A Simple and Efficient Approach for Estimating Recovery of a Preparative Reversed Phase HPLC Purification

The widely applied reversed phase high-performance liquid chromatography (RP-HPLC) is an indispensable purification technique in drug discovery. During drug discovery, recovery was usually calculated based on the weight of the purified product after drying over the weight of the crude material multiplied by the assumed purity from HPLC/UV area percent of the product. Such a purity assumption can be off significantly when the crude material contains water, solvents, other UV-inactive impurities and inorganic salts. In this paper, we report a simple and efficient way to estimate recovery of preparative HPLC purification process. It is based on the ratio of the HPLC/UV peak area measured for the product in the crude solution and that in the final collected fraction with both accounted for their volumes. This approach eliminates not only the need for drying of the collected fraction to calculate recovery but also the inaccuracy associated with the true content in the crude sample using the traditional method. A systematic study was conducted to verify this method using caffeine mixed with various UV-active and -inactive impurities. The calculated recoveries using this approach were found to be consistent within 4% with the true recoveries based on dry weight estimation. The approach has been successfully applied for our in-house purifications. Furthermore, the approach was extended to library purifications, where in many cases heart-cutting the desired peaks is used to meet the purity requirements.

     

Enhanced Production of Laccase from Coriolus versicolor NCIM 996 by Nutrient Optimization Using Response Surface Methodology

Plackett and Burman design criterion and central composite design were applied successfully for enhanced production of laccase by Coriolus versicolor NCIM 996 for the first time. Plackett and Burman design criterion was applied to screen the significance of ten nutrients on laccase production by C. versicolor NCIM 996. Out of the ten nutrients tested, starch, yeast extract, MnSO(4), MgSO(4) x 7H(2)O, and phenol were found to have significant effect on laccase production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Plackett-Burman design. The optimized medium composition for production of laccase was (g/l): starch, 30.0; yeast extract, 4.53; MnSO(4), 0.002; MgSO(4) x 7H(2)O, 0.755; and phenol, 0.026, and the optimum laccase production was 6,590.26 (U/l), which was 7.6 times greater than the control.