## 2 Photo-Grafting Reactions
### _Grafted Monolithic Columns for Biomolecule Immobilization_  
The immobilization of a biomolecule to the polymeric monolithic surface may firstly require the hydrophilization of the monolith's surface [8, 13, 71, 72, 73] by grafting a neutral hydrophilic monomer [7]. [PERSON] _et al_. prepared a dual function monolith incorporating a \(\upmu\)-solid phase extraction (SPE) section, followed by an enzymatic reactor [68]. A generic hydrophobic monolith was prepared (poly(BuMA-_co_-EDMA)), to which a 20 mm section of monolith was grafted with VAL for further reaction with trypsin, thus creating the enzymatic reactor segment of the monolith. The remaining length of the hydrophobic monolith was dedicated to \(\upmu\)SPE. In addition, a co-polymerized monolith incorporating VAL was also prepared (poly(EDMA-_co_-2-hydroxyethyl methacrylate-_co_-VAL)). The photo-grafted and co-polymerized columns exhibited similar porosity, and so were compared directly. The photo-grafted column demonstrated a higher enzymatic activity compared to the co-polymerized monoliths. The effect of time upon the graft density was investigated and measured by enzymatic activity. Increasing graft time showed a proportional increase in graft density and a pressure drop across the column within the limits of the experiment, _i.e._, between 1 and 3 min of grafting. The column was used successfully in the digestion of a peptide consisting of fluorescent fragments, facilitating the visualization of the SPE zone. [PERSON] _et al_. produced a column expressing three segments of differing enzymatic systems, each specifically placed using photo-grafting of the reactive monomer, VAL [8]. The monolith (poly(BuMA-_co_-EDMA)) was grafted first with polyethyleneglycol methacrylate (PEGMA) along the column, producing a hydrophilized surface. Sequential grafting of three discreet zones of VAL was used to immobilize each of the enzymes (glucose oxidase, horseradish peroxidase, and invertase). Using VAL grafts and green fluorescent protein (GFP), the group determined that the use of sequential grafting had little or no effect on the activity of the initially immobilized GFP, and thus immobilized enzymes. This demonstrates the versatility of the photo-masking technique to graft monolithic columns within a single column housing, with a variety of different functionalities via subsequent steps. Monomers such as acrylamide, vinyl pyrrolinone, 2-hydroxyethyl methacrylate (HEMA), and polyethyleneglycol methacrylate (PEGMA) were grafted upon poly(BuMA-_co_-EDMA) monoliths in an effort to study their resistance to protein adsorption [7]. Fluorescent bovine serum albumin (BSA) and GFP were flushed across the test columns, where zones of hydrophilic monomer were grafted, resulting in a fluorescent pattern across the column. On the non-grafted sections of the monolith, the protein probes were adsorbed, and the grafted monomers showed reduced fluorescence intensity (HEMA and PEGMA only). The fluorescence assay provided a qualitative validation of the grafted groups, both in location and ability to resist protein adsorption. The cross-validation of grafted moieties was also probed by [PERSON] _et al_. [70], wherein poly(VAL) grafts were generated on the surface of poly(BuMA-_co_-EDMA) monolithic columns. The grafts facilitated the immobilization of GFP for fluorescent visualization and also bovine serum albumin. The spatial location of these groups was visualized non-destructively using the fluorescence of immobilized GFP and sC\({}^{4}\)D profiling. Using the sC\({}^{4}\)D technology, the zones of grafted moieties were easily confirmed and visualized. [PERSON] _et al._ used photo-grafting to introduce hydrophilic surface chemistry to an otherwise hydrophobic monolithic column [71], similar to the work by [PERSON] _et al._[7], and [PERSON] _et al._[8]. In the report, poly(PEGMA) chains were grafted to poly(GMA-_co_-EDMA) monolithic columns to reduce the adsorption of hydrophobic proteins to the surface.