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 p I  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 ( M r ) compounds, in the M r interval 205–965 Da, for a total of 446 isoforms. Bio‐Lyte pH 3–5 consists of 84 different M r species, in the M r range 216–965 Da, for a total of 383 isoforms. Servalyt pH 2–4 is made of 227 different M r compounds, in the M r interval 204–929 Da, for a total of 1201 isoforms. Pharmalyte pH 2.5–5 comprises 245 amphoteres, in the M r 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 p I position and almost no ‘poor’ species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the p I 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%).