The formation of a highly adapted high-E zone is critical to isotachophoresis separation and focusing. Recently, we discovered that the high-E zone is present only in a small portion of electrophoresis channel in the presence of EOF (Liu, S. Q. et al. J. Am. Chem. Soc. 2013, 135, 4644− 4647). Accordingly, a much narrower high-E zone is presumably present in t-ITP. If so, it is hard to achieve efficient t-ITP focusing. Indeed, by online coupling t-ITP with CE-LIF immunoassay, the immunocomplexes of carcinogenic BPDE-dG adducts are not efficiently focused using a freshly prepared background electrolyte. Intriguingly, we observed that 20-day stored background electrolyte displays a 10-fold better focusing efficiency. We hypothesize that the unexpected phenomenon is associated with the dissolution of aerial carbon dioxide, which is mainly converted to ionic HCO 3 − in the weak alkaline background electrolyte. Consequently, HCO 3 − of high electrophoretic mobility will be continuously injected into the capillary along with the background electrolyte and act as an alternative leading ion to improve the focusing. By addition of dry ice (without causing significant pH decrease, ΔpH < 0.4) to freshly prepared background electrolytes, we immediately observed the enhanced focusing of immunocomplexes of the DNA adducts. NH 4 HCO 3 and Na 2 CO 3 , included in the background electrolyte, also improve the focusing efficiency and reproducibility. All these consistently support our hypothesis. To understand the underlying mechanism, an advanced CE-SMFI was exploited to monitor in real time the motion of single DNA molecules and the E change throughout t-ITP. We uncovered that t-ITP can induce a local high-E zone, but the presence of HCO 3 − in the background electrolyte could greatly increase the E value in the high-E zone, which allows more DNA molecules to rapidly move backward and to be efficiently stacked at LE/TE boundary. This study provides new insight into nonuniform electric field-induced electrophoresis focusing.I sotachophoresis (ITP) has been widely applied for separation and preconcentration of DNA, 1,2 proteins, 3−5 as well as charged small molecules. 6,7 To simplify the applications, a variant of ITP, transient ITP (t-ITP), is often used instead. 8−11 By the use of discontinuous buffer, it is convenient to generate ITP and t-ITP formats coupling with other electrophoresis formats such as gel electrophoresis, 12−14 capillary electrophoresis, 15−21 and chip electrophoresis. 22,23 In these applications, no special instrument is required.Basically, ITP can be constructed by the use of two distinct electroloytes containing leading and terminating ions with highest and lowest electrophoretic mobility, respectively. The samples are electrokinetically or hydrodynamically injected into electrophoretic channel by being placed between the leading (LE) and terminating (TE) electrolytes. 24,25 Ideally, target ions are focused at the LE/TE boundary while the leading and terminating ions are migrating at same speed b...