[70] High-voltage electrophoresis and thin-layer chromatographic separation of vitamin B6 compounds

This chapter describes the high-voltage electrophoresis and two dimensional, thin-layer chromatographic methods for the separation of vitamin B6 compounds. To 30 g of cellulose powder, MN 300 is added in 160 ml of water. The slurry is mixed with a mechanical stirrer for 30 minutes, then poured into a Shannon Unoplan Thin-Layer Spreader set at 500 μm thickness. The slurry is sufficient to make three cellulose-coated 46 × 20 cm glass plates. The plates are air-dried for 24 hours before use. The separation of radiolabeled vitamin B6 compounds in tissue extracts can be achieved with electrophoresis and two dimensional methods. An application of tissue extract without standard is applied to determine whether substances are present that would interfere with the migration of standards. To one of the spaces, 2μl of standard alone is applied. The plates are placed in the horizontal Savant electrophoresis apparatus and run in the dark as described for standard solutions.