Anal. Chem. 1996, 68, 2753-2757
Fritless Packed Columns for Capillary Electrochromatography: Separation of Uncharged Compounds on Hydrophobic Hydrogels Chuzo Fujimoto* and Yutaka Fujise
Department of Chemistry, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan Eiji Matsuzawa
Hamamatsu Photonics K.K., Joko, Hamamatsu 431-31, Japan
A novel column is described that does not require frits to keep packing material within a capillary. A continuous bed is prepared in situ in aqueous solution by radical copolymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropanesulfonic acid (the resultant gel is denoted poly(AMPS-co-IPAAm). N,N′-Methylenebisacrylamide is used for cross-linking. On the application of an electrical field, electroosmotic flow (EOF) is developed in the bed along the capillary, where fluid propulsion would be otherwise difficult to achieve. The resultant EOF transports neutral compounds through the column without forcing the gel out of the capillary. Examination of the fluid motion in the continuous bed using a video microscope system and an image processor shows a relatively flat flow profile of EOF. The bed functions as the stationary phase for reversed-phase capillary electrochromatography (CEC). This new approach is an alternative to packed capillary columns which have been used previously in CEC. A high efficiency is obtained for a steroid which is separated on a 4.0% total monomer concentration (T), 10.0% degree of cross-linking (C), and 10.0% mole fraction of AMPS in the total monomer (S), poly(AMPS-co-IPAAm) column. A mixture of polyaromatic hydrocarbons is separated on a 6.9% T, 5.8% C, and 5.5% S poly(AMPS-co-IPAAm) column. The capacity factor of benzo[a]pyrene increases from 0.63 to 1.91 as the acetonitrile content in a Tris-boric acid buffer is decreased from 45 to 30% (v/v). The run-to-run RSD of analyte migration time is less than 0.73%, and the dayto-day RSD is acceptable. Potential benefits of this approach are also mentioned. Capillary electrochromatography (CEC) is a microcolumn separation technique that combines features of both microcolumn liquid chromatography and capillary zone electrophoresis. (The term “capillary electrochromatography” is used here to refer to electrically driven liquid chromatography (LC), although its nomenclature has been a problem recently1-3.) Electrochromatographic separations are based on partitioning and electrophoretic mobility. Therefore, the separation mechanisms for uncharged (1) Fujimoto, C. Anal. Chem. 1995, 67, 2050-2053. (2) Fujimoto, C.; Kino, J.; Sawada, H. J. Chromatogr. A 1995, 716, 107-113. (3) Tsuda, T. LC-GC Int. 1992, 5, 26-36. S0003-2700(96)00177-1 CCC: $12.00
© 1996 American Chemical Society
solutes are the same as those for pressure-driven LC. CEC, however, is superior to pressure-driven LC with respect to separation efficiency. The high efficiency of CEC arises from the flat flow profile of electroosmotic flow (EOF) induced by an applied electrical field. One further item is important in CEC: the instrumentation is considerably simplified, since CEC does not necessarily require a high-pressure LC pump, a mechanical injector, fittings, and plumbing. In practice, however, LC pumps have frequently been used to purge bubbles, to control the velocity and direction of the eluent, and to condition the packed capillary columns. Two types of capillary columns have, so far, been popular in CEC. The first one is packed with small LC packing particles.3-23 The use of this type of column offers us several choices of commercially available LC packings. Needless to say, this type of column requires frits to keep the packing particles within the capillary column. Unfortunately, the packed capillary columns are not routinely used in capillary electrochromatography because of the difficulty of fabricating narrow on-column frits in a reproducible manner. The second type of column is being used in only a few laboratories.24-33 The stationary phases are chemically bonded (4) Pretorius, V.; Hopkins, B. J.; Schieke, J. D. J. Chromatogr. 1974, 99, 2330. (5) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209-216. (6) Stevens, T. S.; Cortes, H. J. Anal. Chem. 1983, 55, 1365-1370. (7) Knox, J. H.; Grant, I. H. Chromatographia 1987, 24, 135-143. (8) Knox, J. H.; McCormack, K. A. J. Liq. Chromatogr. 1989, 12, 2435-2470. (9) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 135-143. (10) Yamamoto, H.; Baumann, J.; Erni, F. J. Chromatogr. 1992, 593, 313-319. (11) Yan, C.; Schaufelberger, D.; Erni, F. J. Chromatogr. A 1994, 670, 15-23. (12) Yan, C.; Dadoo, R.; Zhao, H.; Zare, R. N.; Rakestraw, D. J. Anal. Chem. 1995, 67, 2026-2029. (13) Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1982, 248, 241-247. (14) Tsuda, T. Nippon Kagakukaishi 1986, 937-942. (15) Tsuda, T. Anal. Chem. 1987, 59, 521-523. (16) Tsuda, T. Anal. Chem. 1988, 60, 1677-1680. (17) Tsuda, T.; Muramatsu, Y. J. Chromatogr. 1990, 515, 645-652. (18) Soini, H.; Tsuda, T.; Novotny, M. V. J. Chromatogr. 1991, 559, 547-558. (19) Li, S.; Lloyd, D. K. Anal. Chem. 1993, 65, 3648-3690. (20) Li, S.; Lloyd, D. K. J. Chromatogr. A 1994, 666, 321-335. (21) Smith, N. W.; Evans, M. B. Chromatographia 1994, 38, 649-657. (22) Behnke, B.; Bayer, E. J. Chromatogr. A 1994, 680, 93-98. (23) Schmeer, K.; Behnke, B.; Bayer, E. Anal. Chem. 1995, 67, 3656-3658. (24) Bruin, G. J. M.; Tock, P. P. H.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1990, 517, 557-572. (25) Pfeffer, W. D.; Yeung, E. S. Anal. Chem. 1990, 62, 2178-2182. (26) Pfeffer, W. D.; Yeung, E. S. J. Chromatogr. 1991, 557, 125-136. (27) Garner, T. W.; Yeung, E. S. J. Chromatogr. 1993, 640, 397-402.
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directly onto the capillary wall; therefore, there are no problems associated with the frit construction. The open tubular columns, however, suffer from the disadvantage that the phase ratios are small and provide low retention times and low sample capacities. We previously reported a third type of column for the electrochromatographic separations,1,2 where the stationary phase was made of cross-linked polyacrylamide with attached sulfo groups that was synthesized directly in the capillary in the form of a continuous bed. The bed is impermeable to a hydrodynamic flow. Instead, it is charged over a wide range of pH and generates enough EOF to transport uncharged solutes under the influence of a high voltage, yet the stationary phase is never expelled from the column. By using such a column with a capillary electrophoresis system, we were able to separate various neutral compounds without the aid of an LC pump. With the polyacrylamide column, molecular sieving was the dominant separation mechanism: smaller molecules are eluted earlier than larger ones, in contrast to exclusion chromatography. It is noteworthy that the concept of an in situ formed column was introduced in the 1970s for LC34,35 and is still under development in several laboratories.36-41 Packings have been prepared in a large-diameter tube and made up of either agglomerated particles34-39 or silica skeletons.40,41 The permeabilities of these columns were so high as to allow them to be operated with conventional LC pumps. In this report, we describe the preparation of hydrophobic stationary phases for CEC separation of uncharged compounds. A continuous bed is prepared in such a way that N-isopropyl groups are incorporated into polyacrylamide matrix during the in situ polymerization process. The stationary phases give high column efficiencies, sufficient retention, and good column stability. Over the past two decades, steady efforts have been made to miniaturize LC columns and related instrumentation. To further miniaturize the chromatographic system, however, it is preferable to omit an LC pump and an injection valve. The use of such a fritless packed column with EOF seems the most promising for this purpose. EXPERIMENTAL SECTION Apparatus. The CEC experiments were carried out using a laboratory-made apparatus as described in refs 1 and 2. The inner diameter of the columns used is either 75 or 50 µm. UV (28) Mayer, S.; Schurig, V. J. High Resolut. Chromatogr. 1992, 15, 129-131. (29) Mayer, S.; Schurig, V. J. Liq. Chromatogr. 1993, 16, 915-931. (30) Mayer, S.; Schurig, V. Electrophoresis 1994, 15, 835-841. (31) Meyer, S.; Schleimer, M.; Schurig, V. J. Microcolumn Sep. 1994, 6, 43-48. (32) Armstrong, D. W.; Tang, Y.; Ward, T.; Nichols, M. Anal. Chem. 1993, 65, 1114-1117. (33) Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511-2516. (34) Lynn, T. R.; Rushneck, D. C.; Cooper, A. R. J. Chromatogr. Sci. 1974, 12, 76-79 (35) Hansen, L. C.; Sievers, R. E. J. Chromatogr. 1974, 99, 123-133. (36) Hjerte´n, S.; Li, Y.-M.; Liao, J. L.; Nakazato, K.; Mohammad, J.; Pettersson, G. Nature 1992, 356, 810-811. (37) Hjerte´n, S.; Nakazato, K.; Mohammad, J.; Eaker D. Chromatographia 1993, 37, 287-294. (38) Hjerte´n, S.; Mohammad, J.; Nakazato, K. J. Chromatogr. 1993, 646, 121128. (39) Nakazato, K.; Mohammad, J.; Hjerte´n, S. Chromatographia 1994, 39, 655662. (40) Nakanishi, K.; Soga, N. J. Am. Ceram. Soc. 1991, 74, 2518-2530. (41) Abe, H.; Motokawa, O.; Kobayashi, M.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Tanaka, N. Paper presented at the 44th Annual Meeting of the Japan Society for Analytical Chemistry, Sapporo, Japan, Sept 28-30, 1995, paper 2B12.
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absorption detection was performed at 254 nm. A constant voltage was applied to the column placed in a Plexiglas box with an interlock system. Sample injection was done by the electrokinetic method. The solutes tested are all neutral and are pulled into the column due to the EOF. A video microscope system was used to visualize the font profile of EOF generated in the gel-filled capillary. The column, whose polyimide coating was removed over 30 mm at the inlet end, was fixed in position and imaged by a stereomicroscope (Vanox AHBS3, Olympus Optical, Tokyo, Japan). Images were taken with a 3CCD camera (DXC930, Sony, Tokyo, Japan) and recorded with a Hi-8 video recorder (Sony). Pictures of the video screen (Sony) were obtained using a video printer (Sony). To examine the flow profile further, the raw images were digitized by an ARGUS-50 image processor (Hamamatsu Photonics, Hamamatsu, Japan). Some digital image processing techniques were used to enhance the contrast of the images. Preparation of Columns. Part of the external polyimide coating of a fused silica capillary was removed to create an optical window for UV detection. The inner wall of the capillary was then treated with a bifunctional reagent, (γ-methacryloxypropyl)trimethoxysilane. Next, the mixture of 2-acrylamido-2-methylpropanesulfonic acid (AMPS), N-isopropylacrylamide (IPAAm), N,N′methylenebis[acrylamide] (Bis), ammonium persulfate (APS), and N,N,N′,N′-tetramethylethylenediamine (TEMED) in 100 mM Tris150 mM boric acid (pH 8.1) solution was forced into the capillary with a plastic syringe connected to it by a Teflon tube. After filling, the syringe was carefully removed, and both ends of the capillary were dipped into vials containing the polymerization solution. The capillary was kept there for a few hours. The progress of the reaction was not monitored. Finally, the capillary ends were placed in buffer reservoirs and preelectrophoresed for a few hours to remove the monomers, un-cross-linked polymers, initiator, and catalyst and to ensure constant run conditions. The injection end of the capillary was cleanly cut before use. We use the nomenclature introduced by Hjerte´n42 to refer to the total monomer concentration, % T, and the degree of crosslinking, % C. These definitions have been widely adopted, particularly in the field of gel electrophoresis. Added to these, we define % S as the mole fraction of AMPS in the total monomer.1,2 Chemicals. Acrylamide (AAm), Bis, APS, and TEMED were of electrophoresis grade, obtained from Nacalai Tesque (Kyoto, Japan). IPAAm was obtained from Wako Pure Chemical Industries (Osaka, Japan). AMPS, (γ-methacryloxypropyl)trimethoxysilane, and polyaromatic hydrocarbons (PAHs) were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Steroids were obtained from Sigma (St. Louis, MO). The water used was obtained from an Autostil WG 23 (Yamato, Tokyo, Japan). RESULTS AND DISCUSSION The hydrogel prepared by radical copolymerization of AMPS and IPAAm, denoted poly(AMPS-co-IPAAm), has an appearance and elasticity similar to those of polyacrylamide, which has been utilized in gel electrophoresis. Using a video microscope system, the flow profile of EOF in the presence of such a hydrogel inside a capillary was examined. A few groups have observed EOF in a (42) Hjerte´n, S. Arch. Biochem. Biophys. 1962, (Suppl. 1), 147-151.
Figure 1. Digitized images of zone front of EOF. A single image recorded on video tape was digitized using two different image processing techniques (A and B). Sample solution, 1.5 mM Rhodamine B in methanol; applied field strength, 285 V/cm; current, 7 µA.
capillary,43,44 but those were for free solution electrophoresis. The inner diameter of the capillary used is 75 µm. A 4.0% T, 10.0% C, and 10.0% S gel column was prepared, into which Rhodamine B in methanol was continuously introduced at a field strength of 285 V/cm. In a separate experiment, we have found that the dye is neither charged under these conditions nor significantly adsorbed onto the stationary phase. Therefore, the concentration profile of the dye at the front is supposed to represent the flow profile of the EOF. Several pictures were taken from the video tape, but the front profile was indistinct in the pictures. To enhance the contrast of the images, some digital image processing was done on the raw images. The resultant images are shown in Figure 1. Although the front is slightly uneven across the capillary radius, the flow profile is expected to be much flatter than that observed with pressurized flow. According to Hjerte´n,45 the EOF profile should be flat in any type of CEC (regardless of the un-uniformity of the packings on a microscopic scale), provided there exist no interactions between the sample and the stationary phase. From this result, at any rate, we can expect a substantial increase in separation efficiency or theoretical plate number when using the electroosmotic pump with the continuous bed. (43) Tsuda, T.; Ikedo, M.; Jones, G.; Dadoo, R.; Zare, R. N. J. Chromatogr. 1993, 632, 201-207. (44) Taylor, J. A.; Yeung, E. S. Anal. Chem. 1993, 65, 2928-2932. (45) Hjerte´n, S. Personal communication.
Our next concern was rough estimation of the phase ratio of the column, that is, the volume ratio of stationary to mobile phase in the column. Its estimation for this column is not as straightforward as for open-tubular columns, generally because there is water bound to the polymer chains as well as free water in hydrogels. We measured the gain in weight of the outlet reservoir during different periods of the run and also measured the density of the running solution (100 mM Tris-150 mM boric acid buffer). The weight of the effluent increased linearly with time. This means that the EOF is stable. Next, we measured the linear velocity of EOF using a non-interactive marker. Although it is highly speculative, we expect that there are a large number of channels that align themselves in the direction of the field upon the application of an electrical field and that are partially interconnected. We shall suppose that a single, large passageway is substituted for all the narrow channels and that the eluent can pass through this imaginary passageway. From the volumetric flow rate and linear velocity of the EOF, the diameter of the imaginary passageway was calculated to be 68 µm. Assuming that the remainder, obtained by subtracting the internal volume of the imaginary passageway from that of the capillary, serves entirely as the stationary phase, the void ratio is calculated to be 0.82. Therefore, the phase ratio can be estimated to be 0.22. We compared the poly(AMPS-co-IPAAm) column with the poly(AMPS-co-AAm) column, which was investigated before,1,2 with regard to the separation of ketones (Figure 2). Both columns are the same in size and in gel composition. The field strength and the mobile phase composition were also the same. We can see that substantial improvement in the separation is achieved with the poly(AMPS-co-IPAAm) column, where the solutes are likely to be separated on the basis of hydrophobicity. On the other hand, the separation on the poly(AMPS-co-AAm) hydrogel has been found to be based mainly on size selectivity.1 With regard to the poly(AMPS-co-AAm) column, it may be worth pointing out that increasing the total monomer concentration and/or decreasing the cross-linker concentration has been found to give baseline separation of these solutes.1 Figure 3 demonstrates the high efficiency attained with the fritless hydrogel column. A steroid mixture was separated on a 4.0% T, 10.0% C, and 10.0% S poly(AMPS-co-IPAAm) column with a Tris-borate buffer. The field strength was 308 V/cm. The mobile phase was the same as that used for polymerization. The theoretical plate numbers obtained are 79 850 (159 700/m) and 79 900(159 800/m) for hydrocortisone and testosterone, respectively; these values are much higher than those obtained by pressure-driven LC and even higher than those obtained by electrochromatography using a conventional packed column.11 We examined the separation of more hydrophobic compounds using the hydrogel column. A PAH mixture was separated on a 6.9% T, 5.8% C, and 5.5% S poly(AMPS-co-IPAAm) column (Figure 4). The mobile phase was 35% (v/v) acetonitrile in an aqueous buffer, and the effective length was 21.9 cm. The field strength was 426 V/cm. The solutes eluted in order of increasing hydrophobicity. Calculation of the theoretical plate number was made on naphthalene, which produces a relatively symmetrical peak. The value obtained, N ) 23 540 (107 500/m), is comparable to those obtainable in packed CEC.11 In LC, the partition ratios of analytes are adjusted by changing the mobile phase composition. We examined the effect of varying the acetonitrile content on the separation of PAHs on the polyAnalytical Chemistry, Vol. 68, No. 17, September 1, 1996
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Figure 3. Chromatogram of a mixture of steroids on a poly(AMPSco-IPAAm) hydrogel column. Mobile phase, 100 mM Tris-150 mM boric acid (pH 8.1); capillary, 65.0 cm × 75 µm i.d. (50.0 cm effective length); %T, 4.0; %C, 10.0; %S, 10.0; applied voltage, 20.0 kV. Peaks: (1) hydrocortisone, (2) prednisolone, (3) hydrocortisone 21acetate, and (4) testosterone.
Figure 2. Separations of ketones using (A) poly(AMPS-co-AAm) and (B) poly(AMPS-co-IPAAm) hydrogel columns. Conditions: %T, 4.0; %C, 10.0; %S, 10.0; capillary, 65.0 cm × 75 µm i.d. (effective length, 50.0 cm); mobile phase, 100 mM Tris-150 mM boric acid (pH 8.1); applied voltage, 15.0 kV. Peaks: (1) dimethyl ketone, (2) methyl ethyl ketone, (3) methyl n-propyl ketone, (4) methyl n-butyl ketone, (5) methyl n-pentyl ketone, (6) methyl phenyl ketone, (7) ethyl phenyl ketone, (8) n-propyl phenyl ketone, and (9) n-butyl phenyl ketone.
(AMPS-co-IPAAm) hydrogel column. The mobile phase was electrokinetically changed. As can be seen from Figure 5, the capacity factor (k′) of each solute decreases with an increase in acetonitrile content. It seems reasonable to suppose that a reversed-phase mechanism governs this separation. The column packings must be able to withstand the effects of exposure to a wide range of solvents. The solvent compatibility was checked in the range of 0-45% (v/v) acetonitrile content in the Tris-borate buffer. In general, a gel shrinks or swells, depending on the osmolarity of the surrounding solution. In fact, a marked shrinkage of the poly(AMPS-co-IPAAm) gel was observed when it was immersed in pure water. In the composition range studied here, however, the mixed solvents did not seem to be harmful to the column, because we were able to obtain restored chromatograms after the mobile phase was changed from one to another and subsequently returned to the initial solvent. Reproducibility is a concern in any analytical technique. We tested the reproducibilities of the analyte migration time (tr) 2756 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
Figure 4. Chromatogram of PAHs on a poly(AMPS-co-IPAAm) hydrogel column. Conditions, %T, 6.9; %C, 5.8; %S, 5.5; capillary, 46.9 cm × 50 µm i.d. (effective length, 21.9 cm); mobile phase, 35% (v/v) acetonitrile in 100 mM Tris-150 mM boric acid (pH 8.1) buffer; applied voltage, 20.0 kV. Peaks: (1) naphthalene, (2) unknown, (3) fluoranthene, (4) benz[a]anthracene, and (5) benzo[a]pyrene.
between runs and over seven consecutive days using a 48.4 cm × 50 µm capillary packed with 4.0% T, 10.0% C, and 10.0% S gel. The mobile phase was 35% (v/v) acetonitrile in 100 mM Tris150 mM boric acid buffer and was changed at the beginning of each day to prevent any change in composition due to preferential evaporation of the more volatile component. Table 1 shows the data for the four PAHs. The run-to-run reproducibility is good, and the day-to-day reproducibility is also acceptable, in spite of the fact that no temperature controller was available. The within-
Figure 5. Capacity factor (k′) of PAHs on a poly(AMPS-co-IPAAm) hydrogel column as a function of the % (v/v) of acetonitrile: (O) naphthalene, (2) fluoranthene, (0) benz[a]anthracene, and (() benzo[a]pyrene. Conditions: varying amounts of acetonitrile in 100 mM Tris-150 mM boric acid (pH 8.1) buffer; capillary, 46.9 cm × 50 µm i.d. (21.9 cm effective length); %T, 6.9; %C, 5.8; %S, 5.5; applied voltage, 15.0 kV. Table 1. Migration Time Reproducibilities for PAH Separationsa run-to-run (n ) 5)b day-to-day, 7 days (n ) 35)b solute
tr (min)
RSD (%)
tr (min)
RSD (%)
naphthalene fluoranthene benz[a]anthracene benzo[a]pyrene
16.59 19.14 20.73 22.51
0.70 0.57 0.45 0.73
16.24 18.75 20.14 21.59
2.71 2.91 2.84 3.56
a Conditions: gel composition, 4.0% T, 10.0% C, 10.0% S; capillary size, 48.4 cm × 50 µm i.d. (23.2 cm effective length); applied voltage, 15.0 kV; mobile phase, 30% (v/v) acetonitrile in 100 mM Tris-150 mM boric acid (pH 8.1). b n represents the total number of measurements carried out.
day reproducibility could be further improved if the running solution was changed more often. Occasional use of this column over a period of 3 weeks with a variety of mobile phase compositions showed no significant degradation in performance. The exact estimation of the column lifetime seems difficult, because the life span depends on many variables, including the gel composition, the pH and composition of the mobile phase, the applied voltage, and the storage temperature. Of these variables, the pH of the mobile phase and the storage temperature could be critical, because they would influence the extent of gel hydrolysis. CONCLUSIONS AND FUTURE DIRECTIONS We were able to develop a simple and reliable method for the production of fritless packed columns. The advantages of the (46) Fujimoto, C.; Fujise, Y. Manuscript in preparation.
elimination of cumbersome procedures such as frit-making and particle-packing, moderate sample retention, no need for an LC pump, and the possibility of preparing tailor-made columns ensure that the fritless packed columns will find widespread use in laboratories. Because of the lack of frits, the column can be cut at any point along the capillary. Even if adsorption of some components occurs at the top of the column, the column performance will be restored by cutting off a few millimeters from the top of the column. It is likely that, instead of the polyacrylamide-based gels, other soft materials such as cellulose and agarose could be used for high-resolution separations, with some chemical modification; they have been regarded as inadequate for pressure-driven LC. The magnitude of EOF can be easily varied by changing the AMPS content in the polymerization mixture and by changing the voltage applied to the column. Using an AMPS-introduced gel, the EOF is toward the cathode. We found that the direction of electroosmotic flow is reversed by using [(N,N-dimethylamino)propyl]acrylamide or N-(2-acrylamidoethyl)triethylammonium iodide in the place of AMPS.46 The gels are likely to be positively charged at the pH used, and the walls of the channels in the gel appear to behave like a quaternary amine modifier. The choice of the EOF direction should be important when analyzing samples containing charged analytes. Bubble formation has often been a problem in capillary gel electrophoresis using the cross-linked polyacrylamide gel, since it leads to interruption of the current flow. The bubble formation can take place with the poly(AMPS-co-IPAAm) gel column during preparation and use of the column. During the course of our investigation, we found that bubbles become smaller and finally disappear on applying a high voltage on the column. Due to the extinction of bubbles from the gel, the rate of success in preparation was almost 100%. It appears that the gel would be somewhat liquefied inside the capillary, perhaps due to Joule heating effect, yet the gel exhibits an extremely high viscosity and behaves essentially as a solid. Further investigation to better understand the hydrogel is underway in our laboratory. ACKNOWLEDGMENT We gratefully acknowledge fruitful discussions on the EOF profile with Professors S. Hjerte´n and T. Tsuda. The material was presented in part at the 15th Symposium on Capillary Electrophoresis, Hiroshima, Japan, Dec 6-8, 1995, Paper 35, and at the 8th International Symposium on High Performance Capillary Electrophoresis, Orlando, FL, Jan 22-25, 1996. Received for review February 26, 1996. Accepted June 10, 1996.X AC9601775 X
Abstract published in Advance ACS Abstracts, July 15, 1996.
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