Mass distribution, polydispersity, and focusing properties of carrier ampholytes for IEF. Part V: pH 9–11 interval

Abstract

As a last part of an investigation on all 2-pH-unit intervals of carrier ampholytes (CAs) for IEF (see Electrophoresis 2006, 27, 3919-3934; 2006, 27, 4849-4858; 2007, 28, 715-723) two different lots of Servalyt CAs, in the pH 9-11 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by capillary electrophoresis mass-spectrometry of 10 out of 20 fractions harvested, in the second dimension. The findings: the two lots contain 65 and 69 different M(r) compounds, in the M(r) interval of 232-667 Da, for a total of 341-387 isoforms, respectively. Since this is a chaotic organic synthesis, the high reproducibility (here demonstrated for the first time during the 40 years of existence of CAs) of the synthetic process (for two batches produced at 6 years of distance) is remarkable, considering that a 94% agreement for the individual chemicals and 88% agreement for the total number of isoforms for the two lots is found. It is additionally demonstrated that the lower pI species are accompanied by considerably more isoforms than the high pI forms and that in all cases such isoforms consist of family of compounds clustered around the pI of the parental form, with a pI spread of ca. 0.1-0.2 pH units.

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

“…In fact, as we hope this review will prove, experiments by our group [43][44][45][46][47], in the last year, led to a much deeper understanding on the complexity of CAs, their focusing behavior and their M r distribution.…”

Effect of electrolyte pH on CIEF with narrow pH range ampholytes

“…Similarly, we should conclude that some ampholyte components with p I s down to 5 can be found in all the three ampholyte mixtures, since the p I 5.3 dye did never leave the gradient. Previously it has been shown that the quality of the carrier ampholyte solution might influence the CIEF separation, when studying the focusing behavior and mass distribution of the narrow range (2–3 pH unit) carrier ampholytes from different commercial sources, as described by Righetti and co‐workers (see a review in ).…”

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection

“…The broad pH range ampholyte solutions have ca. 4000–6000 isomers , while the narrow pH range ampholyte solutions, which are prepared from the broad ones, have, consequently, a lower number of components. We showed that the broad pH range ampholytes are usable in CIEF separation after “sandwich injection”, but the resulting isoelectrofocusing pattern depends on the applied pH range and the pI values of the analytes.…”