One‐Step Focusing of Ampholytes With Electrophoretic Mobilization: Concepts Assessed by Computer Simulation

ABSTRACT The dynamics of three one‐step focusing protocols described in the literature for IEF‐MS analyses of proteins are assessed by computer simulation. Focusing of 101 carrier ampholytes (pI range 3.0–11.0) and 9 analytes (pI range 3.9–10.4) with concurrent electrophoretic mobilization in an electroosmosis‐free environment is studied via use of the following: (1) an acid as anolyte and catholyte, (2) a plug of a base between the sample and the acid as catholyte, and (3) a base incorporated into the acid‐based catholyte. The data reveal that the first approach is of limited applicability and separation efficiency as it provides separated transient foci of acidic ampholytes only. The second system largely extends the applicability range, separation, and sensitivity and can be applied to ampholytes with pIs as high as 10. The same is true with the base incorporated into the catholyte. Dynamic simulation provides for the first time insight into the complexity of these one‐step focusing systems with buffer components that are compatible with MS detection. Simulation is an attractive and simple tool for characterization of the involved electrophoretic processes and for assay optimization.