Isolation and quantitative analysis of B1, B2, B6 AND B12 vitamins using high-performance thin-layer chromatography

A new, simple and accurate high-performance thin-layer chromatography (HPTLC) method has been developed for the separation and simultaneous analysis of B1, B2, B6 and B12 vitamins. Standard and sample solutions of vitamins were applied onto precoated aluminum-backed silica gel G 60 F254 HPTLC plate. The plates were developed with about thirty different solvent systems, and the ethanol – chloroform – acetonitrile – toluene – ammonia – water (7 : 4 : 4.5 : 0.5 : 1 : 1, v/v) mixture was chosen as the optimum mobile phase. UV detection was performed densitometrically at 254 nm. The retention factors of B1, B2, B6 and B12 vitamins were 0.36, 0.60, 0.85 and 0.46, respectively. The linearity range for indicated compounds was 10 – 2500 ng per spot and the correlation coefficients were R2 = 0.97 – 0.98. The limits of quantification (LOQ) were 141.72, 42.41, 100.31 and 11.50 ng and the limits of detection (LOD) were 42.52, 12.72, 30.09 and 3.45 ng for B1, B2, B6 and B12 vitamins, respectively. Analyses of standard solutions showed good precision and repeatability.