High Performance Liquid Chromatographic Analysis of Cytokinins in Sorghum bicolor Leaves

High performance liquid chromatography with octadecylsilica (Bondapak C₁₈/Poracil B) column packing was used to purify and separate cytokinins in Sorghum leaf extracts. The column size was 56 × 0.21 cm i.d. By gradient elution, using acidified water with increasing amounts of methanol, the major peaks of cytokinin activity, as determined by the callus tissue bioassay. were effectively separated from large amounts of extraneous impurities. These cytokinins were further separated on a microoctadecylsilica column (μBondapak C₁₈, 30 × 0.4 cm i.d.) with a gradient of acidified water-acetonitrile. Zeatin and zeatin riboside gave distinct ultra violet absorption peaks which could be used for quantitative estimation. Biological activity corresponded to the elution of these peaks. These two cytokinins are the major cytokinins in Sorghum leaves.