## 2 Various PPF Strategies  
### Monoliths with GMA Functional Monomers  
poly(glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA)) monoliths grafted with poly(2-acrylamide-2-methyl-1-propanesulfonic acid) chains were prepared and used for the separation of proteins by ion-exchange HPLC mode [1]. The precursor monolith was synthesized via in situ polymerization of a mixture consisting of GMA as functional monomer and EDMA as cross-linker in a binary porogenic solvent containing cyclohexanol and dodecanol in the presence of AIBN as the thermal initiator. This was followed by converting the epoxy monolith to a diol monolith using acid hydrolysis at 60 \({}^{\circ}\)C (route I in Scheme 1). The diol monolith thus obtained constituted the substrate onto which a grafting can be performed via the transformation of the surface hydroxyl groups to free radicals by the Ce(IV) initiation which yield a single radical on the oxygen atom of the hydroxyl group according to the following simplified mechanism:  
\[\text{Ce(IV)}+\text{RCH}_{2}\text{OH}\rightarrow\text{Ce(III)}+\text{H}^{+}+ \text{RCH}_{2}\text{O}.\]  
The free radicals allowed the grafting of poly(2-acrylamide-2-methyl-1-propanesulfonic acid) onto the internal surface of the porous diol monolith (see route I in Scheme 1). The optimization of the experimental conditions was investigated, and the study demonstrated that the method successfully separated three proteins (i.e., myoglobin, chymotrypsinogen A and lysozyme) within 1.5 min. Recently, PPF allowed the covalent attachment of iminodiacetic acid (IDA) to a poly(GMA-co-EDMA) continuous rod column [2]. In this case, the precursor monolith was prepared via in situ polymerization of a mixture containing GMA and EDMA in the presence of a binary porogenic mixture of dodecyl alcohol and cyclohexanol under thermal initiation condition. The continuous rod column thus obtained was then subjected to PPF with IDA (see route II in Scheme 1) and was used in immobilized metal affinity chromatography (IMAC) with IDA chelated with a bivalent metal ion, e.g., Cu\({}^{2+}\), Ni\({}^{2+}\) or Zn\({}^{2+}\). The monolithic rod in the form of naked IDA or Cu\({}^{2+}\)-IDA confined in a stainless steel tubing of 5 cm \(\times\) 4 mm I.D. allowed the purification of lysozyme from egg white and human serum albumin (HSA) from the commercially available HSA solution as was confirmed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In a rather complex PPF process, a poly(GMA-co-EDMA) monolithic capillary was first modified with sodium hydrogen sulfide to obtain a monolith with reactive thiol groups whereby about 17% of the total epoxide moieties of the monolithic precursor were converted to thiol as was determined by a disulfide-exchange reaction [3]. In a further PPF step, a quinine derivative was attached to the thiol activated monolith and the column thus obtained was assayed by CEC in the separation of the enantiomers of _N_-3,5-dinitrobenzoyll-leucine (see Route III in Scheme 1). Using the thiol activation of poly(GMA-co-EDMA) monolith, the resulting monolith was further functionalized with (_S_)-N-(4-allylov-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutanephosphonic acid as chiral selector to yield enantioselective strong cation exchanger (SCX) capillary columns of 100 \(\upmu\)m I.D. (see Route IV in Scheme 1). However, and due to the lipophilic character of the poly(GMA-co-EDMA), the enantioseparations were not satisfactory. In order to overcome the lipophilicity of the precursor monolith, 2-hydroxyethyl methacrylate (HEMA) monomer was co-polymerized with GMA and EDMAleading to the poly(GMA-_co_-HEMA-_co_-EDMA) precursor of less lipophilicity, which then allowed as good enantioseparations as those with silica-based enantioselective SCX by CEC [4]. A monolithic capillary column made of poly(GMA-_co_-EDMA) and coated with gold nanoparticles (GNP) was suggested for proteomics analysis that would enable the capture and separation of cysteine containing peptides [5].