HPLC Chiral Separation Study Of Some Mannich Enantiomers

The chiral separations of Mannich enantiomers were studied by using Vancomycin (Chirobiotic V) and Cellulose (Sumichiral OA 2500s) Chiral Stationary Phase (CSP), Chiral Mobile Phase Additive (CMPA) and Chiral Ligand-Exchange Chromatography (CLEC) in this study. Influencing factors such as pH, type and concentration of buffer solution, type and concentration of organic modifier and column temperature were investigated to develop the best separation conditions. The separation mechanism of each method was also discussed.1. Chiral separation of some active Mannich enantiomers by CSPThe chiral separation of eight pairs of Mannich enantiomers was studied by using Vancomycin (Chirobiotic V) and Cellulose (Sumichiral OA 2500s) CSP. The separation mode, the mobile phase and column temperature were investigated in order to develop the best separation conditions with Chirobiotic V as the stationary phase. The best conditions were as follows: acetonitrile: ammonium nitrate(35mmol/mL, pH3.8)=15:85(v/v), column temperature 15℃, at a flow rate of 0.6mL/min,λ=300nm for compounds A and B; tetrahydrofuran: ammonium nitrate(20mmol/mL, pH4)=22:78(v/v), column temperature 15"C, at a flow rate of 0.6mL/min,λ=300nm for compounds C, D, G and H,λ=350nm for compounds E and F o Compounds A, C and G enantiomers got baseline separation, compound E enantiomers got partial separation. Comparing the chiral separation results and the structures of compounds, the separation mechanism was discussed. It was also found that the main interactions between the analyte and chiral stationary were p-p complex, hydrogen bonding and steric interactions. When Sumichiral OA 2500s was used, the effect of mobile phase and column temperature were also investigated. And the best conditions were as follow: n-hexane: ethanol=98: 2 (v/v), column temperature 15℃, flow rate 1.0mL/min,λ=300nm. Compound C enantiomers got baseline separation, and compound A enantiomers got partial separation.2. Chirai separation of some active Mannich enantiomers by CMPACMPA was adopted for the chiral separation of Mannich enantiomers. The factors affecting the resolution such as type and concentration of chiral additive, type and concentration of buffer solution, type and concentration of organic modifier and pH were investigated. At last, conditions of 5mmol/L potassium dihydrogen phosphate (SBE-β-CD 10mg/ml, pH4)-acetonitrile=80:20 (v/v), at a flow rate of 1.0mL/min, detection 300nm were developed for the chiral separation of compound C. Conditions of 30mmol/L citrate sodium (SBE-β-CD 3mg/ml, pH4)-acetonitrile=80:20 (v/v), at a flow rate of 1.0mI_/min, detection 350nm were developed for the chiral separation of compound F. Conditions of 20mmol/L citrate sodium (SBE-β-CD 15mg/ml, pH3)-acetonitrile=80: 20 (v/v), at a flow rate of 1.0mL/min, detection 300nm were developed of the chiral separation of compound G. Compounds F, G and C enantiomers were partial separated under the best condition, because the size of molecule, status of molecule and the electrostatic interaction between sulfobutyl ether-β-cyclodextrin and the analyte.3. Chiral separation of some active Mannich enantiomers by CLECCLEC was used for the chrial separation of Mannich enantiomers. The type and concentration of chiral ligand, type and concentration of organic modifier, the concentration ratio of chiral ligand to metal ion, pH and column temperature were studied for the optimization of separation condition, and the separation mechanism was also discussed. At last, the developed conditions were 2mmol/L L-APM, lmmol/L copper acetate in water (pH3.0)-methanol (75:25, v/v), at flow rate of 1.0mL/min, UV detectionλ=350 nm, temperature 30℃. Compound E enantiomers got a very good resolution. The developed method was extensively validated. The sample stability, linearity, precision (method repeatability and intermediate precision), accuracy and the limit of detection and limit of quantitation of the developed method were validated. The results shown that: the developed method had a good linearity in the range of 0.008mg/mL-4.0 mg/mL, the racemic solution was stable in 8 hours, the limits of detection of both enantiomers were 0.0035mg/mL and 0.0040mg/mL, the limits of quantitation of both enantiomers were 0.0076mg/mL and 0.0080mg/mL, and the method had a good precision and accuracy.