Study On Short Capillary-based High Speed Capillary Electrophoresis And Its Application In Sequentially On-line Peptides Sequencing

In 1991, Jorgenson established the High Speed Capillary Electrophoresis system (HSCE) on the basis of the conventional capillary electrophoresis system. HSCE system can perform fast separations within several seconds or minutes by either applying high electric field strength or shortening the length of capillary. HSCE system is commonly built on the basis of micro-fluidic chips or short capillary. Microchip-based HSCE system usually requires fussy micro-fabrication and complicated operation, which may limit their widely application in routine analysis. In contract to microchip, capillary-based HSCE system has obvious merits of commercially available at a low cost, easy to build and convenient to use and have been widely used in many areas especially in life sciences. Due to the shortened separation distance, the improvement of the resolving power of capillary zone electrophoresis (CZE) has been the subject for capillary-based HSCE system to be used in analyzing complex samples. To improve the separation efficiency of HSCE and its application in amino acids analysis as the main goal, the present paper mainly focused on the following work:(1) We present a novel easy-to-operate and efficient method to improve the separation efficiency in short-capillary electrophoresis by introducing steady backflow to counterbalance electro-flow without the use of any external pressure. The backflow was easily generated by tapering the capillary end, which was achieved by heating a straight capillary and stretching it with a constant force. Good run-to-run repeatability and capillary to-capillary reproducibility of the present method were obtained with RSD less than 1.50%, indicating the stability of the fluid transport rate in the tapered capillary. Enhanced separation of the tapered short capillary electrophoresis was demonstrated by CZE analyzing amino acids and positional isomers. Baseline separations were achieved in less than 60 s using a tapered capillary with the effective length of 5cm, while no separation was achieved using a normal capillary without a tapered tip.(2) We developed an efficient method based on a multiple buffer additive strategy for simultaneous CE analysis of various OPA-labeled AAs using in-capillary derivatization. Ionic liquid was used for the first time for CE analysis of AAs with in-capillary derivatization. Several additives, including ionic liquid, SDS,/?/?-CD, and ACN, as well as key parameters on CE separation (buffer pH value, separation voltage), were investigated. Under optimum conditions,17 OPA-labeled AAs, except for two pairs of AAs (His/Gln and Phe/Leu), which were separated with resolutions of 1.1 and 1.2, respectively, were baseline separated and identified within 23 min using the present multiple additive strategy. Our present method allowed simultaneous quantification of different OPA-labeled AAs in a large concentration range of 50 ?mol/L to 3.0 mmol/L with LOD down to 10 ?mol/L. The method was successfully applied for simultaneous analysis of AAs in seven beer samples and as many as eleven trace-amount AAs were detected and quantified, indicating the valuable potential application of the present method for food analysis.(3) We constructed a HSCE device which was combined on-line derivatization with optically gated capillary electrophoresis (OGCE) with laser-induced fluoresencence (LIF) detection. In order to realize on-line derivatization of amino acids, the optically gated system was interfaced to a digital syringe pump with two channels which could simultaneously pump the OPA/?-ME derivatization reagent and amino acids solution into a same capillary. Optically gated sample introduction was easily achieved by controlling the shutter for a period which could deliver narrow and reproducible sample plug. The combination of optically gated sample introduction and LIF detection improved the separation efficiency of HSCE. The presented method allowed thirteen OPA-labeled AAs to be separated within 80 s. The RSDs of peak height and migration time of each amino acid obtained from 20 sequential OGCE-LIF analyses of the standard 13 amino acids were in the range of 0.81%-1.50% and 1.55%-3.28%, indicating the high repeatability and stability of the presented method.(4) The HSCE device which was combined on-line derivatization with OGCE-LIF has been successfully applied for automatic online determination of C-terminal sequencing. Using the coupling systems of HSCE with on-line derivatization and OGCE-LIF, we investigated the CPY digestion reactions by automatic monitoring of the released amino acids during the reaction time to obtain the releasing order of amino acids. The C-terminal amino acids sequence obtained by the present method were consistent with the known sequence of the two custom peptides, indicating the valuable potential application of the present method for peptides sequencing. As complementary to Edman degradation method and mass spectrometry method, the application of HSCE in enzyme reaction, amino acids, peptides and proteins analysis were further expanded.