Water soluble and fat soluble vitamins are vital to human health making it imperative to have a method to detect and quantify them in dietary supplements, drinks etc. Current methods of separation described in the United States Pharmacopeia are complicated and require ion pair reagents. Ion pairing irreversibly changes the chromatographic column making its use for other analyses impossible. Furthermore these methods need to be updated due to emergence of new technologies. The main goal of the project was to create a single High Performance Liquid Chromatography method that would allow separation of the water soluble vitamins: Ascorbic Acid, Biotin, Cyanocobolamin, Niacinamide, Panthothenic Acid, Pyridoxine, Riboflavin and Thiamin.

Analyses of the mixtures were performed on the Agilent 1200 LS Rapid Resolution LC system. The mobile phase was made of two components; A: 25 mM KH2PO4 buffer at pH = 2.5 and B: Methanol. The analytes were separated on Zorbax Eclipse Plus C18 (4.6 x 150 mm, 5 �m), (4.6 x 100 mm, 3.5 �m), and (4.6 x 50 mm, 1.8 �m) columns. As the column particle size decreased; the phase interaction, equilibrium, and separation became faster and more efficient. Identification of the vitamins in the standard mixture, supplements and vitamin drinks was confirmed using the Diode Array Detector with high-speed full spectral UV-Vis detection. This separation was completed in under 3.5 minutes under the conditions stated.