Investigation Of A New Sheathless Interface For Capillary Electrophoresis Mass Spectrometry

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) has become a popular technique with high efficiency and sensitivity. Till present, electrospray ionization (ESI) has been the most frequently used interface for CE-MS coupling. However, there are two major challenges when interfacing CE with ESI-MS. One is to maintain constant electrical contact. Both CE separation and ESI process need the application of high voltage. The other is to match the flow rate of conventional ESI with CE electroosmotic flow (EOF). The EOF in CE often is 50-250 nL/min which is lower than the optimal flow rate (500-700 nL/min) in conventional ESI. To deal with these two problems, researchers developed many interfaces which could be divided into sheath flow interfaces and sheathless interfaces.The frequently used methods to maintain electrical contact include direct contact of CE terminal electrode and solution, coating conductive material at the capillary tip, or using separation voltage for spray. However, problems such as air bubbles formation may occur because of the electrochemical reactions thus affect the separation efficiency. To match the flow rate between ESI and EOF, using sheath flow, narrowed capillary tips, or extra pressure may be effective. But it may broaden the peaks, reduce the separation efficiency and the narrowed tips would be easily clogged. Therefore, it is urgent to develop a new CE-MS interface without compromising either capillary i.d. (inner diameter) or separation efficiency and sensitivity.According to our previous study of induced electrospray ionization (iESI), we found that stable spray was achieved when the flow rate was 0.2μL/min. This feature was suitable for CE-MS interface and thus we developed an iESI-based interface. An intact and regular sized capillary with 50 or 75μm i.d. was used and an alternating current (ac) HV was applied on the metal electrode which was attached outside the separation capillary outlet end. Because there is no physical contact between the electrode and solution in the new interface introduced here, it could avoid the electrochemical reactions. By using this device, stable total ion chromatograms by continuous CE infusion were obtained with regular sized capillaries, which implied the new interface introduced here could match EOF with electrospray and it could avoid using narrowed capillary tips or compromising separation efficiency and sensitivity. Normal CE separations with different buffer solutions (NH4OAc and PBS) by this regular sized capillary-based interface were completed. This might expand the applicable range of buffers for current CE-MS interface. In addition, the reproducibility of this interface gave satisfactory results with relative standard deviation (RSD) in retention time ranged between 1% and 3%.On the other side, the existence of the electrical potential difference in ESI may result in corona discharge (CD) which is a common gas discharge phenomenon caused by the difference of potential between two electrodes. However, CD in ESI may bring in lower signal intensity. There are also many researchers that use the reactive oxygen species generated in CD to react with the disulfide bond in peptide and protein which is good for the sequencing analysis of protein and peptide.Although CD has attracted people’s attention for a long time, the study on the influence factor of CD in ESI has not done yet. So, we investigated the effect of voltage, electrolyte species, flow rate, the distance between the emitter and the counter-electrode, and the emitter size on the CD during ESI. We used a photomultiplier tube (PMT) to record the light emission by electrospray under different conditions. The results showed that increasing the voltage, adding an electrolyte to the solution or increasing the emitter size would elevate the degree of CD, while using a high flow rate or increasing the distance between the emitter and the counter-electrode would reduce the degree of CD. Thus, we could facilitate the occurrence of CD just by adding an electrolyte to the solution in ESI to generate much more reactive oxygen species such as HO· which could dissociate the disulfide bond in protein and peptide by oxidative cleavage reaction. And the experiments in this article proved that the cleavage of the disulfide bond in oxidized glutathione (GSSG) was possible by adding an electrolyte in the solvent. It could be a good choice for in situ analysis of protein and peptide.