Analysis of water soluble vitamins by high pressure liquid chromatography

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, muBondapak C18 and muBondapak NH2. The effect on the retention times of individual vitamins and the separation of a multivitamin 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 muBondapak 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 muBondapak 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 muBondapak 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 muBondapak C18.