Australian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, GPO Box 252-75, Hobart, Tasmania 7001, Australia.
Analyst. 2010 Aug;135(8):1970-8. doi: 10.1039/c0an00010h. Epub 2010 Jun 1.
An electrophoretic method for the separation of derivatised mono- and di-saccharides with on-line concentration via dynamic pH junction has been developed and optimised in capillaries. Dynamic pH junction is perfectly suited for on-line concentration of derivatised sugars due to the acidic derivatisation conditions, however, most reagents for carbohydrates are not ionisable, requiring the use of the novel reagent, O-2-[aminoethyl]fluorescein. Optimisation of the separation selectivity yielded best separations with 170 mM ammonium borate buffer at pH 8.60 in an acrylamide coated capillary. When using an injection comprising 7% of the capillary volume and detection via laser induced fluorescence (LIF) with an argon ion laser, limits of detection as low as 0.13 nM for maltose were obtained, which was 10 times lower than could be achieved without on-line concentration. In order to implement this system in a glass/PDMS microchip, the low pH sample was introduced into the microchannels via a cathodic pH independent electro-osmotic flow (EOF) generated using a poly(dimethyldiallylmethyl-ammonium chloride) (PDADMAC)/poly(styrene sulfonate) (PSS) polyelectrolyte multilayer coating. Optimisation of the injection volume in capillaries greatly simplified translation to the microchip platform, with the optimum capillary sample volume of 7%, dictating the use of an off-set cross with a volume 7% of the separation channel. Microchip separations of maltose, glucose, galactose and allose with dynamic pH junction, were achieved within 120 s, with the limit of detection of maltose using a light emitting diode induced fluorescence (LEDIF) detection system being 790 nM. This is 10 times lower than that achieved without concentration, and is lower than other reports of derivatised sugars using LEDIF detection. This is the first implementation of on-line concentration via a dynamic pH junction in a microchip, and significantly, the improvement in sensitivity achieved when translated to the microchip was equivalent to that achieved in capillaries.
一种用于分离衍生单糖和二糖的电泳方法，通过动态 pH 结实现了在线浓缩，该方法已经在毛细管中得到了开发和优化。由于衍生化条件呈酸性，因此动态 pH 结非常适合于衍生糖的在线浓缩，但大多数碳水化合物试剂不可离子化，需要使用新型试剂 O-2-[氨基乙基]荧光素。通过在涂覆有丙烯酰胺的毛细管中使用 170 mM 硼酸铵缓冲液在 pH 8.60 下进行分离选择性优化，获得了最佳分离效果。当使用包含毛细管体积 7%的进样量并通过激光诱导荧光 (LIF) 进行检测，使用氩离子激光器时，对于麦芽糖的检测限低至 0.13 nM，这比没有在线浓缩时的检测限低 10 倍。为了在玻璃/PDMS 微芯片中实现此系统，将低 pH 样品通过使用聚（二甲二烯丙基氯化铵）（PDADMAC）/聚（苯乙烯磺酸盐）（PSS）聚电解质多层涂层产生的阴极无关电渗流（EOF）引入微通道。在毛细管中优化进样量大大简化了向微芯片平台的转化，最优毛细管样品体积为 7%，这决定了使用体积为分离通道 7%的偏移交叉。使用动态 pH 结在微芯片上实现了麦芽糖、葡萄糖、半乳糖和阿洛糖的分离，在 120 s 内完成，使用发光二极管诱导荧光 (LEDIF) 检测系统检测麦芽糖的检测限为 790 nM。这比没有浓缩时的检测限低 10 倍，也低于使用 LEDIF 检测报告的其他衍生糖的检测限。这是首次在微芯片中通过动态 pH 结实现在线浓缩，重要的是，当转化到微芯片时，灵敏度的提高与在毛细管中获得的提高相当。
