Preparation of New HPLC Stationary Phase and Study of Its Chromatographic Performance Toward The Separation of Some Water-Soluble Vitamins

New stationary phase for high performance liquid chromatography has been prepared by the interaction of crystal violet solution with silica gel. The stability of the new attached functional group on the new stationary phase was studied against different solvents such as benzene, ethanol, acetonitrile, acetone, methanol, hexane, chloroform, and petroleum ether as well as NaOH and HCl solutions. The new prepared stationary phase was found stable and no depletion of the crystal violet moiety from the stationary phase was noticed with the above materials .The FTIR analysis has configured the attachment of crystal violet on the stationary phase as well as the physical appearance of the new stationary phase. The new stationary phase has been packed into stainless steel column. The chromatographic performance of the packed column was characterized. The number of plate numbers N, height equivalent of a theoretical plates H, capacity factors Ḱ, selectivity factors α, and peaks asymmetry, and Resolution Rs were measured by analyzing different analytes on the new columns using different mobile phase compositions and flow rates. These parameters were measured from analysis of some water-soluble vitamins such as vitamin C, vitamin B2, vitamin B6 and vitamin B12. The analysis of vitamins using silica gel-crystal violet column has performed using dichloromethane and hexane (5:95, (v/v)) as the mobile phase. The average value of plate numbers N was (356.4). The plate heights H was averaged (0.042) cm, capacity factors Ḱ were ranged from (1.83– 4.96). Separation factors α were ranged from (1.27-1.63), and peak asymmetry value were range from (0.99-1.18). Resolution (Rs) were ranged from (0.86-1.56). R.S.D were ranged from (0.025-0.065) %, recovery were ranged from (96.44-97.96) %, the relative errors were ranged from (-2.04_-3.56) %.
Calibration curves for all analyzed compound were linear from their detection limits to at least 50 ppm with correlation coefficient ranged from (0.9992- 0.9997).