Anal. Chem. 1995, 67,2050-2053
Charged Polyacrylamide Gels for Capillary ElectrochromatographicSeparations of Uncharged, Low Molecular Weight Compounds Chwo Fujimoto Department of Chemistry, Hamamatsu University School of Medicine, Hamamatsu 43 1-31, Japan
The polyacrylamide gels described here have the ability to separate uncharged substances of comparatively low molecular weight due to the built-in, negatively charged functional groups. The functional groups permit the development of electroosmotic flow under the influence of a high voltage, which makes possible capillary electrochromatographic separations of uncharged compounds. The effects of gel compositions on the column performance were examined. Migration time reproducibilities of 0.43%RSD or less were found with the gel-filled capillary columns. Possible insight into the separation mechanisms is discussed. Due to its potential to increase chromatographic performance, the feasibility of electoosmotic pumping of the mobile phase in capillary liquid chromatography has been extensivelyinvestigated. Recently, Yan et al.’ and Knox and Grant2Jdescribed a chromatcgraphic method in which the mobile phase is transported through a stationary phase in a microbore or capillary column by electroosmotic flow (EOF) using an electric field, termed capillary electrochromatography (CEC). Originally, the use of the term electrochromatographywas restricted to electrophoretic procedures where separation is effected by sorptive interaction with the stationary phase.4 According to this original delinition, separation of uncharged analytes with the aid of EOF, that is, electroosmotically driven chromatography (or electroosmotic chromatography), is excluded. Tsuda approved of the inclusion of electroosmotic chromatography in the term electrochromatography as one of its extreme cases.5 Further, Tsuda introduced the concept of pressurized flow in electrochromatography6-8 and collectively called these techniques “high-performanceelectrochromatogra~ h y ” Every . ~ capillary liquid chromatography performed under the influence of an electric field will henceforth be termed CEC, without any consideration to the presence of EOF or analyte charges. In CEC, the stationary phase can be polymer networks used in capillary gel electrophoresis (CGE) and capillary affinity gel electrophoresis (CAGE) as well as packing material utilized for high-performanceliquid chromatography (HPLC). (This paper has few references to CGE and CAGE in that the EOF of the gel columns is suppressed in these types of chromatography or it is (1) Yan, C.; Schaufelberger, D.; Emi, F. J. Chromatogr. A 1994,670, 15-23. (2) Knox, J. H.; Grant, I. H. Chromatographia 1987,24, 135-143. (3) Knox,J. H.; Grant, I. H. Chromatographia 1991,32, 317-328. (4) Wieme, R. J. In ChromatographrA Laboratory Handbook of Chromatographic and Electrophoretic Methods, 3rd ed.; Heftmann, E., Ed.; Van Nostrand Reinhold Co.: New York, 1975; Chapter 10, and references therein. (5) Tsuda, T. LC-GC 1st. 1992,5, 26-36. (6) Tsuda, T. Anal. Chem. 1987,59, 521-523. (7) Tsuda, T. Anal. Chem. 1988,60, 1677-1680. (8) Tsuda, T.; Muramatsu, Y. J. Chromafogr. 1990,515, 645-652.
2050 Analytical Chemistry, Vol. 67,No. 73, July 7, 7995
insufficient for effective separation of uncharged compounds, unlike the columns described here.) Alternatively, some interactive materials attached to the capillary wall may be used in the open-tubular format. Normal chromatographic distribution (including size-selectivedistribution) between a mobile phase and a stationary phase is utilized to separate charged and uncharged compounds with higher resolution and superior efficiency, compared with micro-HPLC using an identical column.1-3,5-23 CEC has been performed with several types of columns. As early as 1974, Pretonus et al.9 proposed the use of EOF with a packed column and an open tube (both 1 mm i.d. x 50 cm) to improve the efficiencies of the chromatographic system, but the idea remained dormant until Jorgenson and Lukacslo reported the results obtained with a glass capillary (170 pm i.d. x 68 cm) packed with a 10;um reversed-phase packing. In 1982, Tsuda” employed an octadecylsilane-bonded, open-tubular capillary column (30 pm i.d.) for the separations of aromatic compounds. The use of packed fused-silica capillary columns in CE was demonstrated by Knox and Grant2,3and Tsuda.6-*J1~12 Recently, Jacobson et al.13 reported CEC on a glass microchip having a narrow channel (5.6 pm x 66 pm). In this article, we report the results of our investigations on CEC with charged polyacrylamide gel columns. In contrast to polyacrylamide gels which have been used in CGE, the gels are prepared using three kinds of acrylamide monomers, that is, straight acrylamide (AA),N,”-methylenebisacrylamide @IS),and 2-acrylamido-2-methyl-1-propanesulfonicacid (AMPS); the last component is incorporated into the common binary acrylamide gel to give an EOF. In the case of conventional CEC, the surfaces of HPLC packings or capillary walls can exhibit sufficient SiOgroups to generate an EOF in our approach, the sulfo group of AMPS immobilized into the cross-linked polymer is ionized in solutions of a wide range of pH so that the generated EOF (9) Pretorius, V.; Hopkins, B. J.; Schieke J. D.]. Chromtogr. 1974,99, 23-30. (10) Jorgenson, J. W.; Lukacs, K D.J. Chromatogr. 1981,218,209-216. (11) Tsuda, T.; Nomura, IC;Nakagawa, G. J. Chromafogr. 1982,248,241-247. (12) Tsuda, T. Nippon Kagakukaishi, 1986, 937-942. (13) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369-2373. (14) Pfeffer. W. D.; Yeung, E. S. Anal. Chem. 1990,62, 2178-2182. (15) Pfeffer, W. D.; Yeung, E. S. J. Chromatogr. 1 9 9 1 , 5 5 7 , 125-136. (16) Gamer, T. W.; Yeung. E. S. J, Chromatogr. 1993, 640, 397-402. (17) Ymamoto, H.; Baumann, J.; Emi, F. J. Chromatogr. 1992,593, 313-319. (18) Mayer, S.: Schurig, V.J High. Resolut. Chromatogr. 1992,15, 129-131. (19) Bruin, B. J. M.; Tock. P. P. H.: Kraak, J. C.; Poppe, H. J. Chromafogr.1990, 517, 557-572. (20) Li, S.; Lloyd, D. K Anal. Chem. 1993,65, 3684-3690. (21) Ng, C. L.; Ong. C. P.; Lee, H. K.; Li, S. F. Y.]. Chromatogr. Sci. 1994,32, 121-125. (22) Martin, M.; Guiochon, G.: Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1985,57, 559-561. (23) Martin, M.; Guiochon, G. Anal. Chem. 1984. 56, 614-620.
0003-2700/95/0367-2050$9.00/0 0 1995 American Chemical Society
transports uncharged compounds as well as charged compounds through a bed of polyacrylamide gel. The presence of such ionized groups in polyacrylamide gels has been unfavorable to CGE because of the possible electrostatic interactions between ionized analytes (usually, large molecules such as proteins and DNA fragments) and the built-in, ionized groups, resulting in ruined separations. However, it should be possible to advantageously use EOF when uncharged molecules are being separated. We examined the separation performance of the gels using uncharged, low molecular weight compounds (
