title: Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results.
abstract: In 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 M(r) 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 M(r) 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 alpha,beta-unsaturated acids.
authors.affiliation: Politecnico di Milano, Department of Chemistry, Materials and Engineering Chemistry Giulio Natta, Milano, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Sebastiano
authors.forename: Carolina
authors.initials: C
authors.lastname: Simó
authors.forename: Martha Elena
authors.initials: ME
authors.lastname: Mendieta
authors.forename: Paolo
authors.initials: P
authors.lastname: Antonioli
authors.forename: Attilio
authors.initials: A
authors.lastname: Citterio
authors.forename: Alejandro
authors.initials: A
authors.lastname: Cifuentes
authors.forename: Gabriel
authors.initials: G
authors.lastname: Peltre
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Righetti
mesh_terms: D000665:Ampholyte Mixtures / Q000737:chemistry* / Q000302:isolation & purification; D005591:Chemical Fractionation; D019075:Electrophoresis, Capillary; D007525:Isoelectric Focusing / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000665:Ampholyte Mixtures