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Counterflow in isotachophoresis: computer simulation and experimental studies.

A computer model has been developed to predict the effects of co- and counter-current flows on electrophoretic transport. Model predictions are compared with experimental data obtained with the large-scale recycling isotachophoresis apparatus, recently developed in our laboratory. There were no significant changes in the steady state properties or the transient development of the Kohlrausch-adjusted profiles with and without flow. The application of counterflow in free flow isotachophoresis in early stages, before a steady state is achieved, can result in selective wash-out of slower sample components. Thus, the magnitude of counterflow can be used as a variable in optimizing separations. The characteristic features of the model render it also suitable for the simulation of electroosmotic flow in capillary electrophoresis in open tubes, where plug-type flow prevails.

Anions↗

Implementation of a semiclosed large scale counterflow centrifugal elutriation system.

A semiclosed counterflow centrifugal elutriation system that minimizes the risks of contamination and cell loss is presented. A detailed description of the configuration of the system and its sterilization and assembly is provided along with examples of its application to large-scale separation of leukapheresis buffy coat and cadaveric bone marrow. We are currently using this system to deplete lymphocytes from human bone marrow prior to use in allogeneic bone marrow transplantation. The implementation of a semiclosed system increases the safety, ease of operation, and reproducibility of a technique that has the potential for a wide range of clinical applications.

Cell Separation↗

A preparative isolation and purification of arctigenin and matairesinol from Forsythia koreana by centrifugal partition chromatography.

Centrifugal partition chromatography was applied to separate arctigenin and matairesinol from Forsythia koreana extract with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:5:5:5 v/v). Using this method, arctigenin and matairesinol were successfully separated from partially purified F. koreana extracts in only one step. The purities of isolated compounds were determined to be over 90% by HPLC analysis.

Chromatography, High Pressure Liquid↗

Isolation and purification of oridonin from Rabdosia rubescens using upright counter-current chromatography.

A preparative counter-current chromatography (CCC) method for isolation and purification of oridonin, a new cancer chemoprevention agent, from the Chinese medicinal plant Rabdosia rubescens was successfully established. The crude oridonin was obtained by elution with a light petroleum/acetone solvent mixture from ethanol extracts of R. rubescens using column chromatography on silica gel. With a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:2:1:2, v/v), 120 mg of oridonin at the purity of 97.8% was obtained from 200 mg of the crude sample in a single-step CCC separation. The structure of oridonin was identified by ESI-MS, 1H NMR, and 13C NMR.

Chromatography, High Pressure Liquid↗

Isolation and purification of canthaxanthin from the microalga Chlorella zofingiensis by high-speed counter-current chromatography.

Certain microalgae are considered to be a potential source of canthaxanthin, which possesses strong antioxidant and anticancer activities. A high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of canthaxanthin from the microalga Chlorella zofingiensis. The crude canthaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative HSCCC, with a two-phase solvent system composed of n-hexane-ethanol-water (10:9:1 v/v), was successfully performed yielding canthaxanthin at 98.7% purity from 150 mg of the crude extract (2.1% canthaxanthin) in a one-step separation. The recovery of canthaxanthin was 92.3%. This was the first report that canthaxanthin was successfully separated and purified from microalgae.

Animals↗

Optimisation of the derivatization in cellulose-type chiral selectors for enantioseparation by centrifugal partition chromatography.

Cellulose was chemically modified with hydrophobic dodecanoyl groups followed by 3,5-dimethylphenylcarbamoyl substituents forming mixed ester/carbamate derivatives in order to improve the solubility in lipophilic solvents compared to the corresponding homosubstituted cellulose tris(3,5-dimethylphenylcarbamate). Two mixed derivatives of different degree of substitution were prepared, tested as chiral selectors (CSs) in counter-current chromatography (CCC) and compared to the homo-substituted derivative. Alternatively, cellulose tris(3,5-dichlorophenylcarbamate), was synthesised and tested with the same purpose. The racemic drugs pindolol and warfarin were used as test compounds. Biphasic organic/aqueous solvent systems, composed of methyl isobutyl ketone, t-butyl methyl ether or ethyl acetate and aqueous ammonium acetate or sodium phosphate buffer, were used. Centrifugal partition chromatography, a variety of CCC, in the classical elution mode and the pH-zone-refining displacement mode was applied. The enantioseparation of pindolol and warfarin was achieved in the latter conditions. The presence of dodecanoyl chains on the CS increased solubility in the organic solvents used. The selectivity of the mixed dodecanoyl/3,5-dimethylphenylcarbamoyl cellulose derivative with low degree of dodecanoyl groups (degree of substitution 0.3/degree of substitution 2.6, respectively) was similar to that of the homo-substituted derivative. Furthermore, the loading capacity for pindolol was increased by a factor of three compared to cellulose tris(3,5-dimethylphenylcarbamate). Nevertheless, the increasing degree of substitution with dodecanoyl groups on the CS, although improved solubility in the stationary phase, contributed negatively to the enantioselectivity, where warfarin was more affected than pindolol.

Cellulose↗

Preparative isolation of hydrolysable tannins chebulagic acid and chebulinic acid from Terminalia chebula by high-speed counter-current chromatography.

As a chromatographic column, the high-speed counter-current chromatography system was equipped with a preparative HPLC series, enabling the successful isolation of hydrolysable tannins from the fruits of Terminalia chebula, a traditional Chinese medicine. The two-phase solvent system was composed of n-hexane-ethyl acetate-methanol-water (1:20:1:20 v/v). As a result, 33.2 mg chebulagic and 15.8 mg chebulinic acids were obtained in one step from 300 mg of crude extract. Their purities were determined by HPLC to be 95.3 and 96.1%, respectively. The chemical structures were identified by their MS and 1H NMR spectra.

Benzopyrans↗

One step purification of corilagin and ellagic acid from Phyllanthus urinaria using high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) has been successfully applied to the preparative separation of corilagin and ellagic acid in one step from the Chinese medicinal plant Phyllanthus urinaria L. by use of direct and successive injections of a crude methanolic extract. Some aspects concerning the practical use of this technique in the described application are considered.

Countercurrent Distribution↗

High-speed countercurrent chromatography as a valuable tool to isolate C-glycosylflavones from Cecropia lyratiloba Miquel.

A new apigeninglycoside, apigenin 6-C-galactosyl-6"-O-beta-galactopyranoside (1), isoorientin, and a mixture of orientin and isovitexin were isolated from leaves of Cecropia lyratiloba by high-speed countercurrent chromatography using a solvent system containing ethyl acetate, butanol, methanol and water. The structural elucidation of 1 was based on NMR spectroscopy.

Apigenin↗