Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2000-08-23
2003-09-09
Nguyen, Nam (Department: 1753)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S469000, C210S656000, C522S006000, C526S256000, C526S259000, C526S260000, C526S318440, C526S320000
Reexamination Certificate
active
06616825
ABSTRACT:
TECHNICAL FIELD
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.
BACKGROUND
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 technologies—open-tubular and monolithic columns—that 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; Hjertén (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 “superporous” 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 “shield”) undesired electroosmotic flow (EOF) along the interior wall of a column or channel. In addition, Peters et al. teach the use of ionic (or “pre-ionized”) 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 “pockets”. 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 enanti
Frechet Jean M. J.
Lämmerhofer Michael
Svec Frantisek
Nguyen Nam
Noguerola Alex
Reed Dianne E.
Reed & Eberle LLP
The Regents of the University of California
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