ONLINE SOLID-PHASE EXTRACTION OF SOIL AUXINS PRODUCED FROM EXOGENOUSLY-APPLIED TRYPTOPHAN WITH ION-SUPPRESSION REVERSE-PHASE HPLC ANALYSIS

The isolation and purification of plant hormones such as auxins from biological matrices has long been complicated by the need to use liquid-liquid solvent partitioning to isolate and concentrate the neutral, basic and acidic auxin compounds from matrix interferences. To eliminate this solvent partitioning step, a column switching method was devised for use with on-line solid phase extraction and separation by ion-suppression reverse-phase high performance liquid chromatography (IS-HPLC) with ultra-violet (UV) detection (280 nm). The on-line purification of auxins with isocratic HPLC separation is applicable to neutral, basic and acidic auxins and auxin conjugates in a single 40-minute analysis. The method allows direct injection of aliquots from 5 to 100-mu-l with the same precision as loop injection. The method also allows for the analysis of tryptophan and its transformed intermediates into secondary auxin products in a single chromatographic run. Bacterial fermentation broths and soil extracts were analyzed by this method to monitor the conversion of exogenously-applied tryptophan into auxin derivatives.