A SMALL-SCALE HPLC METHOD FOR DETERMINATION OF ALPHA-TOCOPHEROL IN FRESH PLANT-TISSUE

This paper presents an high-performance liquid chromatographic (HPLC) method for the determining of alpha-tocopherol in small samples of fre sh plant material. Ryegrass was used as the test material. The samples of 5-100 mg are saponified for 30 min at 60 degrees C. After evaporat ing the n-hexane extract of the unsaponifiables and dissolving the res idue in n-hexane, the sample is ready for the HPLC analysis. The HPLC system involves an amino column, n-hexane-isopropanol (99:1) flowing a t the rate of 2 ml/min as the mobile phase and a fluorescence detector . The recovery of alpha-tocopherol added to ryegrass samples was 110 % when the quantification was based on an alpha-tocopherol standard run through the whole sample preparation procedure. The variation in the alpha-tocopherol contents was 11 % (coefficient of variation CV %) whe n 18 samples ranging 5-100 mg were analysed within one day. The day-to -day variation was only 4.6 % when samples of 50-100 mg were analysed. The results confirm that this simple, rapid and sensitive method is s uitable both for routine analysis of fresh plant tissues and for plant physiological studies where live plants are to be monitored by repeat ed sampling.