## 5 Hypercross-Linking  
A pre-cursor material or substrate is solvated in a thermodynamically good solvent, where a cross-linking reaction locks the polymer chains in a state similar to that in the solvated state [106]. Following the removal of the solvent, the remaining structures are the new pores within the material, adding significantly to surface area in polymer monolithic columns. The origins of hypercross-linking are connected with [PERSON] _et al_. from the 1970's to the early 2000's [107, 108, 109, 110, 111]. Generic monolithic columns with relatively low specific surface area (usually \(<\)10 m\({}^{2}\)/g [2]) can be improved upon by a straightforward post-polymerization modification, the hypercross-linking method. [PERSON] _et al_. highlighted the main theoretical considerations of hypercross-linking by Friedel-Crafts reaction [112]. The extent of DVB in the monomer precursor and the ratio to vinyl benzyl chloride (VBC) controls the degree of cross-linking, as well as the ratio of VBC to STY, which controls the distance between reactive chloromethyl sites and the extent of hypercross-linking. Hypercross-linking was performed in monolithic columns, using poly(STY-_co_-VBC-_co_-DVB) monolithic columns [112]. The porosity of the monolith was not altered due to the minimal mass of the cross-linked moieties, however, the surface area of the monolith was increased from 29 m\({}^{2}\)/g to 663 m\({}^{2}\)/g, which is twice that of silica monolithic columns [113]. The isocratic separation of small molecules before modification showed little efficiency. Following the cross-linking reaction, the separation efficiency for alkylbenzenes performed under the same conditions (see Figure 5), was increased to 73,000 N/m. In the gradient separation of proteins, however, the efficiency was significantly reduced, as the introduction of meso-pores resulted in the higher impedance of mass transfer of the proteins. The group investigated the effects of the reaction conditions (temperature, time) upon the cross-linking [114]. The surface area increased dramatically for reaction times of up to 2 h, however, no significant changes in surface area were obtained at greater reaction times. The column was successfully used in three separation types: small molecules (alkylbenzenes), biomolecules (a tryptic digest of the cytochrome c protein), and size exclusion (toluene with polystyrene standards of varying molecular size). A column temperature of 80 \({}^{\circ}\)C produced columns with the best efficiencies and lower resistance to mass transfer when compared to lower column temperatures. Separations performed with the column hypercross-linked at 90 \({}^{\circ}\)C for 2 h, produced plate heights in excess of 80,000 N/m. Using the procedure outlined by [PERSON] _et al._[114], [PERSON] _et al._ produced hypercross-linked columns for applications in CEC [115]. Poly(STY-_co_-DVB-_co_-VBC) and poly(MST-_co_-DVB-_co_-VBC) columns were prepared and hypercross-linked. The hypercross-linked poly(STY-_co_-DVB-_co_-VBC) demonstrated a 34-fold increase in EOF, while the hypercross-linked poly(MST-_co_-DVB-_co_-VBC) exhibited a 21 fold increase, relative to the unmodified generic monolith. The increase in EOF was  
Figure 5: Separation of uracil and alkylbenzenes using a (**a**) generic precursor poly(STY-_co_-VBC-_co_-DVB) monolithic column, and (**b**) the hypercross-linked counterpart. Separation conditions were 80% ACN, a flow rate of 1.5 \(\upmu\)L/min, and UV detection at 254 nm. Peaks: uracil (1), benzene (2), toluene (3), ethylbenzene (4), propylbenzene (5), butylbenzene (6), and amylbenzene (7). Reprinted with permission. Adapted with permission from [112]. Copyright (2010) American Chemical Society. attributed to the increase in surface area in the column due to the formation of new meso-pores. Another use of the column design by [PERSON] _et al_. [114] was demonstrated by [PERSON] _et al_. who coupled NMR to on-line micro-HPLC separations [116].