反相HPLC离子对色谱法测定植物组织中的甜菜碱

Quantitative Determination of Glycinebetaine in Plant Tissues by Reverse Phase Ion-pair HPLC

A reverse phase ion-pair HPLC assay has been developed for screening glycinebetaine content in plant tissues in studies of its relation to salinity tolerance. Separation was performed on a 250 mm×4.6 mm stainless steel column (packed with 10 μm irregular-H) eluted with 50　mmol/L KH 2PO4 (pH 4.45) containing ion-pair agent 0.1% PIC B-8 (1-octane sulfonic acid, Waters). Detection was performed by UV absorbance at 192 nm. The recovery rate of glycinebetaine in tissue extracts ranged from 85% to 96%. Glycinebetaine concentrations in leaf and root of Populus euphratica and P.`popularis 35-44' varied between 0.06 and 0.75 μg/g fresh weight (FW) with higher values in leaf tissue. Glycinebetaine level in suspension cells of P. euphratica increased from 0.45 to 0.77 μg/g FW following NaCl stress. The main assay procedure described here offers a number of advantages over other methods of estimating betaines: a) It increases precision and accuracy as compared with the periodide. b) Using a reverse HPLC column thus it decreases the expense for purchasing special equipment (such as strong cation-exchange columns). c) It avoids the need to generate derivatives thus allows rapid assay for betaines. This technique is not, however, suitable for use on crude, unpurified extracts and an ion-exchange clean up procedure is required. In the author's experiment, the extracts were passed through a 5 mL column of Dowex 1×8 (100-200 mesh, OH- form, Sigma) in series with a second column of a 5 mL Dowex 50W×2 (50-100 mesh, H+ form, Serva). Bound betaine was recovered from Dowex 50W×2 with 10mL of 2mol/L NH4OH and the eluate was evaporated to dryness at 65℃ in vacuum. 1 mL solution for the HPLC elution was used for dissolving betaine samples before separation on column. 