## 2 Various PPF Strategies
### PPF Involving Click Coupling of Monoliths  
In a study focused on the separation of proteins, poly(GMA-_co_-divinylbenzene (DVB)) and poly(4-vinylbenzyl chloride (VBC)-_co_-DVB) were prepared and then modified with azide functionalities to achieve the reactive sites for a subsequent click chemistry [19]. The azide modified monoliths were then subjected to PPF with a solution of 1-decyne and CuI to obtain alkylated monoliths via the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, see Scheme 9. The chromatographic performances of the \"clicked\" stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min. A novel thiol-ene \"click\" strategy has been applied for the preparation of hybrid organic-inorganic monolith-based trypsin microreactor [20]. The preparation of hybrid organic-inorganic monolithic columns consisted of tetramethyloxysilane and \(\gamma\)-methacrylopropyltrimethoxysilane. Then, the trypsin immobilization solution, which contained free thiol groups, was passed onto the obtained capillary and then the capillary was kept at 25 \({}^{\circ}\)C for 5 h to form trypsin microreactor, see Scheme 10. The study involved on-line capillary zone electrophoresis and the results showed that the trypsin microreactor could give highest activity with poly(\(N\),\(N^{\prime}\)-methylenebisacrylamide). The thiol-ene \"click\" strategy is a promising method for the selective immobilization of proteins. An organic-inorganic hybrid silica monolithic column containing n-octadecanethiol (C\({}_{18}\)), 3-mercapto-1-propane-sulfonate (MPS), and vinyltrimethoxysilane (VTMS) has been prepared through thiol-ene \"click\" strategy (Scheme 11) for CEC [21]. The obtained monolithic column showed high hydrophilic property compared to unmodified VTMS monolithic column. The proposed column can also be used in different separation modes, such as RPC. It showed successful separations of benzenes, aromatic amines, acids and peptides. The preparation of a monolithic-immobilized enzyme reactor by click-chemistry for micro-LC was recently reported [22]. It involved the preparation of a poly(GMA-_co-_EDMA) monolith via in situ thermal polymerization in capillary format. The epoxypropyl groups of monolith were  
Figure 11: Scheme for the modification of the vinyltrimethoxysilane (VTMS) hybrid monolith with functional monomer compounds. Taken with permission from [21]. converted to diol form by a well-established acidic hydrolysis process. Terminal chlorine moiety was obtained on the monolith by following a silane coupling chemistry based on the reaction between trimethoxysilyl groups of the reagent 3-chloropropyltrimethoxysilane (CPTMS), and diol groups of hydrolyzed monolith (Scheme 12). NaN\({}_{3}\) was then reacted with terminal chlorine of the monolith to generate azide functionality (Scheme 13). On the other hand, alkyne-carrying enzyme was obtained by reacting an alkyne-functionalized ligand, pentynoic acid (PA) with \(\alpha\)-chymotrypsin (CT) via amide bond formation between carboxyl group of PA and amine groups of enzyme via water-soluble carbodiimide activation (Scheme 13B). In the last stage, the alkyne-carrying enzyme was covalently attached onto azide-functionalized monolith via triazole ring formation according to click-coupling reaction (Scheme 13C). The obtained monolithic capillary column was then modified with the enzyme \(\alpha\)-chymotrypsin through the covalent attachment by triazole-ring formation. The activity behavior of the proposed monolith was studied using benzoyl-L-tyrosine ethyl ester as the synthetic substrate. The results revealed that the final substrate conversion increased with increasing the feed flow rate and substrate feed concentration due to the increase of the interaction between the substrate and the enzymatic monolith reactor. The preparation of porous polymer monoliths with dodecyl and zwitterionic functionalities via \"thiol-ene\" click chemistry for the chromatographic separations in RPC and HILIC modes was studied [23].