HPLC Separation of Two Vitamins: Different Polarity – Isocratic Methods

Primesep B and B2 columns separate Vitamins C (ascorbic acid) and B2 (riboflavin) with tunable selectivity. Peak order reversal is exhibited on these two columns with the same mobile phases due to their different polarity. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and either formic or acetic acid (HOAc) with UV detection at 250 nm.


Column Primesep B, Primesep B2 , 3.2x50 mm, 5 µm, 100A
Mobile Phase MeCN/H2O
Buffer Formic Acid, Acetic Acid
Flow Rate 0.5 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:
Ascorbic Acid
Riboflavin (Vitamin B2)
Vitamin B2 (Riboflavin)
Vitamin C