Separation and Retention of Vitamins in Mixed-Mode HPLC

Primesep B and B2 columns separate Vitamins C (ascorbic acid) and B2 (riboflavin) with tunable selectivity. Both columns allow peak order reversal by simply changing the acetonitrile concentration in the mobile phase. Vitamin B2 is especially sensitive to acetonitrile concentration. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and either trifluoroacetic acid (TFA) 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:
Vitamin B2 (Riboflavin)
Vitamin C