Head-column field-amplified sample stacking in binary system capillary electrophoresis: A robust approach providing over 1000-fold sensitivity enhancement
Head-column field-amplified sample stacking in binary system capillary electrophoresis: A robust approach providing over 1000-fold sensitivity enhancement
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DOI：
10.1021/ac951250e
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发表时间：
1996-08-01
影响因子：
7.4
通讯作者：
Thormann, W
中科院分区：
文献类型：
--
作者：
Zhang, CX;Thormann, W
描述了在毛细管入口处发生的有效电动场放大样品注入，其样品体积相当于毛细管体积的10倍以上，并证明可使灵敏度提高1000倍以上。将这种柱头场放大样品堆积方法成功应用于二元体系毛细管电泳中带正电荷的疏水化合物分析，结果表明需要初始引入长度>1 mm的低电导率区（水塞）、样品注入电压<20 kV以及注入时间间隔<60 s。在50μm内径和约20 cm有效长度的毛细管上按照这些条件进行1500多次运行后，可以防止在柱入口处的低电导率区内电注入过程中产生有害的热量（溶剂沸腾以及可能的溶质沉积或毛细管壁熔化）。进一步表明，柱头场放大样品堆积注入的溶质量取决于样品溶液中的有机成分和缓冲液。高含量的有机溶剂、低电导率以及少量H⁺（50 - 100μM）的存在为分析带正电荷的模型物质（包括胺碘酮和去乙基胺碘酮）提供了最高的灵敏度。由此，纳摩尔水平的溶质可以从约4μL的等效样品体积中积累（将约20 nL的样品溶剂注入毛细管），并通过紫外吸收检测进行测量。为防止电解引起的干扰，样品瓶应仅使用一次。所获得的数据进一步表明，使用基于峰高的内标法可以可靠地进行定量（日间和日内测定的相对标准偏差在2%水平）。然而，由于毛细管壁的粗糙度和切口的变化、操作步骤之间的时间间隔以及毛细管壁上的微量吸附，入口侧毛细作用吸入的水区长度不是恒定的，因此外标法不能用于定量。
Effective electrokinetic field-amplified sample injection occurring at the capillary inlet from a sample volume equivalent exceeding that of the capillary up to 10-fold is described and demonstrated to provide over 1000-fold sensitivity enhancement. Successful application of this head-column field-amplified sample stacking approach to the analysis of positively chargeable, hydrophobic compounds in binary system capillary electrophoresis is shown to require an initially introduced low-conductivity zone (water plug) of > 1 mm length, a sample injection voltage < 20 kV, and an injection time interval < 60 s, Following these conditions for more than 1500 runs with capillaries of 50 mu m i.d. and about 20 cm effective length, damaging heat production during electroinjection within the low-conductivity zone at the column inlet (boiling of solvent and possible deposition of solutes or fusing of capillary walls) could be prevented, The solute amount injected by head-column field-amplified sample stacking is further shown to be dependent on the organic fraction and the buffer in the sample solution, High content of organic solvent, low conductivity, and the presence of a small amount of H+ (50-100 mu M) provides the highest sensitivity for analysis of positively chargeable model substances, including amiodarone and desethylamiodarone, Solutes present at the nanomolar level can thereby be accumulated from a sample volume equivalent of about 4 mu L (with injection of about 20 nL of sample solvent into the capillary) and measured by UV absorption detection, To prevent disturbances caused by electrolysis, sample vials should be employed only once, The data obtained further show that quantitation can be reliably performed using internal calibration based on peak height (RSDs for inter- and intraday determinations are on the 2% level), However, due to variation of the roughness of the capillary walls and cuts, the time interval between operational steps, and trace adsorption onto the capillary walls, the length of the water zone drawn by capillary action on the inlet side is not constant, and external calibration therefore cannot be employed for quantitation.