Chiral ligand exchange countercurrent chromatography: Enantioseparation of amino acids.

This work deals with the enantioseparation of α-amino acids by chiral ligand exchange high-speed countercurrent chromatography using N-n-dodecyl-l-hydroxyproline as a chiral ligand and copper(II) as a transition metal ion. A biphasic solvent system composed of n-hexane/n-butanol/aqueous phase with different volume ratios was selected for each α-amino acid. The enantioseparation conditions were optimized by enantioselective liquid-liquid extractions, in which the main influence factors, including type of chiral ligand, concentration of chiral ligand and transition metal ion, separation temperature, and pH of the aqueous phase, were investigated for racemic phenylalanine. Altogether, we tried to enantioseparate 15 racemic α-amino acids by the analytical countercurrent chromatography, of which only five of them could be successfully enantioseparated. Different elution sequence for phenylalanine enantiomer was observed compared with traditional liquid chromatography and the proposed interactions between chiral ligand, transition metal ion (Cu2+ ), and enantiomer are discussed.

Application of pH-zone-refining countercurrent chromatography in the chiral separation of two ß-adrenergic blocking agents.

Two ß-adrenergic blocking agents, 1-[(1-methylethyl)amino]-3-phenoxy-2-propanol (1) and 1-[(1-methylethyl)amino]-3-(3-methylphenoxy)-2-propanol (2; Toliprolol), were enantioseparated by pH-zone-refining countercurrent chromatography. A two-phase solvent system composed of chloroform containing 0.10 mol/L of di-n-hexyl l-tartrate/0.10 mol/L of boric acid aqueous solution (1:1, v/v) was selected, in which 20 mmol/L triethylamine was added in the organic phase as a retainer and 2 mmol/L HCl was added in the aqueous phase as an eluter. Fifty milligrams of each racemate was completely enantioseparated by pH-zone-refining countercurrent chromatography to yield each enantiomer with a purity of more than 98%, and the recovery of each separated enantiomer reached around 76-82%.

Silver ion coordination countercurrent chromatography: Separation of ß-elemene from the volatile oil of Curcumae Rhizoma.

In this work, the antitumor constituent ß-elemene was selectively separated from the volatile oil of the Curcumae Rhizoma by countercurrent chromatography with silver nitrate as selective reagent based on the formation of coordination complexes. A biphasic solvent system composed of n-hexane/methanol/water (2:1.5:0.5, v/v/v) was selected, in which 0.15 mol/L of silver nitrate was added to the aqueous phase. The aqueous phase was used as the stationary phase for separation of ß-elemene by countercurrent chromatography after it was partially purified from the volatile oil by silica gel column chromatography. An enriched ß-elemene fraction was obtained by silica gel column chromatography to improve the percentage of ß-elemene from 16.5 to 46.1%. Subsequently, ß-elemene was further purified from 445 mg of the partially purified sample of volatile oil by countercurrent chromatography with silver nitrate as a selective reagent, yielding 145 mg of ß-elemene with greater than 99% purity, as determined by gas chromatography mass spectrometry. The recovery of ß-elemene from the crude volatile oil through two steps was around 63.6%.

Separation of epimeric aromatic acid (-)-menthol esters by countercurrent chromatography using hydroxypropyl-ß-cyclodextrin as an additive.

The separation of ten epimeric aromatic acid (-)-menthol esters by countercurrent chromatography with hydroxypropyl-ß-cyclodextrin as the mobile phase additive was investigated, and methods for the analysis of all the epimeric esters by reversed-phase high-performance liquid chromatography were established. A biphasic solvent system composed of n-hexane/20-70% methanol containing 50 mmol/L of hydroxypropyl-ß-cyclodextrin (1:1, v/v) was selected, which provided high separation factors for five of the epimeric esters, and successful separations by countercurrent chromatography were achieved. The complete separation of five pairs of epimeric ester was obtained with the purity being over 98% for each peak fractions, as determined by high-performance liquid chromatography. The recovery of each analyte from the eluted fractions reached around 80-88%.

pH-zone-refining elution-extrusion countercurrent chromatography: Separation of hydroxyanthraquinones from Cassiae semen.

Seven hydroxyanthraquinones were successfully separated from the traditional Chinese medicinal herb Cassiae semen by conventional and pH-zone-refining countercurrent chromatography with an environmentally friendly biphasic solvent system, in which elution-extrusion mode was investigated for pH-zone-refining countercurrent chromatography for the first time. A two-phase solvent system composed of n-hexane/ethyl acetate/ethanol/water (5:3:4:4, v/v/v/v) was used for the conventional countercurrent chromatography while the same system with a different volume ratio n-hexane/ethyl acetate/ethanol/water (3:5:2:6, v/v/v/v) was used for pH-zone-refining countercurrent chromatography, in which 20 mmol/L of trifluoroacetic acid was added in the organic phase as a retainer and 15 mmol/L of ammonia was added to the aqueous phase as an eluter. A 400 mg crude sample could be well separated by pH-zone-refining countercurrent chromatography, yielding 53 mg of aurantio-obtusin, 40 mg of chryso-obtusin, 18 mg of obtusin, 24 mg of obtusifolin, 10 mg of emodin, and 105 mg of the mixture of chrysophanol and physcion with a purity of over 95.8, 95.7, 96.9, 93.5, 97.4, 77.1, and 19.8%, as determined by high-performance liquid chromatography. Furthermore, the difference in elution sequence between conventional and pH-zone-refining mode was observed and discussed.

Enantioseparation of 3-phenyllactic acid by chiral ligand exchange countercurrent chromatography.

3-Phenyllactic acid is an antimicrobial compound with broad-spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3-phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3-phenyllactic acid with a synthesized chiral ligand. A two-phase solvent system was composed of n-butanol/hexane/water (0.4:0.6:1, v/v/v) to which N-n-dodecyl-l-hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid-liquid extraction. Baseline enantioseparation of racemic 3-phenyllactic acid by analytical high-speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3-phenyllactic acid reached 99.0%, as determined by chiral high-performance liquid chromatography.

Stereoselective separation of ß-adrenergic blocking agents containing two chiral centers by countercurrent chromatography.

Four ß-adrenergic blocking agents, including 1-[(1-methylethyl)amino]-3-phenoxy-2-propanol (1), 1-[(1-methylethyl)amino]-3-(3-methylphenoxy)-2-propanol (2), 1,1'-[1,4-phenylenebis(oxy)]bis[3-[(1-methylethyl)amino]-2-propanol (3) and 1,1'-[(4-methyl-1,2-phenylene)bis(oxy)]bis[3-[(1-methylethyl)amino]-2-propanol (4), were stereoselectively separated by countercurrent chromatography using di-n-hexyl l-tartrate and boric acid as chiral selector. The compounds (3) and (4) have four optical isomers since they contained two chiral centers. A two-phase solvent system composed of chloroform-0.05molL-1 of acetate buffer containing 0.10molL-1 of boric acid (1:1, v/v) was selected, in which 0.10molL-1 of di-n-hexyl l-tartrate was added in the organic phase as chiral selector. 20-42mg of each racemate was stereoselectively separated by countercurrent chromatography in a single run with high purity of 96-98%, and the recovery of each separated compound reached around 87-93%. This is the first time report on successful stereoselective separation of optical isomeric compounds containing two chiral centers by countercurrent chromatography. At the same time, a chiral stationary phase was screened for analytical stereoselective separation of compounds (3) and (4) by high performance liquid chromatography.

[Separation and purification of acteoside from Rehmannia glutinosa by combining macroporous resin with high-speed countercurrent chromatography].

Acteoside,a chemical component of the traditional Chinese medicinal herb Rehmannia glutinosa,was separated from the plant by a combination of macroporous resin column chromatography and high-speed countercurrent chromatography.The static adsorption and desorption of acteoside in the crude extract were in-vestigated using four kinds of macroporous resin,among which the D101 macroporous resin presented the best adsorption and desorption rates toward the compound.The crude extract was then gradient-eluted with increa-sing volume percentages of ethanol,where it was found that the highest content of acteoside was obtained when using 10%(v/v) ethanol eluent,in which the purity of acteoside was increased from 4.9% to 32.6%.Then,the partially purified crude extract (165 mg) was further purified by high-speed countercurrent chroma-tography using a two-phase solvent system consisting of ethyl acetate-n-butanol-water (1:4:5,v/v/v),yielding 45 mg of acteoside with 96% purity.