Carrier ampholytes for IEF, on their fortieth anniversary (1967-2007), brought to trial in court: the verdict.

The present review summarizes the data accumulated in 1-year work, by exploring, via a 3-D methodology (Rotofor fractionation followed by CE-MS), all the narrow (2 pH unit wide) carrier ampholyte (CA) compounds for IEF, produced by three companies (Pharmacia with Pharmalyte and Ampholine, BioRad with Bio-Lyte and Serva with Servalyt). All species have been assessed by measuring the types of pH gradient produced, the total number of individual chemicals (with M(r) values) and isoforms and their focusing behavior ('good' or 'poor' ampholytes). Servalyt contains a grand total of 686 chemical entities and no less than 3899 isoforms; Pharmalyte 643 and 2211; Bio-Lyte 255 and 1192; Ampholine 294 and 1182, respectively. In terms of M(r) distribution, although all 2-pH-unit ranges start with the same low M(r) values (ca. 200) their upper limits are quite different. Thus, Pharmalyte reaches an upper M(r) value of 1179 (in the pH 4-6 range), versus 907 for Servalyt, 835 for Bio-Lyte and 893 for Ampholine. In general, in going towards the more alkaline pH intervals (e.g. pH 8-10) the molecular mass of carrier ampholytes (CAs) is reduced to as low as 491 (Bio-Lyte), indicating that the alkaline species are probably made with shorter oligoamines and are, in general, less substituted. All acidic pH intervals (up to pH 6-8) appear to be constituted by a very large proportion of well focusing species, indicating small values of DeltapK across their pI. Above pH 8, all brands of CAs worsen, the vast majority being unable to focus properly and sustain adequately the pH gradient. General guidelines are given for the synthesis of new alkaline species for improving the basic pH ranges.