Quantitative studies on the adsorption of proteins to the bare silica wall in capillary electrophoresis. II. Effects of adsorbed, neutral polymers on quenching the interaction

Barbara Verzola, Cecilia Gelfi, Pier Giorgio Righetti

Research output: Contribution to journalArticle

Abstract

A novel method is reported for quantifying protein adsorption to naked silica tubings and for assessing the efficacy of polymers added to the background electrolyte as dynamic wall modifiers. It consisted of flushing a fluorescently-labelled protein (myoglobin) into a capillary equilibrated in Tris-acetate buffer, pH 5.0, until full saturation of the potential adsorbing sites. Desorption was then affected by electrophoretically driving sodium dodecyl sulphate micelles into the capillary from the cathodic reservoir: the peak of eluted material is quantified by using a dual laser beam instrument able to read the fluorescein isothiocyanate-derivatized myoglobin at 520 nm and the internal standard (sulphorodamine) at 630 nm. Four polymers have been assessed as potential quenchers of interaction of proteins with the silica wall: hydroxypropylmethylcellulose (HPMC, M(r)=1 000 000), hydroxyethylcellulose (HEC, M(r)=27 000), poly(vinyl alcohol) (PVA, M(r)=49 000) and short-chain poly(dimethylacrylamide) [poly(DMA)] (average M(r) ca. 150 000). HPMC, poly(DMA) and PVA were effective in the 0.005 to 0.02% (w/v) range, whereas HEC was active in the 0.1 to 0.8% concentration range. All polymers, however, except for poly(DMA), exhibited a rather poor performance in suppressing protein interactions with the siliceous surface, and could inhibit adsorption only by, at most, 50% (contrary to oligoamines which can quench such interactions by >90%). It is hypothesized that dynamically adsorbed polymers leave ample regions of the capillary inner surface unmasked, thus allowing strong interactions of proteins with the silica wall. This is also confirmed by the modest reduction of electroendoosmotic flow upon polymer adsorption, as compared with an untreated silica surface. Although poly(DMA) can inhibit protein adsorption by as much as 85%, its hydrophobic nature could in turn provide more adsorption sites for less hydrophilic proteins than myoglobin. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)293-303
Number of pages11
JournalJournal of Chromatography A
Volume874
Issue number2
DOIs
Publication statusPublished - Apr 7 2000

Keywords

  • Adsorption
  • Buffer composition
  • Capillary columns
  • Proteins

ASJC Scopus subject areas

  • Analytical Chemistry

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