Noncovalently bilayer-coated capillaries for efficient and reproducible analysis of proteins by capillary electrophoresis.
The suitability of noncovalently bilayer-coated capillaries for the analysis of proteins by capillary electrophoresis (CE) at medium pH was investigated. Fused-silica capillaries were coated simply by successively flushing with a polybrene (PB) and a poly(vinyl sulfonate) (PVS) solution. A protein test mixture was used to evaluate the performance of the coated capillaries. Comparisons with bare fused-silica capillaries were made. Several background electrolytes (BGEs) were tested in combination with the PB-PVS coating, showing that optimum performance was obtained for the proteins using high BGE concentrations. With a 300 mM Tris phosphate buffer (pH 7.0), good plate numbers (150,000-300,000), symmetrical peaks, and favorable migration-time repeatabilities (RSDs below 0.8%) were obtained for the proteins. Using bare fused-silica capillaries, the protein peaks were significantly broadened and the migration-time RSDs often exceeded 5%. It is concluded that the PB-PVS coating effectively minimizes adverse protein adsorption and provides a very stable electroosmotic flow (EOF). We also investigated the potential of a commercially available bilayer coating (CEofix) for protein analysis. It is demonstrated that with this coating, good plate numbers and peak symmetries for proteins can be achieved when the CEofix BGE ("accelerator") is replaced by a common BGE such as sodium or Tris phosphate. Apparently, the negatively charged polymer present in the "accelerator" interacts with the proteins causing band broadening. The utility of the bilayer coatings is further illustrated by the separation of proteins such as interferon-alpha 2b, myoglobin and carbonic anhydrase, by the analysis of a degraded insulin sample in time, and by the profiling of the glycoprotein ovalbumin. In addition, it is demonstrated that even in the presence of concentrations of human serum albumin in the sample of up to 60 mg/mL, the PB-PVS coating still provides reproducible protein separations of good performance.
Catai
Jonatan R
JR
Department of Biomedical Analysis, Utrecht University P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
Tervahauta
Heli A
HA
de Jong
Gerhardus J
GJ
Somsen
Govert W
GW
D020099:Coated Materials, Biocompatible; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D005240:Feasibility Studies; D006583:Hexadimethrine Bromide; D007328:Insulin / Q000302:isolation & purification; D007768:Lactalbumin / Q000302:isolation & purification; D007782:Lactoglobulins / Q000302:isolation & purification; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D013451:Sulfonic Acids; D013499:Surface Properties
D020099:Coated Materials, Biocompatible; D007328:Insulin; D007782:Lactoglobulins; D011145:Polyvinyls; D011506:Proteins; D013451:Sulfonic Acids; C008959:lyapolate; D006583:Hexadimethrine Bromide; D007768:Lactalbumin