| Isoelectric focusing (IEF) is a very useful high-resolution electrophoresis technique in biomolecule analysis. Carrier ampholyte (CA) free IEF eliminates the use of CAs that are difficult to remove in preparative IEF and interfere with UV or mass spectrometric (MS) detection in analytical capillary IEF (CIEF). Better understandings of the natural pH gradient IEF mechanism facilitates understanding and optimization of both CA IEF and CA-free IEF. Microfabrication utilizing reachable instrumentations provides microfluidic devices, all of which are beneficial for more efficient applications of the IEF method.; A novel CIEF of proteins in pure water, in the absence of CAs, was demonstrated. It was found that the migration of protons and hydroxyl ions produced from the electrode reactions creates the pH gradient required for protein concentration and separation.; The behavior of the anolyte, catholyte, and ampholytes in the IEF systems were clarified, taking into account the unique electrochemical process in IEF systems. The impact of the initial concentration of the anolyte, catholyte, ampholytes, and the existence of salt in the IEF system on the separation of proteins in CA-free CIEF and on the pH gradient formed by CAs is presented.; Transitional CA IEF process was explained as the result of the migration of protons into the anodic end, and hydroxyl ions into the cathodic end, of the separation channel. The correlation of the focusing time with the CA concentration was found to be the result of the dependence of the charge transfer rate on the amount of charged CAs during focusing. The electric field distribution along the focused CA bands was found to be the result of the dependence of the local conductance on local protons and hydroxyl ions.; By introducing the concept of dynamic pHs, the stability of pH gradients was linked to experimental conditions (the applied voltage, the CA concentration, and the dimensions of the separation channel). The proposed dynamic pH concept illustrates whether and when IEF is a steady state process, and illustrates the parameters that affect the stability of pH gradients in IEF.; A novel application of the screen-printing technique to the mass production of microfluidic cartridges for whole column detection CIEF was demonstrated to provide a simple and convenient way of microfabrication. The qualitative determination of low molecular weight pI markers and protein samples using the fabricated microfluidic device was demonstrated.; Thermally generated pH gradient IEF, utilizing non-uniform Joule heating in tapered channels, was explored, in which the parameters (in particular power supply mode and sample injection and restrain) that determine the success of thermally generated pH gradient IEF were proposed. |
