Folic acid is a water-soluble vitamin. It belongs to Group B of vitamins. Folate is composed of the aromatic pteridine ring linked to para-aminobenzoic acid and one or more glutamate residues. Folic acid (Vitamin B9) and related impurities were analyzed on the Primesep 100 mixed-mode HPLC column. This column can be used for analysis of this and other water-soluble vitamins. Several impurities and folic acid are well separated. The method is robust and can be used for analysis of folic acid in supplements and other compositions.