A major difficulty with the study of endogenous gibberellins (GAS) in plants is to remove substances interfering with the subsequent qualitative or quantitative analyses, for a general review see, e.g., ref. 1. Since vegetative plant tissues usually contain small amounts of GAS together with large amounts of closely related impurities, the efficiences of the chromatographic procedures employed are of great importance. Therefore, high-performance liquid chromatography (HPLC) is now increasingly preferred to other chromatographic techniques's2.
Reversed-phase HPLC has proved to be very suitable for the separation of GAs~-~. If even higher purity is required, or as an alternative, normal-phase HPLC, can be used. The combination of a reversed-phase HPLC purification step with a normal-phase HPLC purification step increases the probability that the impurities are separated from the GAS and thereby facilitates the subsequent qualitative and quantitative analyses2.
A frequently used normal-phase HPLC method for GAS was developed by Reeve et al.* from an open column system described by Powel and Tautvydasg. The stationary phase of this system was aqueous formic acid adsorbed to a microparticulate silica gel. The mobile phase used consisted of gradients or certain proportions of n-hexane saturated with aqueous formic acid, and ethyl acetate saturated with aqueous formic acid. This system is difficult to handle since the liquid stationary phase is easily stripped off the silica support, resulting in adsorption chromatography rather than partition chromatography. The development of HPLC has, however, resulted in covalently bonded polar stationary phases and such a system would be preferable. Bonded normal-phase HPLC of free GAS has been reported by Yamaguchi et al.'O who used Nucleosil N(CH3)2 as a stationary phase, isocratically eluted with methanol plus 0.05% acetic acid.
The purpose of the present investigation was to develop a normal-phase HPLC method for semi-preparative and analytical separations of GAS. The method should be used for the purification of GAS in extracts of vegatative conifer tissues.