Water-soluble vitamins were analyzed using RSpak DM-614, a reversed phase polymer column for chromatography. Six components including caffeine were separated by isocratic elution. Since orotic acid is difficult to dissolve in water, it was dissolved in 0.1 M NaOH aqueous solution at 500 µg/mL, then mixed and diluted with other vitamines.


Sample : 4 µL
5 μg/mL each
(in 2.5 mM H₃PO₄ aq.)
1. Ascorbic acid, Vitamin C
2. Orotic acid
3. Pyridoxine, Vitamin B₆
4. Thiamin, Vitamin B₁
5. Nicotinamide
6. Caffeine

Column       : Shodex RSpak DM-614 (6.0 mm I.D. x 150 mm)
Eluent       : 0.055 M Na2HPO4 + 0.045 M KH2PO4 aq.
Flow rate    : 1.0 mL/min
Detector     : UV (254 nm)
Column temp. : 30 °C