Beattie J H, Richards M P
Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, Scotland.
J Chromatogr A. 1995 May 12;700(1-2):95-103. doi: 10.1016/0021-9673(94)01167-d.
Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 microns I.D. columns give better separations than 50 or 75 microns I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C1 or C18 hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress.(ABSTRACT TRUNCATED AT 250 WORDS)
毛细管电泳（CE）技术已成功应用于金属硫蛋白（MT）异构体的分离，并已证明具有快速、实用和经济的特点。对多种不同电解质和毛细管的研究表明，电解质缓冲液组成和毛细管内壁表面修饰对异构体分离和分辨率有相当大的影响。离子表面活性剂如十二烷基硫酸钠（SDS）在高浓度时会形成胶束，分子在疏水胶束相和水相之间的分配以及它们在电场中的迁移是被称为胶束电动毛细管色谱法（MECC）的技术基础。在本研究中，我们使用绵羊和兔子的MT来优化MECC条件，以分析MT异构体。57厘米长的毛细管比短柱分离效果好得多，尽管分析时间增加到了约12分钟。改变缓冲液、SDS浓度或pH值会影响异构体分离的选择性，绵羊MT中多达5种异构体以及兔子MT中6种异构体被完全或部分分离。比较不同内径的毛细管，我们得出结论，内径25微米的柱子比内径50或75微米的柱子分离效果更好，尽管灵敏度分别降低了约3倍和5倍。使用我们的MECC条件，涂有C1或C18疏水材料的柱子在改善MT分离或分辨率方面并无作用，不过对这些柱子的进一步评估正在进行中。（摘要截于250字）
