Evaluation of photografted charged sites within polymer monoliths in capillary columns using contactless conductivity detection

Capacitively coupled contactless conductivity detection (C⁴D) is presented as a novel versatile means of visualising discrete zones of charged functional groups grafted onto polymer based monoliths. Monoliths were first formed within 100 μm UV-transparent fused silica capillaries. Photografting methods were subsequently used to graft a charged functional monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid, onto discrete regions of the monolith using a photomask. Post-modification monolith evaluation involves scanning the C⁴D detector along the length of the monolith to obtain a profile of the exact spatial location of grafted charged functionalities with millimetre accuracy. The methodology was extended to the visualisation of several zones of immobilised protein (bovine serum albumin) using photografted azlactone groups to enable covalent attachment of the protein to the monolith at precise locations along its length. In addition, the extent of non-specific binding of protein to the ungrafted regions of the monolith due to hydrophobic interactions could be monitored as an increase in background conductivity of the stationary phase. Finally, the technique was cross-validated using digital photography in combination with a UV light source by immobilising green fluorescent protein in discrete zones and comparing the results obtained using both complementary techniques.