Analysis of Water Soluble Vitamins by High Pressure Liquid Chromatography

Abstract

Resolution of the water soluble vitamins—pyridoxin, riboflavin, niacinamide, vitamin B₁₂, thiamin, ascorbic acid, niacin and folic acid—by high pressure liquid chromatography was examined on two bonded phase columns, μ Bondapak C₁₈ and μ Bondapak NH₂. The effect on the retention times of individual vitamins and the separation of a multlvitamin sample was determined using varying proportions of water/methanol as the eluting solvent and by addition of various salts, buffer solutions and PIC reagents to the water/methanol. Each vitamin was able to be eluted satisfactorily from μ Bondapak C₁₈. It was found that seven vitamins could be resolved from a multivitamin mixture in a single analysis in several solvent systems with the total time for the analyses being always less than 40 min. With μ Bondapak NH₂, all the vitamins except folic acid were eluted and six vitamins could be resolved from a mixture in a single analysis. The speed of analysis was greater with μ Bondapak NH₂ with all compounds eluted in 15 min and the peaks were sharper. The order of elution was essentially the reverse of that obtained with μ Bondapak C¹⁸.