A Note on the Synthesis of
Tritiated
Abscisic Acid
The tritiated abscisic acid was required as a marker in the development of an improved method for the analysis of abscisic acid in potato tubers by gas-liquid chromatography.
Published methods for the analysis of abscisic acid in plant material quote recovery figures of from 30 to 70%. Since the recovery figure may be affected by other solutes present in the tissue, it was clear that for a physiological study of the changes in the dormancy of potato tubers at various temperatures it was essential to develop a method which gave recovery figures of 90% or better under all physiological conditions. Addition of tritiated abscisic acid to the initial extraction mixture served not only as a check on the overall recovery but also located the exact stages of the procedure at which losses of abscisic acid occurred.
Attempts to synthesise tritiated abscisic acid by catalytic exchange in aqueous media (Code TRI, the Radiochemical Centre, Amersham, England) required considerable purification of the crude product and gave very poor yields. The yield in an alternative method (1) was only 5% and the product had to be purified by thin layer chromatography. In contrast the method described in the present paper is easy to manipulate, gives almost quantitative yields and because of the high yield, the tritiated abscisic acid requires very little purification. The principle of the method is the same as that described earlier (2) for the preparation of hexadeuterio abscisic acid in which six of the skeletal hydrogen atoms of abscisic acid were exchanged in N sodium deuterioxide for deuterium. These deuterium atoms were found to be stable provided that the isolated deuteriated abscisic acid was not subjected in aqueous solution to a pH higher than 8. An extraction procedure for plant tissues has been described (3) in which acid conditions are employed throughout.
The reaction with tritiated water was carried out using a vacuum manifold fitted with taps so as to isolate various vessels as required.
Transfer of tritiated water (specific activity 5 Cilml; 4 ml) was effected by warming and cooling the appropriate vessels in VCICUO. For cooling, a bath of solid CO,/acetone was convenient and, for warming, an infrared lamp. The procedure was as follows: Tritiated water (1 ml) was distilled from C into the reaction vessel B which contained sodium metal (23 mg; 1 mmol). The water which condensed as ice was allowed to thaw and when the sodium had dissolved, the displaced tritiated hy-543