-4 The efficiencyof the BSA-based LC methods has, however, been relatively poor.5 In addition, the large size of the protein chiral selector limits the capacity of the stationary phase. Capillary electrophoresis (CE) is a technique which has developed considerably during the past years.6-* CE distinguishes itself from other liquid-phase separation methods in that extremely high efficiency is obtained. Furthermore, CE can be viewed as a microanalytical procedure and is advantageous when, for example, the availability of sample, mobile phase, or separation phase are limited. In order to circumvent the low efficiency displayed by protein-based liquid chromatography separation of enantiomers, we have investigated CE as a means of achieving enhanced theoretical plate number characteristics for proteinbased chiral separation. Though we solely report here the use of BSA as chiral selector, we are firmly convinced that the method is generally applicable for other protein-based affinity separations using capillary electrophoresis. In addition, the method used here to prepare the separation phase involves no inert carrier support; only the chiral selector BSA cross-linked with glutaraldehyde is employed, thus maximizing the potential capacity of the separation phase. In this note, we describe the preparation, utilization, and performance characteristics of high-performance electro-
* T o whom correspondence should be addressed at the Department
of Technical Analytical Chemistry. t Department of Pure and Applied Biochemistry. (1) Kirkland, K. M.; Neilson, K. L.; McCombs, D. A. J. Chromatogr. 1991,545,43-58. (2) Hermansson, J. Trends Anal. Chem. 1989,8, 251-259. (3) Andersson, S.; Allenmark, S.; Erlandsson, P.; Nilsson, S. J. Chromatogr. 1990,498, 81-91. (4) Erlandsson, P.; Nilsson, S. J . Chromatogr. 1989, 482, 35-51. (5) Erlandsson, P.; Hansson, L.; Isaksson, R. J. Chromatogr. 1986, 370,475-483. (6)Hjbten, S. J. Chromatogr. 1983, 270, 1-6. (7) Karger, B. L.; Cohen, A. S.;Guttman, A. J.Chromatogr. 1989,492, 585-614. .~~ (8)Ewing, A. G.; Wallingford, R. A,; Olefirowicz, T. M. Anal. Chem. 1989,61, 292A-303A. ~~~
0003-2700/92/0364-2S72$03.00/0
phoresis performed in capillaries filled with gels consisting of BSA cross-linked with glutaraldehyde for the separation of tryptophan enantiomers. The methodological description significantly extends the application area of CE to separation methods based on affinity interactions, exemplified here by the optical resolution of D- and L-tryptophan on immobilized BSA as the separation phase. We term this method as capillary affinity gel electrophoresis(CAGE). Electrophoretic separation methods based on ligands included in polyacrylamide gel-filled capillaries have been reported for enantiomeric separation of dansylated amino acids as well as for DNA restriction fragment separations.QJ0
EXPERIMENTAL SECTION Apparatus. During these studies, a Waters Quanta 4000 capillary electrophoresis instrument (Millipore Inc., Bedford, MA) was employed. Analytes were detected on-column at a wavelength of 214 nm. The temperature was 25 "C. The electropherograms were recorded and stored on a NEC personal computer with the MAXIMA 825 software program (Millipore Inc., Bedford, MA). Procedure. A detection window was prepared on the polyimide-cladfused-silica capillary tubing (75pm X 40 cm,Polymicro TechnologiesInc., Phoenix,AZ) 7 cm from one end of the capillary by quickly flaming an approximately 1-cmportion of the tubing. A typical preparation of the BSA-filled capillary was done aa followsand is summarized in Figure 1. The capillary was washed with 10 mL each of water, 0.5 M NaOH, water, and finally 0.05 M potassium phosphate/acetate buffer pH 5.0 with a peristaltic pump. Five pars 5% (w/v) BSA in 0.05 M phosphate/acetate, pH 5.0, was mixed with two parts 25% (v/v)glutaraldehyde and eight parts 0.05 M phosphate/acetate buffer, pH 5.0. The mixture was immediately pumped (circa 10pL/min) into the buffer-filled capillary from the injection end of the capillary. The capillary was disconnected from the peristaltic pump when the BSA/ glutaraldehyde mixture had reached approximately 2 mm from the detection window. The capillary ends were then held level until after gelation (usually circa 10 min). Thus, the detection window region of the capillary contained phosphate/acetate buffer and not BSA gel. The gel-filled capillary was subsequently preconditioned by exposing the capillary to a potential of 2 kV overnight with the cathode at the injection side and the anode at the detection side of the capillary in 50 mM potassium phosphate buffer, pH 7.5. Electrophoresis of samples was subsequently performed (6kV) with the capillary in reverse direction to that used during preconditioning. Samples were electrokinetically introduced (5 s, 4 kV) from a buffer with half the ionic concentration of the running buffer (50 mM potassium phosphate, pH 7.5). Chemicals. BSA (Product No. A7030, Lot No. 571;'-0404)was purchased from Sigma (St. Louis, MI). Glutaraldehyde (Product (9) Guttman, A.; Paulus, A.; Cohen, A. S.;Grinberg, N.; Karger, B. L.
J. Chromatogr. 1988,448,41-53. (10) Guttman, A,; Cooke, N. Anal. Chem. 1991, 63, 2038-2042.
0 1992 American Chemical Society
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75 km capillary washed (H20, 0.5 M NaOH, HzO, 50 mM Phosphate-acetate pH 5.0)
J. J. J.
5 parts 5 % BSA mixed with 2 parts 25 % GA and 8 parts 50 mM Phosphate-acetate pH 5.0
BSA-GA mixture pumped into buffer filled capillary Capillary detached from pump when BSA-GA is ca. 2 cm from detection window BSA-GA aliowed to gel
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BSA-gel capillary preconditioned overnight (electrophoresis in reverse direction ie. cathode at injection end)
Fburr 1. Flow diagram for Preparation of BSA-filled capillary.
No. 121791, potassium phosphate, and sodium acetate were obtained from Merck (Darmstadt,FRG). RESULTS AND DISCUSSION Preparation of Capillaries Filled with BSA Gel. Initially, conditions were investigated for gelation of BSA cross-linked with glutaraldehyde. Various BSA and glutaraldehyde concentrations as well as various pH and ionic strengths were tested. It was found that an opaque off-white gel formed after approximately 3 min at room temperature when 50 pL of BSA (50 mg/mL) in 0.05 M potassium phosphate/acetate buffer (pH 5.0) was mixed with 100 p L of 5% (v/v) glutaraldehyde in 0.04 M potassium phosphate/ acetate buffer (pH 5.0). Our first trials at running electrophoresis through the gelfilled capillaries resulted in the formation of bubbles within the capillaries, primarily within the buffer region of the capillary, soon after current was applied. After various attempts to alleviate this difficulty, we finally found that reversing the direction of current (simply by inverting the capillary so that the anode was now at the detection window end of the capillary) and exposing the BSA-filled capillary overnight to a potential of 2 kV eliminated the incessant Occurrenceof bubble formationin the capillaries when samples were subsequently electrophoresed in the correct direction. Enantiomeric Separation of Tryptophan with Capillary Affinity Gel Electrophoresis. The optical isomers of tryptophan were introduced by electrophoretic migration and separated, as shown in Figure 2A. We presume that the migration of tryptophan depends on electroosmotic flow in the gel-filled capillary, as the isoelectric point of tryptophan is 5.9 and of BSA is 4.9, the pH of the mobile phase was 7.5, and the electrophoresis was performed with the cathode at the detection end of the Capillary. Identification of the enantiomers was subsequently accomplishedby introduction and electrophoresis of solely the D isomer under similar conditions (Figure 2B). The theoretical plate number, N, calculated using the peak width at the base, obtained in this case was circa 85 OOO.ll The N values for the present version of a capillary affinity gel electrophoretic separation of the isomers are thus significantlyhigher than those obtained from high-performanceliquid affinity chromatographyseparations (11) Snyder, L. R.;Kirkland, J . J . Introduction to Modern Liquid Chromatography; J. Wiley: New York, 1979.
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Flgure 2. (A) Capillary affinity gel electrophoretic separation of tryptophan enantiomers. Sample was w-tryptophan (10 pM In 25 mM potassium phosphate pH 7.5). (6)Identlflcatlonof racemate. Sample was o-tryptophan (5 pM in 25 mM potasslum phosphate pH 7.5). Conditions: constant applied electric field, 150 V/cm, 80 MA;gel length = 32 cm, total length = 40 cm; buffer 50 mM potassium phosphate (pH 7.5); sample injection, 100 V/cm, 5 8.
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Flgure 3. Capillaryaffinity gel electrophoretic separationof tryptophan enantiomers. Sample was m-tryptophan (10 pM in 25 mM potassium phosphate pH 7.5). Conditions: constant applled electric field, 125 Vlcm; gei length = 32 cm,totallength = 40 cm;buffer 50 mM potasslum phosphate (pH 7.5); sample Injection, 100 V/cm, 3 s.
on immobilized BSA.5 The resolution, R,, defined as
R, =
t2
- tl
(w1+ W J / 2
where t is the elution time and w ,the peak width at the base (i.e. the length of the baseline intercepted by the tangents drawn to the inflection points), was 3.0 for the separation shown in Figure 2A which is comparable to BSA-based LC separation. Using another capillary preparation, an even better resolution was obtained, as shown in Figure 3, with a resolution value of 6.0 and an N value of 91 O00. One of the limitations of protein-based chiral stationary phases in LC is their limited capacitydue to the high molecular weight of the selector. Using the quantities employed here, we see that the BSA concentration within the capillary is circa 0.25 mM. For satisfactory separation, the concentration of the molecules to be separated should be considerablylower than 0.25 mM (assuming that a one to one interaction between BSA and the optical isomer exists and, in addition, that probably not all BSAmoleculesare available for interaction). Generally, at least 1 order of magnitude less is prescribed. Thus, a capacity of 25 pM sample is possible, which we also observed in practice. With UV detection at 214 nm, as used here, the lowest L-tryptophan concentration which can be
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detected is 1pM at a signal to noise ratio of 2. The range of concentrations separated and analyzed was thus 1-25 pM. Sample Injection and Separation Conditions. DLTryptophan was introduced electrophoretically, and the enantiomers were subsequently separated at 6 kV typically in 10-11 min (Figure 2A). The pH and ionic strength of the running buffer were varied. As the pH of the running buffer was changed from 7.0 to 8.0, we found no significant change in separation performance except at pH close to 8.0 when the difference in migration time for the enantiomers decreased. The best separation results were obtained at an ionic strength of 25-50 mM for the running buffer. The ionic strength of the sample buffer was also varied, and we found that sample injection from a buffer with an ionic strength half that of the separation buffer gave better resolution and higher efficiency, presumably due to sample stacking.12 In conclusion, we have shown the feasibility of employing protein gels for affinity separation in capillary electrophoresis. Specifically, we have used the protein, in this case BSA, as the chiral selector for the separation of tryptophan enantiomers with very high efficiency compared to the analogous protein-based LC separation^.^ In addition, the method employedfor preparing the separation phase, i.e. gel formation through chemical cross-linking, allows a high concentration of the affinity selector to be immobilized. The protein (BSA) itself is the sole component (except for bifunctional cross-
We gratefully acknowledge the financial support from the Crafoord, Lars Hiertas Memorial, and Carl Tryggers Foundations. We thank Hans Olof Nilsson for his technical assistance. The results described here were, in part, presented at the Euchem Conference on Capillary Electroseparations, December 9-11, 1991, Storlien, Sweden, and at the 4th International Symposium on High-Performance Capillary Electrophoresis, February 9-13, 1992, Amsterdam, The Netherlands.
(12) Burgi, D. S.;Chien, R.-L. Anal. Chem. 1991,63, 2042-2047. (13) Zopf, D.; Ohlson, S. Nature 1990,346, 87-88.
RECEIVED for review April 7, 1992. Accepted September 1, 1992.
linking agent glutaraldehyde) in the separation phase without any other inert support material. In the future, we see that the interactive methods described here in which immobilized protein phases are used in CE expands the application field of CE to new practical separation endeavors (such as antibody-based separations) as well as to fundamental protein interaction studies, particularly with respect to weak affinity interaction.13 The minimal volumes and amounts of components used in CE induce the initial screening of exclusive proteins (Le. proteins of limited availability) as potential chiral selectors, for example for drug enantiomer separation. The method described here has the potential to be applicable for all types of affinity-based separations which include cross-linkable specific binding partners.
ACKNOWLEDGMENT
