Abstract
A coating method using comb-like polymers with poly(ethylene oxide) (PEO) grafts is described and studied in order to use porous silica as phase in size exclusion chromatography (SEC) of proteins. The coating is carried out by in situ polymerization and crosslinking of methacrylates of PEO with OH end groups (macromonomers) after deposition from a solution. A series of phases was prepared using two different graft lengths and two different crosslinking agents in different proportions. The efficiency of the coatings to mask the silanol groups of the matrix is characterized by pH metric titration and the best coating conditions are deduced. From calibration with PEO standards and with proteins, the SEC behavior of each phase is analyzed from column experiments and compared to results of pH metric measurements. A close interdependence between the two methods is observed. The optimized phases exhibit very good performance in SEC, except with low molecular weight basic proteins which interact with some residual unmasked silanol groups. The presence of these groups is attributed to the intrinsic porosity of the polymeric layer which increases with the length of the grafts.
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