## 2 Photo-Grafting Reactions
### Monolithic Columns Grafted with Ionizable Exchange Groups  
Photo-grafting for uses in HPLC and CEC was developed by [PERSON] _et al_. in 2003 [36, 37]. Firstly, a charged monomer 2-acrylamido-2-methyl-1-propanesulphonic acid (AMPS) was grafted to the surface of a \"generic\" poly(butyl methacrylate-_co_-ethyleneglycol dimethacrylate) (poly(BuMA-_co_-EDMA)) monolithic column [37]. The effect of grafting time was investigated, with an increased pressure drop on the monolithic column, resulting from an increase in graft time. This was indicative of the growing dense network of polymer chains, with increasing irradiation time. The electroosmotic flow (EOF) was generated by the charged AMPS graft. In CEC, the EOF was found to plateau once a certain density of grafts was achieved, as only those sites at or near to the surface contribute to EOF. Photo-patterning (the photo-grafting of selective groups in defined areas) was also investigated, where reactive VAL groups were grafted to a \"generic\" monolith. Fluorescent rhodamine 6G was covalently attached to the VAL sites, enabling a visual cross-validation of \"patterned\" sites. Using a fluorescence-based assay, an increased fluorescent intensity was observed for a column grafted with the same irradiation intensity for a longer period of time. [PERSON] _et al_. grafted methacryloyloxyethyl trimethylammonium chloride (META) to a poly(GMA-_co_-EDMA) monolithic column to prepare a grafted anion exchange selectivity [69], using a single step graft polymerization with benzophenone as the initiator. The column was capable of delivering efficiencies of up to 29,500 N/m (for inorganic anions) at a reduced flow rate of 100 nL/min, higher than the latex agglomerated (quaternary ammonium-functionalized) monolithic columns described by [PERSON] _et al_. (26,000 N/m), which were prepared via co-polymerization with nano-particle modification [46]. Improved photo-grafting was accomplished using multi-step initiator immobilization with subsequent grafting by [PERSON] _et al_. [7]. [PERSON] _et al_. used a single step in the surface modification of neutral monolithic columns (poly(BuMA-_co_-EDMA)) with ionized functional monomer, either AMPS or META [67]. Measurements on pre- and post-modified monoliths have shown that the grafting procedure supported the generation of EOF. Increasing the amount of grafting functional monomer in solution (META) from 0.5% to 15% resulted in a ten-fold increase in electro-osmotic mobility. In addition, the group investigated the increased retention of hydrophobic analytes in reversed-phase (RP) CEC. An ionizable layer (META) was grafted to the monolith, followed by 10 min of grafting of a hydrophobic monomer lauryl methacrylate (LMA), leading to a 53% increase of the retention factor in reversed-phase CEC, in agreement with the findings by [PERSON] _et al_. [37], and [PERSON] _et al_. [36]. Changes to surface chemistry following grafting procedures were investigated by [PERSON] _et al_. [70], where they grafted discrete zones of ionized AMPS to a neutral poly(BuMA-_co_-EDMA) monolithic column. Using a contactless conductivity detector (scanning C\({}^{4}\)D, sC\({}^{4}\)D), the relative density of the grafted zones could be mapped, providing a cross-validation of the grafting procedure. Grafted zones of VAL were also produced to immobilize histidine-tagged green fluorescent protein (GFP). Similarly, [PERSON] _et al_. studied the fundamentals in photo-grafting behavior, using poly(AMPS) grafts on poly(BuMA-_co_-EDMA) monolithic columns, whilst using sC\({}^{4}\)D as a characterization technique [29]. The zones of grafted charged monomer were subjected to varying doses of UV energy, and thus density of grafted chains. The detector's response was proportional to the amount of charged monomer grafted in the column, however, it was noted that a plateau in detector response occurred withincreased UV doses. This is supported by the observations made by [PERSON] _et al_. [36, 67], wherein the uppermost layer is attributed to the resulting charge density.