This paper develops a novel procedure of quantitative predictions for the on-column stacking conditions of a zwitterionic analyte by a moving chemical reaction boundary (MCRB) in capillary electrophoresis (CE). The procedure concerns the choice of the weak acidic running and alkaline sample buffers and the velocity design of MCRB created with the two buffers. Based on the theory of MCRB, the theoretical computations are performed. From the computations, the following two predictions are refined for the stacking conditions of zwitterion. (1) The zwitterion velocity in the acidic buffer should be greater than that of MCRB moving toward the cathode, or the zwitterion cannot be well stacked by the MCRB. (2) The gap between pH values of the acidic and alkaline sample buffers ought to comprise the isoelectric point (pI) of zwitterion to be stacked; namely, there exists the relation of pH (acidic buffer) < pI < pH (sample). The predictions are quantitatively proved by the experiments of zwitterionic stacking with two kinds of MCRBs. In addition, the experiments also show the tightly stacked peak of zwitterion existing in the process of MCRB, but not after the MCRB. The theoretical and experimental results hold obvious significances to other zwitterion (such as peptide and protein) on-column stacking in CE.
The paper advanced the theoretical procedures for quantitative design on selective stacking of zwitterions in full capillary sample matrix by a cathodic-direction moving reaction boundary (MRB) in capillary electrophoresis (CE) under control of electroosmotic flow (EOF). With the procedures, we conducted the theoretical computations on the selective stacking of two test analytes of L-histidine (His) and L-tryptophan (Trp) by the MRB created with 30 mM pH 3.0 formic acid-NaOH buffer and 2-80 mM sodium formate. The results revealed the following three predictions. At first, the MRB cannot stack His and Trp plugs if less than 12.5 mM sodium formate is used to form the MRB and prepare the sample matrix. Second, the MRB can stack His and/or Trp sample plugs completely if higher than 50 mM sodium formate is chosen to form the MRB. Third, the MRB can only focus His plug completely, but stack Trp plug partially if 20-50 mM sodium formate is used; this implied the complete MRB-induced selective stacking to His rather than Trp. All the three predictions were quantitatively proved by the experiments. With great dilution of sample matrix and control of EOF, controllable, simultaneous and MRB-induced selective stacking and separation of zwitterions were achieved. The theoretical results hold evident significances to the quantitative design of selective stacking conditions and the increase of detection sensitivity of zwitterions in CE. In addition, the control of EOF by cetyltrimethylammonium bromide (CTAB) can evidently improve the stacking efficiency to both His and Trp.
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