QUASI-LINEAR PH GRADIENTS FOR CHROMATOFOCUSING USING SIMPLE BUFFER MIXTURES - LOCAL EQUILIBRIUM-THEORY AND EXPERIMENTAL-VERIFICATION

Chromatofocusing utilizes internally generated, retained pH gradients to focus proteins into narrow chromatographic bands. One of the charac teristics of current chromatofocusing methods is that they use expensi ve polyampholyte buffers containing large numbers of ill-defined compo nents in order to generate linear or quasi-linear pH gradients. in add ition to being costly to use, polyampholyte buffers also tend to assoc iate with proteins and often yield irreproducible gradient shapes. In order to avoid the various difficulties associated with the use of pol yampholyte buffers, this study investigates the use of mixtures of sim ple buffering species to generate quasi-linear pH gradients on a weak- base ion-exchange column packing. The ability of these gradients to se parate protein mixtures was also investigated. To optimize the conditi ons used, a computer simulation method using a local equilibrium model was developed that predict:; the shape of the pH gradient. Several ex periments were performed that demonstrate the usefulness of the method and the accuracy of the model. (C) 1998 Elsevier Science B.V. All rig hts reserved.