A Simple, Inexpensive, High Pressure Liquid Chromatographic Method for Separating Cytokinins in Plant Extracts

Abstract

Separation of a mixture of the main cytokinins occurring naturally in plant tissues was achieved by high pressure liquid chromatography using insoluble polyvinylpyrrolidone as the solid support. The separation of each cytokinin was first assessed over a range of salt and 1-butanol concentrations and pH using a mixture of borate buffer and 1-butanol as the mobile phase to determine the conditions necessary for optimum resolution. A discrete separation of zeatin, N-6-(A-2-isopentenyl)adenine, their related ribonucleosides, and kinetin was achieved using a simple isocratic elution with 0.025 M borate buffer at pH 6.8 and 4% (v/v) (6, 9,12,17,19).Until recently, GLC has been considered the most reliable technique for separating and analyzing the identification of growth regulators in plant extracts, providing the hormone activity of the extracts is high, such as in immature seeds (10). However, this technique suffers from the disadvantage that volatile derivatives of the hormones must first be prepared and very often the analysis is destructive. This is particularly so with cytokinins where high column temperatures are required and flame ionization detectors are used for the analysis (12,19).The recent use of HPLC (3, 4,13,19) has introduced yet another possible major tool for cytokinin analysis. However C18 on Porasil (Waters Associates Ltd.) for reverse phase chromatography of cytokinins, but again a gradient elution system was necessary to achieve good separation. In our laboratory, we wished to develop a simple, inexpensive HPLC system for separating cytokinins, which would operate under isocratic conditions. It had been shown previously that PVP, a weak hydrogen-bonding material, could be used on a normal column scale to give reasonable separation of some cytokinins using phosphate buffer as the eluting solvent (1, 5, 18). However, a more satisfactory separation of cytokinins can be achieved on PVP TLC plates with borate buffer, since the cytokinin ribonucleosides form quick moving complexes with the borate ions, thus moving away from the free bases (17). On the other hand, since the free bases tend to be more soluble in 1-butanol than their related ribonucleosides, a combination of borate buffer and 1-butanol might be expected to provide the optimum separation conditions in the shortest possible time on LC columns.In this paper, we describe the results of our investigations with borate buffer/1-butanol mixtures on PVP HPLC columns for separating pure cytokinin standards and endogenous cytokinins in extracts of cabbage heads. MATERIALS AND METHODSChromatographic Materials and Procedure. A 2-meter long chromatography column was prepared from 2 mm i.d. precision bore 316 stainless steel tubing obtained from Varian Aerograph Ltd. The column was fed with solvent from a 0 to 750 p.s.i. constant pressure pump and the eluate monitored with a Model 02-001426-00 UV-detector, both purchased from Varian Aerograph Ltd. The pump was pressurized with N2 and all the solvents were degassed before use.In preliminary ...

Concurrent profiling of indole-3-acetic acid, abscisic acid, and cytokinins and structurally related purines by high-performance-liquid-chromatography tandem electrospray mass spectrometry

“…Quantification of CKs has been described previously [37-40,47-52] and some of the important themes discussed in recent work were to separate CK isomers (ie. c Z and Z, (cis) [9R]Z and [9R]Z) and CK groups (ie.…”

High Performance Liquid Chromatography of Natural Products

“…In addition, the separations of certain specific classes of primary metabolites have been reviewed, including amino acids and peptides (3,7), carboxylic acids (3, 8), purine nucleosides (9), and carbohydrates (13)(14). Separations of the cytokinins (modified plant nucleosides) are described in two recent papers (15)(16), while other representative separations of nucleosides (5, 7-21) and nucleotides (6, [22][23][24][25][26] are given in the references cited. The material that follows has been organized along biosynthetic lines as far as possible.…”