Electrophoresis chiral additives

Catechin and epicatechin are two flavanols of the catechin family. They are enantiomers. The capillary zone electrophoresis (CE) methods with UV-detection were developed for quantitative determination of this flavanols in green tea extracts. For this purpose following conditions were varied mnning buffers, pH and concentration of chiral additive (P-cyclodextrin was chosen as a chiral selector). Borate buffers improve selectivity of separation because borate can make complexes with ortho-dihydroxy groups on the flavanoid nucleus. 

Enantioresolution in capillary electrophoresis (CE) is typically achieved with the help of chiral additives dissolved in the background electrolyte. A number of low as well as high molecular weight compounds such as proteins, antibiotics, crown ethers, and cyclodextrins have already been tested and optimized. Since the mechanism of retention and resolution remains ambiguous, the selection of an additive best suited for the specific separation relies on the one-at-a-time testing of each individual compound, a tedious process at best. Obviously, the use of a mixed library of chiral additives combined with an efficient deconvolution strategy has the potential to accelerate this selection. 

Chiral additives have been shown to be very effective for chiral separations by capillary electrophoresis (CE) [4, 5]. Indeed, it may be argued that there has been considerably more research activity in chiral separations by CE than by EC methods since the introduction of the former technique. Chiral additives in CE have several advantages, some of which are highlighted in Table 11-2. 

Stalcup aiid co-workers [14] adapted this method to a continuous elution mini-prep electrophoresis apparatus shown in Fig. 11-3. In this apparatus, the end of the electrophoretic gel is continuously washed with elution buffer. The eluent can then be monitored using an HPLC detector (Fig. 11-4) and sent to a fraction collector where the purified enantiomers, as well as the chiral additive, may be recovered. In this system, the gel configuration was approximately 100 mm x 7 mm, and was aircooled. The number of theoretical plates obtained for 0.5 mg of piperoxan with this gel was approximately 200. A larger, water-cooled gel was able to handle 15 mg of... 

Kang, J. and Ou, Q., Chiral separation of racemic mexilitine hydrochloride using cyclodextrins as chiral additive by capillary electrophoresis,. Chromatogr. A, 795, 394, 1998. 

Lin et al. [95] used capillary electrophoresis with dual cyclodextrin systems for the enantiomer separation of miconazole. A cyclodextrin-modified micellar capillary electrophoretic method was developed using mixture of /i-cyclodextrins and mono-3-0-phenylcarbamoyl-/j-cyclodextrin as chiral additives for the chiral separation of miconazole with the dual cyclodextrins systems. The enantiomers were resolved using a running buffer of 50 mmol/L borate pH 9.5 containing 15 mmol/L jS-cyclodextrin and 15 mmol/L mono-3-<9-phcnylcarbamoyl-/j-cyclodextrin containing 50 mmol/L sodium dodecyl sulfate and 1 mol/L urea. A study of the respective influence of the /i-cyclodcxtrin and the mono-3-(9-phenylcarbamoyl-/i-cyclodextrin concentration was performed to determine the optical conditions with respect to the resolution. Good repeatability of the method was obtained. 

Phinney et al. [Ill] investigated the application of citrus pectins, as chiral selectors, to enantiomer separations in capillary electrophoresis. Successful enantioreso-lution of primaquine and other antimalarials, was achieved by utilizing potassium polypectate as the chiral selector. Changes in pH, chiral additive concentration, and capillary type were studied in relation to chiral resolution. The effect of degree of esterification of pectin materials on chiral recognition was evaluated. 

Capillary electrochromatography-mass spectrometry (CE-MS), 4 641 Capillary electrodes, 14 27 Capillary electrophoresis (CE), 4 602-603, 631-633 6 385 9 751-752 antibody based columns with, 6 402 chiral additives, 6 77-79 applications, 4 641 basic principles, 4 606-609 detectors, 4 634-635 for DNA analysis, 4 636-637 flow profiles generated, 4 608 instrumentation, 4 633 as microfluidic assay technique,... 

With capillary electrophoresis (CE), another modern primarily analytically oriented separation methodology has recently found its way into routine and research laboratories of the pharmaceutical industries. As the most beneficial characteristics over HPLC separations the extremely high efficiency leading to enhanced peak capacities and often better detectability of minor impurities, complementary selectivity profiles to HPLC due to a different separation mechanism as well as the capability to perform separations faster than by HPLC are frequently encountered as the most prominent advantages. On the negative side, there have to be mentioned detection sensitivity limitations due to the short path length of on-capillary UV detection, less robust methods, and occasionally problems with run-to-run repeatability. Nevertheless, CE assays have now been adopted by industrial labs as well and this holds in particular for enantiomer separations of chiral pharmaceuticals. While native cyclodextrins and their derivatives, respectively, are commonly employed as chiral additives to the BGEs to create mobility differences for the distinct enantiomers in the electric field, it could be demonstrated that cinchona alkaloids [128-130] and in particular their derivatives are applicable selectors for CE enantiomer separation of chiral acids [19,66,119,131-136]. 

Fillet, M., Hubert, P., Crommen, J. Method development strategies for the enantiosep-aration of drugs by capillary electrophoresis using cyclodextrins as chiral additives. Electrophoresis 1998, 19, 2834—2840. 

Riekkola, M. L., and Wiedmer, S. K. (1997). Potential of capillary electrophoresis with micelles or chiral additives as a purity control method in pharmaceutical industry. Process Control Qual. 10, 169-180. 

S Cladrowa-Runge, A Rizzi. Enantioseparations of 6-aminoquinolyl-lV-hydrox-ysuccinimidyl carbamate derivatized amino acids by capillary zone electrophoresis using native and substituted /3-cyclodextnns as chiral additives II. Evaluation of complexation constants. J Chromatogr A 759 167-175, 1997. 

M Hedeland, R Isaksson, C Pettersson. Cellobiohydrolase I as a chiral additive in capillary electrophoresis and liquid chromatography. J Chromatogr A 807 297-305, 1998. 

Another method for creating a chiral environment is lo add an optically pure chiral selector to a bulk liquid phase. Chiral additives have several advanlages over chiral stationary phases and continue lo be the predominant mode for chiral separations by tic and capillary electrophoresis (cc). First of all, the chiral selector added to a bulk liquid phase can be readily changed. The use of chiral additives allows chiral separations lo be done using less expensive, conventional stationary phases. A wider variety of chiral selectors are available [ be used as chiral additives than are available as chiral stationary phases, thus, providing the analyst with considerable flexibility. Finally, the use of chiral additives may provide valuable insight into (he chromatographic conditions and/or likelihood ol success with a potential chiral stationary-phase chiral selector. This is particularly important for the development of new chiral stationary phases because of the difficulty and cosl involved. 

Unlike in the case of a chiral column, the selectivity in CEC with chiral additives is determined by both partition and electrophoresis, and the electric field either increases or decreases the selectivity. Table 1 summarizes the relationship between the direction of field... 

Electrophoretic methods are widely used alternatives for the analytical determination of the enantiomeric purity of chiral compounds [194]. Due to the high elTi-ciency of capillary electrophoresis, separations can be achieved even when very low selectivities are observed. At a preparative scale, these methods are well established for the purification of proteins and cells [195] but there is very little published on enantioselective separations. Only recently, some interest in chiral preparative applications has been manifested. Separation of the enantiomers ofterbu-taline [196] and piperoxan [197] have been reported by classical gel electrophoresis using sulfated cyclodextrin as a chiral additive, while the separation of the enantiomers of methadone could be successfully achieved by using free-fluid isotachophoresis [198] and by applying a process called interval-flow electrophoresis [199]. 

In the group of Jung [90], a new approach has been developed for the use of selector libraries for chiral separations. Complex peptide libraries consisting of 183 different cyclo-hexapeptides in each sublibrary have been used as chiral additive in capillary electrophoresis. Three libraries separated enantiomers of a-amino acid derivatives as well as, e.g., Troger s base (Fig. 10.7). Meanwhile, our approach has also been applied successfully by Chiari et al. [91], who described the deconvolution of cyclopeptide libraries down to single selectors. 

Capillary electrophoresis has become extremely important in several application areas (a) enantiomeric separations can be achieved by the simple addition of a chiral additive to the run buffer (b) rapid and distinctive profiles for different isoform groups can be obtained by reversed-charge CE separations of heterogeneous mixtures and (c) inorganic and organic ion determination has improved significantly with new detection systems. 

The enantiomeric separation of some racemic antihistamines and antimalar-ials, namely (+/-)-pheniramine, (+/-)-bromopheniramine, (+/-)-chlorophen-iramine, (+/-)-doxylamine, and (+/-)-chloroquine, were investigated by capillary zone electrophoresis (CZE). The enantiomeric separation of these five compounds was obtained by addition of 7 mM or 1 % (w/v) of sulfated P-cyclo-dextrin to the buffer as a chiral selector. It was found that the type of substituent and degree of substitution on the rim of the cyclodextrin structure played a very important part in enhancing chiral recognition (174). The use of sulfated P-cyclo-dextrin mixtures as chiral additives was evaluated for the chiral resolution of neutral, cyclic, and bicyclic monoterpenes. While there was no resolution of the monoterpene enantiomers with the sulfated P-cyclodextrin, the addition of a-cyclodextrin resulted in mobility differences for the terpenoid enantiomers. Resolution factors of 4-25 were observed. The role of both a-cyclodextrin and sulfated P-cyclodextrin in these separations was discussed (187). The enantiomeric separation of 56 compounds of pharmaceutical interest, including anesthetics, antiarrhythmics, antidepressants, anticonvulsants, antihistamines, antimalarials, relaxants, and broncodilators, was studied. The separations were obtained at pH 3.8 with the anode at the detector end of the capillary. Most of the 40 successfully resolved enantiomers contained a basic functionality and a stereogenic carbon (173). 

M Fillet, I Bechet, Ph Hubert, J Crommen. Resolution improvement by use of carboxymethyl-P-cyclodextrin as chiral additive for the enantiomeric separation of basic drugs by capillary electrophoresis. J Pharm Biomed Anal 14 1107-1114, 1996. 

Ruta, J., Ravelet, C., Grosset, C., Fize, J., Ravel, A., Villet, A., Peyrin, E. (2006). Enantiomeric separation using an l-RNA aptamer as chiral additive in partial-filling capillary electrophoresis. Anal Chem 78, 3032-3039. 

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