HPLC Separation of Vitamin C and Vitamin B2

Vitamin C (ascorbic acid) and Vitamin B2 (riboflavin) are water-soluble vitamins that are not stored in the body and must be replaced each day. Primesep D easily resolves Vitamins C and B2 and the elution order can be reversed by adjusting acetonitrile and acetic acid concentrations in the mobile phase. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and acetic acid (AcOH) and UV detection at 250 nm.


Column Primesep D , 4.6x150 mm, 5 µm, 100A
Mobile Phase MeCN/H2O
Buffer Acetic Acid
Flow Rate 1.0 ml/min
Detection UV, 250 nm


Class of Compounds Drug, Vitamin B₆, Hydrophobic, Ionizable
Analyzing Compounds Vitamins C (ascorbic acid) and B2 (riboflavin)

Application Analytes:

Vitamin B2 (Riboflavin)
Vitamin C