Vitamins are essential components for maintaining physiological functions in living bodies. In particular, five water-soluble essential vitamins, B₁, B₂, C, nicotinic acid and nicotinamide are sometimes lacking in human bodies. The necessary quantities of three water-soluble vitamins, B₆, B₁₂ and folic acid have been determined by the NRC (National Research Council), though these vitamins are seldom lacking in human bodies. This chromatogram shows an example of the analysis of such water-soluble vitamins except folic acid. This analysis has been performed by reversed phase mode with gradient elution. Good calibration curves are obtained for the vitamins using RSpak DE-413 (a column for reversed phase chromatography). As an example, calibration curves for vitamin B₂ and vitamin B₆ are shown below.


Sample : 20 μL
1. Vitamin C
2. Niacin
3. Vitamin B₆
4. Niacinamide
5. Vitamin B₁
6. Vitamin B₁₂
7. Vitamin B₂

Column       : Shodex RSpak DE-413 (4.6 mm I.D. x 150 mm)
Eluent       : (A); 1/30 M Potasium phosphate buffer (pH6.8)/CH3CN=98/2
               (B); H2O/CH3CN=50/50
               0 min to 4 min, 100 % (A) 
               4 min to 15 min, 100 % (A) to 30 % (A)
               after 15 min, 30 %(A) 
Flow rate    : 0.6 mL/min
Detector     : UV (254 nm)
Column temp. : 35 °C