title: Isoelectric point separation of proteins by capillary pH-gradient ion-exchange chromatography.
abstract: In the present work, isoelectric point (pl) separation of proteins by pH-gradient ion-exchange chromatography (IEC) on packed capillary columns is demonstrated. The development of a miniaturized flow-through pH probe for reliable pH monitoring of the column effluent, which was an important technical challenge for adapting this technique to capillary dimensions, was solved by designing a low microliter per minute flow rate housing to a commercially available micro pH probe. Highly linear outlet pH-gradients within the pH range 8.5-4.0 were obtained when applying simple inexpensive buffers consisting solely of piperazine, N-methylpiperazine and imidazole on 10 cm x 0.32 mm i.d. fused silica capillaries packed with anion-exchange poly(styrene divinylbenzene)-based macroporous materials, i.e. 10 microm Mono P from Amersham Biosciences and 10 microm PL-SAX from PolymerLabs. Furthermore, when using a pH-gradient from 6.8 to 4.3, both columns were able to baseline separate the A and B genetic variants of beta-lactoglobulin, which differ with two amino acid residues only, but the PL-SAX column provided almost a two-fold decrease in peak widths compared to the Mono P column. The influence of varying the buffer concentration, injection volume and column temperature on the peak widths and resolution of the beta-lactoglobulins was investigated, e.g. a 100 microl sample of dilute beta-lactoglobulins was injected directly on the column with practically no increase in peak width as compared to what obtained with conventional injection volumes. Finally, a pH-gradient from 6.8 to 4.3 was used to separate proteins in skimmed bovine milk on the PL-SAX column. The milk was simply diluted 1:10 (v/v) with water and filtrated before injection.
authors.affiliation: Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315 Oslo, Norway. than@kjemi.uio.no
authors.forename: Thomas
authors.initials: T
authors.lastname: Andersen
authors.forename: Milaim
authors.initials: M
authors.lastname: Pepaj
authors.forename: Roger
authors.initials: R
authors.lastname: Trones
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point