A pH gradient is established by the use of ampholytes, which are low molecular weight amphoteric molecules. Ampholytes, when present as a mixture, migrate in an electric current to their pI, thereby producing a pH gradient. A protein, when introduced in this pH gradient, will be either positively or negatively charged depending on the local pH. Subsequently, the protein will move towards either the anode or cathode until it encounters that part of the system which corresponds to its pI. It will then have no net charge and will therefore stop migrating. This technique is useful to find pI of unknown proteins. We have developed the expertise in the design and optimization of such systems for pH range, gradient, ampholyte selection, and electric field. The sample problem demonstrates how a pH gradient can be generated inside a microchannel by stacking five different ampholytes of linearly varying pK values and applying electric current.