Rapid Separation and Quantification of Abscisic Acid from Plant Tissues Using High Performance Liquid Chromatography

Abstract

(v/v) methanol (20 ml methanol/ g fresh weight of tissue) at 4 C, transferred to a 250-ml Erlenmeyer flask, and shaken for 24 hr at 4 C. The homogenate was centrifuged at 1,545g for 10 min and the supernatant filtered through methanol-washed Miracloth (Chicopee Mills, Inc.), and taken to dryness in vacuo at 35 C. The sample was then frozen and stored at -20 C. PARTITION COEFFICIENTS FOR ABAThe determination of ABA from plant tissues has been timeconsuming in the past. It usually consists of a preparative and an analytical procedure. The preparative procedure normally involves the partitioning of a plant extract against a number of organic solvents (6, 11) and is followed by either TLC and/or paper chromatography (1,10,13, 20). The analytical procedure has been achieved either by bioassay (12,17,18) or by GLC (4,8,15 Although several organic solvents have been used for partitioning ABA, to our knowledge the literature does not contain a direct comparison of the effectiveness of such solvents. To achieve such a comparison, the partition coefficients were determined for the partitioning of ABA between water at four pH values and seven organic solvent systems commonly used for this purpose.ABA standards (1 ,tg/ml) in charcoal-filtered, deionized, distilled H20 were adjusted to either pH 2.5, 5, 7, or 9 with 0.1 N HCI or 0.1 N KOH. Ten ml of the ABA standards were placed in 60-ml culture tubes and partitioned once against an equal volume of glass-distilled organic solvent. The organic fractions were removed and dried in vacuo at 40 C. The ABA in the control (no partitioning) was determined by taking to dryness a 10-ml ABA standard for each pH treatment. All samples were reconstituted in 1 ml of 5% (v/v) methanol in 0.2 N acetic acid. A 200-,lI aliquot was injected into a 316 stainless steel column (30 cm x 4 mm i.d.) packed with ,uBondapak C18 (Waters Associates). The sample was eluted with 0.2 N acetic acid in 45% (v/v) methanol at a flow rate of 4.5 ml/min, and a pressure of 262 to 290 bars. The ABA was detected with a fixed wavelength UV detector at 254 nm. The instrumentation used in this HPLC procedure is further described below.The ABA eluted with a very narrow peak width which permitted quantification based on peak height.Partition coefficients were determined as:

Purification and Measurement of Abscisic Acid and Indoleacetic Acid by High Performance Liquid Chromatography

“…A little IAA partitioned into hexane used to remove lipids before pH adjustment, but almost none went into hexane after the pH was adjusted to 8 (Table I). Very little ABA partitioned into hexane either before or after pH adjustment, in agreement with results of Ciha et al (6).…”

Changes in the Levels of Abscisic Acid and Its Metabolites in Excised Leaf Blades of Xanthium strumarium during and after Water Stress

“…However, prior to HPLC, the plant extracts were purified by solvent partitioning and/or TLC. The methods described here are based on work in Brenner's laboratory (3), in which acidified H20 and ethanol, rather than methanol (2), were used to elute the series of Bondapak C18 columns. A major advantage of the present method is that a concentrated extract, after lyophilization and Millipore filtration, can be injected without further purification.…”

Identification of Pea Gibberellins by Studying [14C]GA12-Aldehyde Metabolism

“…Next, all of the GA fractions were purified further on a PRP-1 column (system 3, Table I). Samples were taken to dryness under reduced pressure at 32°C, redissolved in 3.0 ml methanol:ether (1:1, vol/vol), and methylated with diazomethane (6). The samples were centrifuged for 15 min at 2,000g to remove an insoluble precipitate of Naphosphate from the solvents used in the PRP-1 chromatography.…”