Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. III: pH 2.5–4 intervals

To study the molecular mass distribution and number of species in narrow-range (2-pH-unit wide, in the nominal pI 2–4 or 3–5 interval) carrier ampholytes from four commercial sources (Bio-Lyte, Servalyt, Ampholine and Pharmalyte), a 2-D technique was adopted, consisting of a preparative focusing step in a Rotofor instrument, followed by analysis of every other collected fraction (10 out of 20) by CE-MS. It was found that Ampholine pH 3.5–5 contains 105 different molecular mass (Mr) compounds, in the Mr interval 205–965 Da, for a total of 446 isoforms. Bio-Lyte pH 3–5 consists of 84 different Mr species, in the Mr range 216–965 Da, for a total of 383 isoforms. Servalyt pH 2–4 is made of 227 different Mr compounds, in the Mr interval 204–929 Da, for a total of 1201 isoforms. Pharmalyte pH 2.5–5 comprises 245 amphoteres, in the Mr range 203–857 Da, for a total of 857 isoforms. Pharmalyte appears to be the best brand, with the vast majority of species focusing sharply at their pI position and almost no ‘poor’ species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the pI value. Due to some overlap with the adjacent acidic pH 4–6 interval, the species in common have been evaluated: the most extended overlaps are found in Ampholine (55% of the species appearing in the two neighbouring intervals) and in Servalyt (47% coincidence). The lowest overlaps are found in Pharmalyte (23%) and in Bio-Lyte (20%)