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Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. Part V: pH 9-11 interval
No full textAs 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 Mr compounds, in the Mr 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 unit
Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. IV: pH 6-8 intervals
No full textFour commercial brands of carrier ampholytes (Ampholine, Pharmalyte, Servalyt, Bio-Lyte), in the pH 6–8 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by CE-MS of 10 out of 20 fractions harvested, in the second dimension. The findings: Ampholine (pH 6–8) contains 80 different Mr compounds, in the Mr interval 216–979 Da, for a total of 326 isoforms. Bio-Lyte (pH 6–8) consists of 62 different Mr species, in the Mr range 341–1048 Da, for a total of 237 isoforms. Servalyt (pH 6–8) is made of 126 different Mr compounds, in the Mr interval 240–785 Da, for a total of 703 isoforms. Pharmalyte (pH 5–8) comprises 123 amphoteres, in the Mr range of 221–992 Da, for a total of 476 isoforms. Pharmalyte appears to be the best brand, with a good proportion of species focusing sharply at their pI position and relatively few ’poor' species, distributed along the entire pH gradient. General conclusions are drawn on the properties of all the pH intervals explored in this series of investigations and some guidelines for possible synthetic routes ameliorating the neutral and alkaline pH intervals are discusse
Carrier ampholytes for IEF, on their fortieth anniversary (1967-2007), brought to trial in court: The verdict.
No full textThe 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 Mr 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 Mr distribution, although all 2-pH-unit ranges start with the same low Mr values (ca. 200) their upper limits are quite different. Thus, Pharmalyte reaches an upper Mr 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 ΔpK 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 range
A proteomic approach to fractionation and analysis of carrier ampholytes for isoelectric focusing
No full textMass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results
No full textIn an attempt to prepare quasi-isoelectric buffers as BGEs for CE, carrier ampholytes (CAs) (Ampholine, pH 7–9; Servalyt, pH 7–9; Bio-Lyte, pH 8–10 and Pharmalyte, pH 8–10.5) have been subdivided with the Rotofor into 20 fractions, of ca. 0.1 pH unit span, whose composition has been studied by CZE-MS. The results have allowed identifying the number of different molecular mass compounds present in every commercial brand, as well as the number of isoforms (having identical mass, but representing positional isomers) associated with a given Mr value. Ampholine is composed of 29 species, for a total of 85 different isoforms; Bio-Lyte is made of 43 compounds, for a total of 136 isoforms; Pharmalyte comprises 58 different Mr chemicals, for a total of 102 isoforms and Servalyt is constituted by 65 species, for a total of 306 compounds (all of these values to be considered as minimum numbers, as detected by the present methodology). Surprisingly, and contrary to theory, a very large proportion (up to 70%) of these species are ‘poor carrier ampholytes’, in that they are unable to focus and are evenly distributed along the generated pH gradient in the electric field. Paradoxically, the pH gradient is created and sustained by the minority of species (30% for three brands, up to 50% for Pharmalyte) that appear to focus at their pI position into reasonably sharp zones. Even in the narrowest pI fraction, up to 20 different compounds can be detected. It is concluded that very few amines with different useful pK values are utilized for the synthesis and that a new generation of CAs with a more diversified population of amines with proper pK values within the given pH intervals should be sought. Ampholine, the poorest of the commercial brands, appears to be still made with the original synthesis devised by Vesterberg, i.e. by reacting a concoction of oligoamines with α,β-unsaturated acid
Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. III: pH 2.5–4 intervals
No full textTo 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%
Determination of mass distribution, polydispersity and focusing properties of carrier ampholytes by capillary electrophoresis-mass spectrometry
No full textUse of capillary electrophoresis-mass spectrometry for the determination of mass distribution, polydispersity and focusing properties of carrier ampholytes
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