Study On Sample Stacking And Separation By Moving Reaction Boundary In Electrophoresis

Capillary electrophoresis (CE) is an analytical technique which has manyadvantages, such as ease of operation, high separation efficiency and short analysistime. However, the low sensitivity hinders the development of CE. The on-linestacking methods that are preformed through controlling the conditions and methodsof the experiments are useful for improving the sensitivity of CE. As one of theon-line stacking methods, the moving reaction boundary (MRB) has already formedsome theories for the stacking of the zwitterions, and can achieve the effective stacking inthe real samples. However, some mechanisms in the stacking of zwitterions by MRBare still unknown, and the method and theoretic research for some real samples, suchas biological matrix and metal ions, have not been developed in MRB.The aim of this paper is to indicate the mechanism of MRB in the stacking ofzwitterion, broaden the scope of MRB in the analysis for the biological matrix andmetal ions, develop the new methods with good sensitivity and selectivity, andsupply alternative mode for the development of electrophoresis. The main researchcontents and gained conclusions are that1. We performed the comparative theoretical and experimental study on samplestacking by MRB formed by weak or strong alkali in CE. The theoretical andexperimental results unveil that:(1) MRB velocity possesses key importance tothe design of MRB stacking conditions;(2) compared with strong alkali, weakalkali is more suitable to be used as the sample buffer;(3) the advanced theorycan be employed to predict the effect of the sample stacking and to design theexperimental conditions. The proposed theoretic and experimental results holdobvious significances for the on-column stacking of low abundance zwitterionsin CE.2. We advanced the concept of moving affinity boundary (MAB) using metal ionNi(II) and histidine (His) as the model inorganic ion and ligand, respectively,developed the simple method of MAB affinity capillary electrophoresis (MAB-ACE). The experiments manifested that (1) based on the selectiveaffinity interaction and the effective control of affinity conditions, a MAB couldbe created with the model metal ion and ligand;(2) the MAB-ACE couldspecifically capture His rather than other amino acids, or numerous metabolitesin the mix solution of twenty amino acids and human urine. The analyses of Hisin raw urine by the MAB-ACE are in agreement with those via the standardamino acid analyzer, indicating the reliability of the developed method.Additionally, the MAB-ACE with UV detector had good sensitivity (LOD=43ng/mL, S/N=3),1.0-150μM linearity and <5%intra-/inter-day variations. Thenovel method has an evident potential application for capture of a targetmetabolite in complex biological sample.3. We advanced a novel separation mode of isotachophoresis (ITP) based on thecontinuous moving chelation boundary (MCB) formed with EDTA and twometal ions of Co(II) and Cu(II). The relevant experimental results verified thefollowing theoretical predictions:(1) there are three boundaries in the wholesystem, viz., a sharp MCB, a wide moving substitution boundary (MSB) and asharp complex boundary (CB);(2) the original low concentration Co(II) andCu(II) are chemically separated according to their stability constants;(3) thenovel ITP is relied on MRB, rather than MBS which is the base for the classicITP. These findings provide guidance for the development of the MRB and theMCB-based ITP separation of metal ions in environmental and biologicalmatrices.4. We developed the relevant model and theory of the novel ITP in the multi-moving chelationboundary (m-MCB) system. The theoretical results indicated that:(i) at the steady state ofm-MCB, the boundary velocities are equal to each other, resulting in a novel ITP stackingand separation mode of metal ions;(ii) the boundary velocity and direction are mainlycontrolled by the fluxes of the chelator and the metal ion with the maximum lgK rather thanother metal ions with smaller lgK values; and (iii) a controllable focusing of metal ionscould be simultaneously achieved in the m-MCB system. All of the experimental resultsverified the validity of the developed model and theory of ITP in m-MCB system. These findings provide guidance for the development of ITP separation and stacking of metal ionsin environmental and biological sample matrices.