This invention relates generally to the field of electrochromatographic separation, and more particularly relates to a novel enantioselective separation medium comprised of a monolithic, chiral, ionizable copolymer.
The original promise of electrochromatography to improve the efficiency of liquid chromatography by using an electrical field to achieve plug-like electroosmotic flow (EOF) for transporting analytes through a chromatographic column has materialized only recently. See, for example, Dittman et al. (1996) J. Chromatogr. A 744:6374; Cikalo et al. (1998) Analyst 123:87R-102R; and Majors (1998) LC-GC 16:96-100. Capillary electrochromatography (CEC) continues to develop rapidly and find applications in a variety of areas, including the separations of enantiomers. See Wistuba, D.; Schurig, V. J.Chromatogr. (2000) 875: 255-276 and references cited therein. Several groups have adapted an HPLC-like bead approach to a capillary column format in an attempt to achieve the high efficiencies predicted by theory. Although packed capillaries are currently the most common column technology, this approach is accompanied by several difficulties. For example, the surface charge often results only from residual surface silanols, making effective control of the magnitude and direction of EOF poor. Additionally, column packing procedures are often tedious, requiring in situ frit fabrication. These frits may have limited stability and/or permeability, and their heterogeneities may initiate spontaneous outgassing and bubble formation. These problems have led to the development of new column technologiesxe2x80x94open-tubular and monolithic columnsxe2x80x94that eliminate many of the drawbacks of packed capillary columns.
In open-tubular electrochromatography (OT-EC), the stationary phase is covalently attached, coated, or adsorbed onto the inner capillary wall. See Tsuda et al. (1982) J. Chromatogr. 248:241-247; Guo et al. (1995) Anal. Chem. 67:2511-2516; and Sawada et al. (1999) Electrophoresis 20:24-30. Since the surface of the open tube is very limited, these columns only afford a low sample capacity. Selective etching of the wall may be used to increase the overall surface area and improve the loadability (Pesek (2000) J. Chromatogr. A 887:31-42). In contrast, monolithic stationary phases often possess much higher surface areas and adsorption capacities. To date, several different approaches to monolithic CEC columns have been reported. Siliceous monoliths for CEC have been prepared by polycondensation of alkoxysilanes using a sol-gel process within the capillary tubing followed by post-functionalization, as reported by Tanaka et al. (2000) J. High Resol. Chromatogr. 23:111-116. In order to minimize the risk of shrinkage typical of sol-gel transitions that can lead to cracks in the bed, the overall volume of the inorganic matrix has been reduced by filling the column with traditional chromatographic particles prior to initiating the sol-gel process (Dulay et al. (1998) Anal. Chem. 70:5103-5107; Tang et al. (1999) J. Chromatogr. A 837:35-50; Chirica et al. (1999) Electrophoresis 20:50-56; Ratnayake et al. (2000) J. High Resol. Chromatogr. 23:81-88). Consolidation of a packed bed by sintering the particles has also been proposed as a method for the preparation of monolithic columns (see Dittman et al. (1997) J. Capil. Electrophoresis 4:201-212 and Asiaie et al. (1998) J. Chromatogr. A 806:251-263) but this technique is even more laborious and the surface chemistry of the stationary phase is often destroyed during the sintering process necessitating post-functionalization. As described by Svec et al. (2000) J. High Resol. Chromatogr. 23:3-18 and Svec et al. (2000) J. Chromatogr. A, 887:3-30, functional monomers have been polymerized in situ within bare or vinylized fused silica tubing in the presence of pore forming solvents to yield continuous porous crosslinked organic polymers. Examples of this approach include polyacrylamide-based gels (Liao et al. (1996) Anal. Chem. 68:3468-3472; Ericson et al. (1999) Anal. Chem. 71:1621-1627; Hjertxc3xa9n (1999) Ind. Eng. Chem. Res. 38:1205-1214; Fujimoto et al. (1995) J. Chromatogr. A 716:107-113; Fujimoto et al. (1996) Anal. Chem. 68:2753-2757) polyacrylamide copolymers prepared in the presence of poly(ethylene glycol) (Palm et al. (1997) Anal. Chem. 69:4499-4507) molecularly imprinted xe2x80x9csuperporousxe2x80x9d monoliths (Schweitz et al. (1997) Anal. Chem. 69:1179-1183; Nilsson et al. (1994) J. Chromatogr. A 680:57-61; Schweitz et al. (1998) J. Chromatogr. A 817:5-13), highly crosslinked polystyrene (Gusev et al. (1999) J. Chromatogr. A 855:273-290; Xiong et al. (2000) J. High Resol. Chromatogr. 23:67-72) and polymethacrylate matrices (Peters et al. (1997) Anal. Chem. 69:3646-3649; Peters et al. (1998) Anal. Chem. 70:2288-2295; Peters et al. (1998) Anal. Chem. 70:2296-2302).
However, only a very limited number of studies have attempted the use of monolith technology for enantiomeric separations. These include Schweitz et al. (1997), supra, Peters et al. (1998) Anal. Commun. 35:83-86, supra, Nilsson et al. (1994), supra, Koide et al. (1999) Anal. Sci. 15:791-794, and Koide et al. (2000) J. High Resol. Chromatogr. 23:59-66. Peters et al. (1998) is a representative reference, and describes an enantiomeric separation medium prepared by copolymerization of multiple monomers including an ionic monomer (2-acrylamido-2-methyl-1-propane sulfonic acid), a chiral monomer, a crosslinking monomer, and a functional monomer. It has been found, however, that using separate ionic and chiral monomers does not allow one to achieve a high content of both monomers in the separation medium simultaneously. In particular, ionic monomers often have poor solubility in the polymerization mixture.
Thus, Peters et al. (1998) and other prior methods proposed for preparing monolithic materials suitable for enantiomeric separations have suffered from several drawbacks. Primarily, no one monolithic material is capable of performing a variety of separate functions, e.g., the ability to carry charge, the ability to consistently and specifically attract a single enantiomer from a racemic mixture, the ability to facilitate electroosmotic flow (i.e., to act as an electroosmotic pump), and the ability to substantially reduce (or xe2x80x9cshieldxe2x80x9d) undesired electroosmotic flow (EOF) along the interior wall of a column or channel. In addition, Peters et al. teach the use of ionic (or xe2x80x9cpre-ionizedxe2x80x9d) monomers to incorporate charge into monoliths. This reduces the versatility of the method as few such monomers exist and those available are often poorly soluble in a largely organic medium, and the high reactivity of the ionic monomer may have a deleterious effect on polymerization.
There is accordingly a need in the art for a monolithic material that is effective in enantiomeric separation and simultaneously provides a variety of functions, namely: (1) acts, as a chromatographic packing material; (2) provides a continuous tortuous path and a large interacting surface for a flowing liquid; (3) performs specific chiral recognition; (4) acts as a charge carrier; (5) acts as an electroosmotic pump; and (6) acts as a surface coating along a column or channel wall. The present invention now provides methods, materials and separation devices that simultaneously fulfill all of the aforementioned requirements. The novel monolithic material has ionizable functionalities as well as multiple interaction sites located within a rigid molecular framework, the interactions sites containing both stereogenic centers and bulky groups to form series of favorable binding xe2x80x9cpocketsxe2x80x9d. The chiral species within the monolithic material provide the surface charges required to generate EOF and therefore eliminate the need for the addition of a charged comonomer, and affords the required stereoselective interactions with complementary chiral analytes, resulting in the separation of enantiomers. Furthermore, capillaries, columns and channels suitable for effecting enantiomeric separations may be readily prepared in a simple and straightforward manner using a simple molding process and in situ polymerization so as to avoid the fabrication of frits and the packing of small beads into capillaries.
Accordingly, it is a primary object of the invention to provide a device for use in conducting chiral electrochromatography using an enantioselective separation medium that overcomes the aforementioned drawbacks in the art.
It is another object of the invention to provide such a device in the form of a capillary tube, microchannel, or the like, i.e., a device that comprises an electrochromatographic conduit containing an enantioselective separation medium of the invention.
It is still another object of the invention to provide such a device wherein the enantioselective separation medium is a monolithic, ionizable copolymer that acts as a continuous separation medium and contains pendant chiral selector groups.
It is yet another object of the invention to provide such a device wherein the enantioselective separation medium is a porous organic polymer.
It is an additional object of the invention to provide a method for making the aforementioned device.
It is a further object of the invention to provide an enantioselective separation medium for use in the aforementioned device.
It is still a further object of the invention to provide a method for making an enantioselective separation medium for use in the electrochromatographic devices of the invention.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
In one embodiment of the invention, then, a device is provided for use in chiral electrochromatography, wherein the device comprises an electrochromatographic conduit containing an enantioselective separation medium comprised of a monolithic, ionizable copolymer that acts as a continuous separation medium and contains pendant chiral selector groups. Because the monolithic copolymer is ionizable and contains stereogenic centers (i.e., in the pendant chiral selector groups), once the separation medium is ionized it acts as charge carrier as well as chiral selector. The electrochromatographic device may comprise a capillary tube containing an enantioselective separation medium of the invention, or the device may be a microfluidics separation device wherein the enantioselective separation medium is contained in one or more xe2x80x9cmicrochannelsxe2x80x9d within the device.
In another embodiment of the invention, an enantioselective separation medium is provided that comprises a chiral copolymer prepared by copolymerization of a mixture comprising (a) an ionizable chiral monomer, (b) a crosslinking comonomer, (c) a polymerization initiator, (d) a porogenic solvent, and optionally (e) a functionalized monovinyl comonomer. In preferred embodiments, the functionalized monovinyl comonomer is employed; typically, the functionalized monovinyl comonomer contains a hydrophilic moiety (e.g., a hydroxyl group) or a precursor to a hydrophilic moiety (e.g., an epoxy group).
In a related embodiment of the invention, an enantioselective separation medium is provided that comprises a chiral copolymer prepared by copolymerization of a mixture comprising (a) a chiral monomer; (b) a crosslinking comonomer, (c) a polymerization initiator, (d) a porogenic solvent, (e) an ionizable comonomer or a precursor thereto, and optionally (f) a functionalized monovinyl comonomer. In preferred embodiments, the functionalized monovinyl comonomer is employed, as above.
In other embodiments of the invention, methods are provided for preparing the aforementioned enantioselective separation medium and electrochromatographic separation device, the methods involving copolymerization of a polymerization mixture comprising (a) an ionizable chiral monomer, (b) a crosslinking comonomer, (c) a polymerization initiator, (d) a porogenic solvent, and optionally (e) a functionalized monovinyl comonomer. In a related embodiment, a separate ionizable comonomer is incorporated into the polymerization mixture, in which case the chiral monomer may or may not be ionizable. Again, in preferred embodiments, the functionalized monovinyl comonomer is employed. Polymerization may be thermally initiated or initiated using radiation, typically ultraviolet (UV) radiation.