## 2 Various PPF Strategies
### PPF of Hypercrosslinked Monoliths  
Hypercrosslinked porous polymer monolithic capillary columns modified with zwitterionic functionalities were developed through the interaction with GNPs in a layered structure for performing separations using the HILIC mode [14]. The proposed monolithic capillary column started with a preparation of a generic hypercrosslinked poly(4-methylstyrene-_co_-vinylbenzyl chloride-_co_-divinylbenzene) via a Friedel-Crafts reaction catalyzed with iron chloride to achieve large surface area of 430 m\({}^{2}\)/g, see Scheme 5. Then, additional surface functionalization using a free radical bromination was obtained to enable their modification as shown in Scheme 5. Afterwards, the brominated monolith was reacted with cystamine under microwave irradiation for 30 min and reached the maximum of sulfur content. The disulfide bond was snipped using tris(2-carboxylethyl) phosphine (TCEP) to liberate the thiol groups. The next step was to pump the GNPs through the monolith to obtain the first layer of GNP-functionalized monolith. To further increase the column efficiency, polyethyleneimine (PEI) was attached onto the GNP-functionalized monolith followed by a second layer of GNPs that were then functionalized with zwitterionic cysteine, see Scheme 6. This proposed monolith showed \"thick\" hydrophilic layer using the dual-layer approach with GNPs that included PEI and cysteine functionalities and was tested with the separation of nucleosides and peptides with separation efficiency of 51,000 plates/m at a flow rate of 0.3 \(\upmu\)L.  
The preparation of porous polymer monolith possessing both large surface area and functional groups for nano-LC was developed [15]. First, the generic monolith was prepared in a capillary by the in situ thermal polymerization of a mixture containing 4-acetoxystyrene, styrene, vinylbenzyl chloride, divinylbenzene and porogenic solvents that consisted of toluene and 1-dodecanol (see Scheme 7). After the polymerization was completed, a solution of hydrazine hydrate was pumped through the monolithic capillary to obtain hydroxyl functional groups resulting from the deprotection of the acetoxy groups. The post-polymerization hypercrosslinking of the monolith was then obtained by adding ferric chloride to offer a large number of small pores thus enhancing the surface area (see Scheme 7). The resulting monolith was evaluated in the separation of small molecules using RPC and normal  
Figure 5: Scheme of preparation of poly(4-methylstyrene-_co_-vinylbenzene chloride-_co_-divinylbenzene) monolith, its hypercrosslinking and free radical bromination with molecular bromine. Taken with permission from [14]. phase chromatography modes. A column efficiency of 40,000 plates/m was obtained for benzene in the RPC mode, while 31,800 plates/m was achieved for nitrobenzene in the normal phase mode. The same research group developed a new approach of the preparation of large surface area monolithic capillary columns using external crosslinkers for the separation of small molecules in nano-LC [16]. The poly(styrene-\(\alpha\)-divinylbenzene) monolith was prepared by an in situ thermal polymerization process followed by a hypercrosslinking process using the catalyst FeCl\({}_{3}\) under Friedel-Crafts reaction. The reaction process included three external crosslinkers, such as 4,4'-bis(chloromethyl)-1,1'-bibhenyl, \(\alpha\)-\(\alpha\)'-dichloro-p-xylene and formaldehyde dimethyl acetal, see the following structures (Figure 1). The results revealed that the 4,4'-bis(chloromethyl)-1,1'-bibhenyl crosslink represented the best separation performance and large surface area that reached 900 m\({}^{2}\)/g. Figure 6: Scheme of modifications of brominated hypercrosslinked poly(4-methylstyrene-\(\alpha\)-vinylbenzene chloride-\(\alpha\)-divinylbenzene) monolith with cystamine, tris(2-carboxylethyl) phosphine (TCEP), gold nanoparticles, polyethyleneimine and cysteine. Taken with permission from [14].