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Improved properties of the non-covalent coating with N,N-didodecyl-N, N-dimethylammonium bromide for the separation of basic proteins by capillary electrophoresis with acidic buffers in 25mum capillaries
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
2006 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1121, no 1, 32-39 p.Article in journal (Refereed) Published
Abstract [en]

Capillaries (25 μm I.D.) treated with the double-alkyl-chain cationic surfactant N,N-didodecyl-N, N-dimethylammonium bromide (DDAB) in an improved coating procedure were used for separation of four basic proteins in volatile buffers (ammonium acetate and ammonium hydroxyacetate) as well as in a non-volatile buffer (sodium phosphate) at pH 4. The DDAB coating was stable enough to, without recoating, permit consecutive separations of the proteins up to 9 h with good precisions in peak areas (RSD = 1.1%) and migration times and with high apparent efficiencies (over 1 million theoretical plates/m) in the presence of a strong anodic electroosmosis. Adsorption of the proteins onto the capillary surface, which in previous studies was found to give a certain contribution to zone broadening, was eliminated with the new modified coating method. Complex formation between the proteins and phosphate buffer was studied and confirmed, and it is proposed that slow protein–buffer component interactions are the main contributions to zone broadening in protein separations by CE.

Place, publisher, year, edition, pages
2006. Vol. 1121, no 1, 32-39 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94575DOI: 10.1016/j.chroma.2006.03.125PubMedID: 16704868OAI: oai:DiVA.org:uu-94575DiVA: diva2:168464
Available from: 2006-05-17 Created: 2006-05-17 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Separation of Proteins with Capillary Electrophoresis in Coated Capillaries with and without Electroosmosis: Studies on Zone Broadening and Analytical Performances
Open this publication in new window or tab >>Separation of Proteins with Capillary Electrophoresis in Coated Capillaries with and without Electroosmosis: Studies on Zone Broadening and Analytical Performances
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins have such structural features that they may interact with different types of surfaces by all possible forces, i.e., electrostatic, hydrogen bonding, hydrophobic. In this thesis two different types of coatings for fused silica capillaries aimed to eliminate such interactions have been studied. The first is a covalent, electroosmosis-free coating with polyacrylamide (PAA) and the second involves a non-covalent coating with the quaternary ammonium compound N, N-didodecyl –N, N- dimethylammonium bromide (DDAB) with a strong anodic electroosmosis. Optimal conditions regarding efficiency and resolution were established by variations of the composition and ionic strengths of buffers at pH below the isoelectric point of the proteins. To achieve high efficiency and resolution the choice of buffer constituents was extremely important.

The PAA coating was very stable at neutral and acidic conditions. Ammonium acetate (0.12 M) and ammonium hydroxyacetate (0.15 M) both at pH 4 provided the best separations with plate numbers up to 1 700 000 plate/m that is among the highest reported in the literature. Capillaries coated with DDAB were stable enough to, without recoating, permit consecutive separations of the proteins up to 9 hours (90 injections). High apparent efficiencies (over 1 million plates/m) were achieved with ammonium acetate (0.07 M), ammonium hydroxyacetate (0.08 M) and sodium phosphate (0.1 M) at pH 4.

Zone broadening was studied by determination of the variance contributions from all main parameters. Significant variances were contributions from longitudinal diffusion, capillary curvature, injection plug, detector time response and detector slit width while other variances, e.g., variances for Joule heat and vertical sedimentation were negligible. The remaining undetermined variance may have its origin in all types of relatively slow interactions including adsorption onto the capillary surfaces and protein-buffer component interactions. The results indicate that the latter is the main cause to zone broadening in protein separations.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 44 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 36
Keyword
Pharmaceutical chemistry, Capillary electrophoresis, capillary coatings, proteins, zone broadening, zone sharpening, efficiency, resolution, protein-surface adsorption, protein-buffer interaction, analytical performance, Farmaceutisk kemi
Identifiers
urn:nbn:se:uu:diva-6913 (URN)91-554-6583-8 (ISBN)
Public defence
2006-06-07, C4:305, Biomedicinska centrum (BMC), Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2006-05-17 Created: 2006-05-17Bibliographically approved

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