Moving Chemical Reaction Boundary Based Urine Profiling And Instability Of PH Gradient In IEF Revisited

The Moving Chemical Reaction Boundary (MCRB) theory can be utilized for the on-line stacking of zwitterions. It had also been manifested that the MCRB technique can achieve effective stacking in a high-salt matrix, which is applicable to biofluids such as urine. On the other hand, the judgment expressions derived from MCRB theory can be used to determine the moving direction of the boundary, which provides new clues for the study of Isoelectric Focusing (IEF) dynamics.Rapid and sensitive profiling of urine holds evident significance for discovery of new biomarkers and for clinical diagnosis. A simple, rapid and sensitive moving chemical reaction boundary (MCRB)-induced stacking technique was described herein for the first time for the analysis of urine profiling by CE. The carefully designed MCRB was formed with acidic Gly-HCl sample buffer and alkaline Gly-NaOH running buffer. The experimental conditions such as the pHs, concentrations of the buffers and the applied voltage were optimized. Compared with the conventional CZE with only ten observable peaks and poor sensitivity, the MCRB could significantly improve the sensitivity of urine profiling: More than 90 stacking peaks had been observed and more than 20 fold sensitivity enhancement achieved. The results indicated that the MCRB-induced stacking is a useful and potential tool for urine profiling.The MCRB theory was also applied to re-investigate the instability of natural pH gradient of ampholytes in classic IEF. By using simple judgment expressions, the Rr value was used for the comparative analyses of the transference numbers of hydrogen ions in the acidic anolyte and hydroxyl ions in the alkaline catholyte in numerous IEF experiments cited from references. The re-investigations showed that (1) in the runs of cathodic drifting of pH gradient, all the values of Rr were evidently or slightly higher than the key important value of zero, namely the transference number of hydrogen ions in the anolyte was greater than that of hydroxyl ions in the catholyte; (2) conversely, in the anodic drifting, Rr < 0, namely the transference number of hydroxyl ions in the catholyte was over that of hydrogen ions in the anolyte. The paper supplied a novel viewpoint for the study of the instability of pH gradient in classic IEF.In the last part, the optimal experimental conditions for the simutaneous stacking of glucose and fructose were investigated. Sodium borate was chosen as the buffer andα- naphthylamine and NaBH3CN as the derivation agents. The optimal conditions were 60 mM pH 11 sodium borate buffer, 1.0 psi pressure injection for 90s. Compared with the normal CZE method, the detection sensitivity of glucose was enhanced by more than 20 times, and that of fructose nearly 60 times.