Direct chiral separation of abscisic acid by high-performance liquid chromatography with a phenyl column and a mobile phase containing γ-cyclodextrin.

Abscisic acid (2-cis,4-trans-abscisic acid) is a plant hormone that has an asymmetric carbon atom. We tried to separate the enantiomers of native abscisic acid by HPLC using a phenyl column and a chiral mobile phase containing γ-cyclodextrin. The optimum mobile phase conditions were found to be 0.8% (w/v) γ-cyclodextrin, 4% (v/v) acetonitrile, and 20 mM phosphate buffer (pH 6.0). It was found that (R)-abscisic acid was earlier detected than (S)-abscisic acid. Since γ-cyclodextrin is hardly retained on a phenyl column, it was suggested that (R)-abscisic acid formed a more stable complex with γ-cyclodextrin than the (S)-abscisic acid. Abscisic acid in an acacia honey sample was successfully enantioseparated with the proposed method and only (S)-abscisic acid was detected. A biologically inactive 2-trans,4-trans-abscisic acid, which was prepared by irradiation of abscisic acid with a light-emitting diode lamp at 365 nm, was partially enantioseparated by the proposed method. Since the irradiation of (S)-abscisic acid-induced cis-to-trans isomerization to produce one 2-trans,4-trans-abscisic acid enantiomer, it is reasonable that racemization did not proceed during the cis-to-trans isomerization. (S)-Abscisic acid and probably (S)-2-trans,4-trans-abscisic acid were detected in a honey sample, where the peak area of (S)-abscisic acid was 7 times larger than that of (S)-2-trans,4-trans-abscisic acid.

Chiral separation of catechin and epicatechin by reversed phase high-performance liquid chromatography with ß-cyclodextrin stepwise and linear gradient elution modes.

Catechin and epicatechin were enantioseparated by high-performance liquid chromatography (HPLC) with a phenyl column and aqueous mobile phases containing 0.05% (w/v) and 0.6% (w/v) of ß-cyclodextrin for catechin and epicatechin, respectively. ß-Cyclodextrin was found to be scarcely retained on a phenyl column. Consequently, it was suggested that catechin, which was eluted earlier than epicatechin, formed more stable inclusion complex with ß-cyclodextrin than epicatechin and earlier eluted enantiomers, (-)-catechin and (+)-epicatechin, formed more stable diastereomer complexes with ß-cyclodextrin than the respective enantiomers. This was confirmed by ß-cyclodextrin-modified micellar electrokinetic chromatography and Benesi-Hildebrand plots by fluorescence spectrophotometry. Effect of sugars (D-sucrose, D-glucose, and D-fructose) on the epimerization of (+)-catechin and (+)-epicatechin by heating was investigated by HPLC with a ß-cyclodextrin stepwise elution mode, in which two kinds of aqueous eluents containing different concentrations of ß-cyclodextrin were used by turns. The epimerization of the two enantiomers was suppressed only when D-fructose was added. Separation of ten kinds of catechins including catechin and epicatechin enantiomers was investigated by a ß-cyclodextrin linear gradient HPLC elution mode without using organic solvents, where two kinds of aqueous eluents containing different concentrations of ß-cyclodextrin were used with changing their ratio gradually. These catechins in a green tea infusion could be separated successfully by this method.

Enantioseparation of phenethylamines by using high-performance liquid chromatography column permanently coated with methylated ß-cyclodextrin.

Cyclodextrins and their derivatives have been used for chiral high-performance liquid chromatography selectors, while they are costly to use as mobile phase additives in high-performance liquid chromatography. Here, we report application of phenyl column coated permanently with methylated ß-cyclodextrin for chiral high-performance liquid chromatography. A 0.1% (v/v) phosphoric acid solution containing 1 M NaCl and 0.5% (w/v) methylated ß-cyclodextrin was subjected to a phenyl column at a flow rate of 0.5 mL/min at 30°C for 2 h. Using the precoating phenyl column, all the enantiomers of the four phenethylamines (norepinephrine, epinephrine, octopamine, and synephrine) were successfully separated simultaneously by high-performance liquid chromatography with a mobile phase without methylated ß-cyclodextrin at a flow rate of 0.2 mL/min at 30°C. The enantioseparation ability was retained for successive analyses during 1 week. It is suggested that inclusion complex of methylated ß-cyclodextrin with a phenyl group on the surface of the stationary phase could be formed and that the inclusion complex could form the ternary complex with the injected analytes. The longer retention time of (S)-enantiomers of analytes than corresponding (R)-enantiomers for high-performance liquid chromatography could be explained from the higher stability of the methylated ß-cyclodextrin complexes with (S)-enantiomers, which were confirmed by capillary electrophoresis and 1 H NMR spectroscopy experiments.

Direct enantioseparation of mandelic acid by high-performance liquid chromatography using a phenyl column precoated with a small amount of cyclodextrin additive in a mobile phase.

Direct enantioseparation of mandelic acid by high-performance liquid chromatography (HPLC) with a reversed phase column and a mobile phase containing a small amount of hydroxylpropyl-ß-cyclodextrin (HP-ß-CD) was studied as an efficient method for saving consumption of the CD additive. As a result, it was proposed that racemic mandelic acid can be analyzed with a phenyl column by using a mobile phase composed of 10 mM ammonium acetate buffer (pH 4.2) and 0.02% (w/v) HP-ß-CD at a flow rate of 1.0 mL/min at 40°C after the passage of 10 mM ammonium acetate buffer (pH 4.2) containing 0.1% (w/v) HP-ß-CD as a precoating mobile phase for 60 min. It is suggested that HP-ß-CD is bound with a phenyl group on the surface of the stationary phase to allow a phenyl column to act as a transient chiral column, and injected mandelic acid can form the ternary complex with the adsorbed HP-ß-CD. The longer retention time of D-mandelic acid than the L-isomer for HPLC can be explained from the higher stability of the HP-ß-CD complex with D-mandelic acid, which was confirmed by CE experiment with HP-ß-CD as a selector. The efficiency of a phenyl column compared with other stationary phases was also discussed.

Effect of ethanol addition on the determination of thiosulfate based on reduction of Ce(IV) and fluorescence detection of Ce(III).

The effect of ethanol addition on the determination of thiosulfate based on the reduction of Ce(IV) and fluorescence detection of Ce(III) was investigated by flow-injection analysis. It was found that the sensitivity of thiosulfate detection was significantly increased by injecting thiosulfate into a mixed solution of Ce(IV) and ethanol, rather than a solution of Ce(IV) alone. This is probably due to trace amounts of thiosulfate accelerating the rate of reduction of Ce(IV) by ethanol: Ce(IV) is slowly reduced by ethanol in the absence of thiosulfate, and thiosulfate serves as a catalyst to the reduction. The detection limits as S/N = 3 for thiosulfate were very low (10(-9) M level).