Comparing capillary coatings for protein separation by capillary electrophoresis

For intact protein separation by capillary electrophoresis, the application of a proper capillary coating is crucial to achieve high separation efficiencies. There are a large number of different capillary coatings and coating protocols, making it crucial to compare the coating performances in identical experimental conditions for intact protein separations. Here, the plate height vs. migration velocity was systematically plotted by doing electrophoretic experiments at different electric voltages, on five model proteins. For the first time, this study allows comparing the performances of seven different neutral and cationic coatings in a 2 M acetic acid background electrolyte. For all tested capillary coatings, i.e. polyvinyl alcohol (PVA), covalent and physically adsorbed polyethylene oxide (PEO), hydroxypropyl cellulose (HPC), linear polyacrylamide (LPA), 5-layers cationic polyelectrolyte coating formed from polydiallyldimethyl ammonium chloride and sodium polystyrene sulfonate (PDADMAC-PSS)_2.5, and dioctadecyldimethylammonium bromide (DODAB), the plate height of all proteins increases when higher voltages are applied, leading to better separation at lower voltages. For neutral coatings, a non-linear increase of H was observed at higher voltages, leading to poor separation efficiency for certain proteins at high voltages. This effect was not observed on cationic coatings and was explained by the stretching of the protein chain, promoting the interaction with the capillary coating. Regarding the average performances of the coatings in terms of separation efficiency, the ranking of coatings followed (best to worst): DODAB > HPC > μSIL-WAX > LPA > PEO > SMIL > PVA. It is worth noting that the resolution depends not only on the separation efficiency, but also on the apparent selectivity, which is strongly dependent on the EOF magnitude relative to the effective mobility of the protein. Therefore, the choice of the coating should also consider the range of desired EOF relative to the targeted proteins.