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.
反相HPLC离子对色谱法测定植物组织中的甜菜碱
陈少良* 毕望富 李金克 王沙生
（北京林业大学生物学院森林生物实验中心，北京100083）
摘要：开发了一种用反相HPLC离子对色谱法测定植物组织中甜菜碱的方法。此法的特点是准确、灵敏、简便、费用低。实验分别以胡杨 (PopuluseuphraticaOliv .)悬浮细胞 ,胡杨和群众杨 (P .‘popularis 35 44’)苗木的叶、根组织为材料。样品提取液经浓缩后依次通过Dowex 1× 8阴离子交换树脂柱和Dowex 5 0W× 2阳离子交换树脂柱进行纯化。最后用NH4 OH洗脱阳离子交换树脂柱 ,洗脱液浓缩干后用流动相溶解。样品用C18反相色谱柱进行分离。流动相为 5 0mmol/LKH2 PO4 (pH 4.45 )和 0 .1%PICB_8(辛烷磺酸 ) ,流速 0 .7mL/min ,192nm波长测定。此色谱条件能使甜菜碱与其他组分很好地分离 ,甜菜碱的保留时间为 6 .3min。甜菜碱的加样回收率达到 85 %～ 96 %。经测定 ,杨树根、叶组织中甜菜碱含量为 0 .0 6～ 0 .75 μg/g鲜重 ,叶中甜菜碱浓度高于根。胡杨悬浮细胞经盐处理后甜菜碱浓度有所增加 ,从 0 .45提高到 0 .77μg/g鲜重。
关键词： 甜菜碱；反相%&’( 离子对色谱；回收率；胡杨；群众杨
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