Strategies for Extracting, Purifying, and Assaying Auxins from Plant Tissues

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Abstract
Rapid progress has been made in the technology for isolating and analyzing plant auxins. The investigator now has several options for each phase of the process, including extraction, purification, identification, and assay. Most attention is directed toward assaying known auxins and auxin conjugates with physicochemical techniques, which have advantages of sensitivity, accuracy, reproducibility, and the inability to be confounded by growth inhibitors. Since many circumstances can differ among species, tissues, and laboratories, investigators must develop extraction, purification, and assay procedures to fit their situations. Acceptable criteria for identification of auxins have become more stringent with the careful application of methods such as GC-MS [gas chromatography-mass spectroscopy] and GC-MS-SICM [selective ion current monitoring]. Chromatographic retention times alone are now recognized to be inadequate for unequivocal identification of suspected auxins. If equipment for unequivocal identification is not available, the investigator has the options of providing the maximum amount of different kinds of information about the compound, of utilizing successive determinations by separate detectors that use different principles of detection, or of some combination of these 2 approaches. Extraction and purification are still the most time-consuming parts of auxin analysis, and several approaches to this problem are discussed. Methanol and acetone are the most frequently used solvents; diethyl ether is no longer commonly used. GC and HPLC [high performance liquid chromatography] are purification systems that also may include detection as a part of their operation, and unique advantages of these systems are discussed. Other detection systems discussed are those employing GC-MS, the indolo-.alpha.-pyrone fluorescence reaction, and immunological techniques. To make comparisons convenient, we present and discuss the details of extraction procedures and of purification and assay procedures from a number of laboratories. Several trends are evident, including more rapid extraction schemes often involving agents to protect against destruction of IAA, increased use of column chromatography for rapid initial purification, use of the extremely high separating capacity and sensitive detectors of HPLC, the almost universal use of internal standards to monitor losses in each analysis, and the reduction in sample size and time necessary to process samples.