Response surface methodology used in the optimization of RP-HPLC condition for separation of carmine and rhodamine B

Abstract

Carmine (CAR) is one of the permitted coloring agents used in cosmetics, whereas Rhodamine B (RDB) is prohibited as a coloring agent. Due to its similarity color, CAR is frequently replaced by RDB. The objective of this study was to optimize reversed phase high performance liquid chromatography (RP-HPLC) using the experimental design approach based on response surface methodology of Central Composite Design (CCD) for the separation and analysis of CAR and RDB in lipstick products. Some factors (independent variables) responsible for RP-HPLC separation including pH of buffer phosphate (X1), the acetonitrile ratio (X2), flow rate of mobile phase (X3), and column temperature (X4) were investigated. On the other hand, the responses (dependent variables) evaluated were resolution between CAR and RDB (Y1), tailing factor of CAR (Y2), tailing factor of RDB (Y3), retention time of CAR (Y4), retention time of RDB (Y5), peak area of CAR (Y6) and peak area of RDB (Y7). CCD showed that separation of CAR and RDB was influenced by those independent variables (factors). The optimum predicted conditions for separation of CAR and RDB based on statistical results was pH buffer of 3.4, ACN 55%, flow rate of 1.1 mL/min and column temperature of 35̊C with desirability of 1. Both CAR and RDB were clearly separated using optimum condition, as suggested by CCD. The developed techniques were effective for optimizing chromatographic separation, therefore, the time consumption and large number of running during RP-HPLC analysis could be hindered.