Vitamin C (ascorbic acid) and Vitamins Group B are separated on Obelisc N mixed-mode column. Method can be used in quantitation and determination of polar vitamins in various formulations and dietary supplements. HPLC method can be based on UV, Evaporative Light Scattering Detection (ESLD), RI or MS detection. Effect of sample matrix can be eliminated by changing mobile phase conditions. Buffer concentration, buffer pH and amount of ACN will affect every vitamin differently due to difference in polar and ionic properties.