Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF II: pH 4–6 intervals

For studying the Mr distribution and number of species in narrow-range (2 pH-unit wide, in the nominal pI 4–6 interval) carrier ampholytes from four commercial sources (Bio-Lyte, Servalyt, Ampholine and Pharmalyte), a 2-D technique was adopted consisting of a focusing step in a liquid phase (Rotofor, yielding 20 fractions) followed by orthogonal CE in both, acidic and basic buffers. As a final step, every other fraction was analyzed by CE-MS. The findings: Ampholine contains 80 different Mr compounds, in the Mr interval 203 to 893 Da, for a total of 325 isoforms. Bio-Lyte consists of 66 different Mr species, in the Mr range 388 to 835 Da, for a total of 436 isoforms. Servalyt is made of 199 different Mr compounds, in the Mr interval 204 to 907 Da, for a total of 1302 isoforms. Pharmalyte pH 4–6.5, comprises 217 amphoteres, in the Mr range 150 to 1179 Da, for a total of 812 isoforms. Pharmalyte appears to be the best brand, with the vast majority of species focusing sharply at their pI position and <5% “poor” species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the pI value