The work describes the preparation of a novel dendronized chromatographic adsorbent for hydrophobic interaction chromatography (HIC). This new matrix was chemically synthesized by covalently attaching polyester dendrons with hydroxyl group (-OH) ends to NHS-activated chromatographic media. The dendronized resin was then functionalized through organic carboxylic acid incorporation by reaction with the available –OH groups. This allowed the creation of a dendronized resin with hydrophobic ligands on the periphery. The obtained branched architecture of the dendron allows the attachment of a higher amount of ligands to the resin and generating hydrophobic clusters throughout it. This results in a higher interaction site number and a stronger hydrophobic interaction between proteins and the chromatographic adsorbent. To the best of our knowledge, this is the first work describing a support of such nature.
The purpose of this work was to evaluate the performance of this new HIC support in the separation of proteins of distinct hydrophobicity. In doing so, five protein models (i.e. ribonuclease A (RNasa A), lysozyme, β-lactoglobulin, α-quimotripsyn and bovine serum albumin (BSA)) were loaded separately into the column. The proteins were eluted by applying a linear decreasing gradient of 1.5 or 2M ammonium sulfate (AS)/20 mM Tris-HCl pH7 buffer. Results showed that at 2M AS concentrations, all proteins were adsorbed onto the support and eluted almost at the end of the gradient, which suggests that all proteins were strongly adsorbed onto the support. When 1.5 M AS was used, only RNase A did not adsorb to the support and was eluted in the follow-through. The other sample proteins eluted earlier in the gradient indicating a weaker binding onto the resin. This innovative dendronized support opens a window to a new chromatographic support generation to develop novel downstream processing strategies.