Porous methacrylate-based polymer monoliths and preparation methods thereof through raft polymerisation

Abstract

A mesoporous, hierarchically porous, and macroporous methacrylate-based polymer monoliths and preparation methods thereof through reversible addition-fragmentation chain-transfer (RAFT) polymerization is disclosed. The polymer monoliths may find use in liquid chromatography applications.

Description

POROUS METHACRYLATE-BASED POLYMER MONOLITHS AND PREPARATION METHODS THEREOF THROUGH RAFT POLYMERISATIONPRIORITY DOCUMENT

[0001] The present application claims priority from Australian Provisional Patent Application No. 2022903776 titled “POROUS METHACRYLATE-BASED POLYMER MONOLITHS AND PREPARATION METHODS THEREOF THROUGH RAFT POLYMERISATION” and fded on 9 December 2022, the content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to porous polymer monoliths and preparation methods thereof. In particular, the present disclosure relates to mesoporous, hierarchically porous, and macroporous methacrylate-based polymer monoliths and preparation methods thereof through reversible addition-fragmentation chain-transfer (RAFT) polymerization.BACKGROUND

[0003] Porous materials are used in a wide range of applications involving interface processes, such as catalysis1, sensors2, adsorption3, separations4, drug delivery5and energy storage6. Both the chemistry and the morphology of materials have a significant impact on their performance and applicability. Surface chemistry directly affects the selectivity of a porous material towards a target compound, while pore size, pore volume and pore shape determine their accessibility, loading capacity and diffusion properties. Therefore, there is a constant interest in developing new technologies to obtain porous materials with different chemistries and pore morphologies.

[0004] According to their chemical nature, porous materials can be classified into organic or inorganic. While inorganic porous structures, such as silica and zeolites, typically present high mechanical resistance, variable pore sizes, including micro- (less than 2 nm), meso- (between 2-50 nm) or macropores (larger than 50 nm) or dual pore size distributions, non-specific interactions with a wide variety of compounds are frequent and can cause undesired adsorption7. Organic porous materials, typically polymer-based, present a higher selectivity towards specific compounds due to the very large variety of precursors that can be used, and a more flexible structure that swells in the presence of a solvating solvent. In particular, methacrylate materials are relatively polar polymers that present interesting properties, making them suitable for separations, biomedical, optical or sensing applications8. For example, because of their relative polar character, methacrylate porous polymers are suitable as stationary phases for the separation of biomolecules. Even though agarose gels have been used for suchpurposes, methacrylate porous polymers present higher mechanical resistance, which is advantageous in chromatographic applications9. Also, methacrylates are optically transparent to UV and visible light, they possess a good thermal stability (ranging from -70 °C to 100 °C) and have a good degree of compatibility with human tissue8.

[0005] Monoliths are a good alternative for stationary phases as they are porous materials synthesised in one piece, which reduces the risk of breakage. A monolith material may have specific macropores, mesopores and / or surface chemistry. Inorganic monoliths are usually associated with non-specific interactions in the surface but possess adequate porosities for efficient separations. Organic monoliths are very versatile in surface chemistries but lack the surface area needed for chromatographic separations. Typically, organic monoliths are obtained by free radical co-polymerization of a vinyl monomer and a divinyl crosslinker in the presence of a mixture of solvents. In order to achieve porosity in organic crosslinked polymers, different methodologies have been used, including use of templates, block copolymers self-assembly and direct synthesis10.

[0006] Templates are inert materials that are present in the polymerization mixture but do not participate in the reaction. In this approach, either the polymerization mixture is introduced into a three- dimensional arrangement of the template or the template is dispersed into the polymerization mixture. In a second step, the polymerization reaction is performed in the voids between the template, and finally the template is removed, leading to a porous polymer11. A wide variety of materials can be used as templates, such as organic12or inorganic particles13, micelles14, sintered salts15or even molecules (as in Molecular Imprinted Polymers (MIPs)16). Templates need to be stable in the polymerization conditions and easy to remove without damaging the porous polymer, which might become a challenge.

[0007] Self-assembly of block co-polymers occurs when a co-polymer contains blocks of immiscible homopolymers. Because the blocks are thermodynamically incompatible, self-assembly takes place to form a bi-continuous structure. In this strategy, one of the blocks is used as a framework for the desired polymer, and the other is used as a sacrificial block and etched to form the pores. The morphology of the porous structure depends on the total length of the co-polymer, the chemical nature of the blocks and the length of each block17.

[0008] Direct synthesis is a simple approach to obtain porous polymers. A reaction mixture containing a monomer, a crosslinker, an initiator and a solvent (also called porogen) is polymerized, obtaining a porous polymer with a total pore volume corresponding to the solvent volume in the initial mixture. When the solvent solvates the monomer but not the final polymer, a phase separation process is observed during the reaction, leading to a completion of the polymerization in a heterogeneous system and resulting in a macroporous structure18. By changing experimental conditions, such as the composition of thepolymerization mixture, reaction temperature or time, macropore size and volume can be tunned19, however, polydisperse pore distributions and low surface areas are obtained20.

[0009] Organic monoliths are typically macroporous and the pore size distribution is broad. Thus, even when high permeability is achieved, the low surface area causes low efficiency as well as poor selectivity in chromatographic applications. Different research groups around the world tried to tackle this issue by exploring a range of approaches to develop organic monoliths with smaller pores while keeping macropores, for example, by using crosslinker as the only monomer or by hypercrosslinking, a postpolymerization modification where functional pendant groups in the monolith surface react with each other. Hypercrosslinking is a very well-established method for styrene-based systems, where a macroporous poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) (poly(Sty-co-VBC-co-DVB)) is first synthesized and in a second step the halogenated groups are crosslinked by a Friedel-Crafts alkylation. For example, hypercrosslinking21provided the introduction of micropores (pore size lower than 2 nm) in styrene-based materials, making organic monoliths suitable only for the separation of small analytes. However, microporosity can be observed in the dry state only if the crosslinker has a rigid molecular structure22, otherwise only the swollen state of the monolith shows a high surface area while in the dry state that microporous structure collapses, which is the case for methacrylate monoliths23.

[0010] Recently, controlled radical polymerizations have been used to change the polymerization mechanism in the synthesis of organic monoliths, changing its resulting pore structure and allowing to perform post-synthesis functionalization on its surface, changing its chemistry and architecture24. In this regard, a poly(styrene-co-divinylbenzene) monolith was synthesised in the presence of 2, 2, 6, 6- tetramethyl-1 -piperidyloxy (TEMPO) as initiator. The presence of TEMPO was observed in the monolith after polymerization and it was active for a grafting process, which was evidenced by the addition of either 2-hydroxyethyl methacrylate (HEMA) or divinylbenzene (DVB). Grafting was performed by heating the monolith in the presence of the second monomer at 130 °C without an additional initiator and the incorporation of the new functionality was confirmed by FT-IR analysis of the resulting monoliths25.

[0011] Moreover, when a monolith is synthesized by controlled polymerization, higher surface areas and lower pore size polydispersity compared to those obtained by free radical polymerization are achieved26. Organotellurium-mediated living radical polymerization (TERP) has been used to synthesize poly(butyl methacrylate-co-ethylene glycol dimethacrylate) (poly(BMA-co-EDMA)) and poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) (poly(LMA-co-EDMA)) monoliths as stationary phases for liquid chromatography. Monoliths obtained by TERP were compared with monoliths synthesized by free radical polymerization and it was observed that columns obtained by controlled radical polymerization were more efficient due to a structure with smaller pores (which dimensions were not informed) and less polydispersity27.

[0012] Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization as a controlled radical polymerization has also been used in conjunction with Polymerization-Induced Microphase Separation (PIMS) for obtaining mesoporous and / or macroporous monoliths. In this approach, a macrochain transfer agent (macro-CTA) is synthesized resulting in a linear homopolymer with a CTA end group. In a second step, the macro-CTA is dissolved in a mixture containing a monofunctional monomer and a bifunctional monomer. A block co-polymer is then generated, where the linear homopolymer domain is encapsulated into the crosslinked domain by microphase separation. Finally, the linear homopolymer is etched and removed from the crosslinked structure, obtaining a mesoporous monolith. In this sense, poly (lactide), poly ( / / -octyl styrene sulfonic ester) or poly(p-vinylbenzyl chloride)28macro- CTAs have been used to obtain mesoporous poly(styrene-co-divinyl benzene) (poly(Sty-co-DVB)) monoliths. This approach was also used to obtain a mesoporous poly(isobornyl acrylate-co-ethylene glycol diacrylate) (poly(IBA-co-EGDA)) film by photopolymerization29. Even when PIMS shows the versatility of RAFT polymerization for the controlled polymerization of a wide variety of monomers, it requires a final step of etching and removal of the sacrificial block, as any other method based in block co-polymers self-assembly, which might take several days30. A styrene-based monolith with macro- as well as mesopores was achieved by combining macro- and microphase separation31. In this approach, mesopores were achieved by introducing in the polymerization mixture a macro-chain transfer agent (macro-CTA), which is a polymer with a transfer agent end group. As the macro-CTA reversibly reacts with the growing chains of the monolith, a final structure of a crosslinked polymer with the macro-CTA encapsulated inside was obtained. Etching and removing the macro-CTA resulted in a mesoporous monolith. If in addition to the macro-CTA, an inert polymer, immiscible with the monolith, was introduced in the polymerization mixture, a polymer blend was formed during the monolith synthesis, resulting in the incorporation of isolated macropores. Both the macro-CTA and the inert polymer promoted porosity in the monolith and no solvents were used. The experimental process was long and complicated as it included the synthesis of the macro-CTA, the polymerization of the monolith and finally the etching and removal of the macro-CTA and inert polymer. There are no reports of the application of this material in chromatography, probably because the lack of interconnected macropores, which would not allow the flowrate of a mobile phase through the material nor allow accessibility of the mesopores for a chromatographic separation.

[0013] Synthesis of mesoporous methacrylate materials remains a challenge. Reports of noncrosslinked mesoporous methacrylate materials30as well as hybrid materials (where mesoporosity is provided by an inorganic nanoparticle32,33or crosslinker34) can be found in literature. However, the mesoporosity is not permanent and the monolith may be subject to a change in structural rigidity and robustness.

[0014] Accordingly, there remains a need for a porous polymer monolith and a preparation method thereof which may solve or alleviate one or more of the above problems.SUMMARY

[0015] In a first aspect, provided herein is a method for preparing a porous methacrylate -based polymer monolith through reversible addition-fragmentation chain-transfer (RAFT) polymerization, wherein the method comprises: introducing a reaction composition into a mold, the reaction composition comprising at least one methacrylate monomer, at least one methacrylate crosslinker, a radical initiator, a chain transfer agent (CTA), and a porogenic mixture of a solvating solvent with a non-solvating solvent and / or an inert polymer; conducting the RAFT polymerisation to form a polymer monolith; and optionally, separating the polymer monolith from unreacted reagents.

[0016] In certain embodiments of the first aspect, the methacrylate monomer comprises a mono vinyl monomer having a methacrylate unit. In some embodiments, the methacrylate monomer is a monovinyl monomer having a methacrylate unit. In some embodiments, the monovinyl monomer having a methacrylate unit is selected from the group consisting of glycidyl methacrylate (GM A), methyl methacrylate, ethyl methacrylate, butyl methacrylate, octadecyl methacrylate, lauryl methacrylate, and poly (ethylene glycol) methacrylate. In some embodiments, the mono vinyl monomer having a methacrylate unit is glycidyl methacrylate (GMA).

[0017] In certain embodiments of the first aspect, the methacrylate crosslinker comprises a multi-vinyl monomer having a methacrylate unit, for example a divinyl monomer having a methacrylate unit. In some embodiments, the methacrylate crosslinker is a multi-vinyl monomer having a methacrylate unit, for example a divinyl monomer having a methacrylate unit. In some embodiments, the multi-vinyl monomer having a methacrylate unit is selected from the group consisting of ethylene glycol dimethacrylate (EDMA), triethylene glycol dimethacrylate (TEGDMA), trimethylol-propane trimethacrylate (TRIM), and poly (ethylene glycol) dimethacrylate. In some embodiments, the multi- vinyl monomer having a methacrylate unit is ethylene glycol dimethacrylate (EDMA).

[0018] In certain embodiments of the first aspect, the crosslinker and the monomer for the monolith are in a molar ratio of about 1:0.9 to about 1:3.3, for example about 1:1.4 to about 1:2.1. In some embodiments, the crosslinker and the monomer for the monolith are in a molar ratio of about 1:1.4.

[0019] In certain embodiments of the first aspect, the molecular chain transfer agent has the following formula (I):wherein Z is selected from an aryl, an alkylthio group, and a carboxyalkylthio group, R is a substituent having a tertiary carbon and optionally a nitrile group connected to the tertiary carbon, and the alkyl has 1 to 20 carbon atoms, for example 2 to 12 carbon atoms, each of the aryl and the alkyl is optionally substituted with a group selected from H, Ci-6 alkyl, halogen, -OH, -SH, -O-Ci 6 alkyl, and -S- Ci-6 alkyl. In some further embodiments, the chain transfer agent is selected from 4-cyano-4- ((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDSTS), 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CETCPA), and 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTBCOOH), 2-cyano-2-propyl benzodithioate, 4-cyano-4- (phenylcarbonothioylthio) pentanoic acid, 2-phenyl-2- propyl benzoditioate, 2-cyano-2-propyl dodecyl trithiocarbonate.

[0020] In certain embodiments of the first aspect, the chain transfer agent and the monomer / the crosslinker is in a molar ratio of about 0.5:100 to about 3:100, for example about 0.75:100 to about 1.25: 100. In some embodiments, the chain transfer agent and the monomer / the crosslinker is in a molar ratio of about 1:100.

[0021] In certain embodiments of the first aspect, the solvating solvent within the porogenic mixture is selected from the group consisting of dioxane and tetrahydrofuran. In some embodiments, the nonsolvating solvent within the porogenic mixture is selected from the group consisting of toluene, cyclohexanol, and dodecanol. In further embodiments, the inert polymer is selected from poly(ethylene glycol) (PEG), poly (aery lie acid) (PAA) and polymethacrylates such as poly (tert-butyl methacrylate) (P(t-BMA)). In some further embodiments, the PEG has a molecular weight of about 6K g / mol to about 35K g / mol, for example 6K g / mol, 10K g / mol, 20K g / mol, and 35K g / mol. In some further embodiments, the PEG has a molecular weight of about 10K g / mol to about 20K g / mol. In some further embodiments, the P(t-BMA) has a molecular weight of about 4.7K g / mol to 21K g / mol, for example 5K g / mol, 10K g / mol and 20K g / mol.

[0022] In certain embodiments of the first aspect, the solvating solvent is in the range of 12-48 %wt, and the non-solvating solvent and / or the inert polymer is(are) in the range of 12-48 %wt, based on the reaction composition. In some embodiments, the porogenic mixture of the solvating solvent with the non-solvating solvent and / or the inert polymer is about 60 %wt based on the reaction composition. In some further embodiments, the weight ratio between the solvating solvent and the non-solvating solvent and / or the inert polymer is(are) in a range of about 20:80 to about 80:20.

[0023] In certain embodiments of the first aspect, the RAFT polymerisation is conducted at a temperature of about 50 °C to about 70 °C. In some embodiments, the RAFT polymerisation is conducted at a temperature of about 50 °C. In some embodiments, the RAFT polymerisation is conducted at a temperature of about 60 °C.

[0024] In certain embodiments of the first aspect, the monolith prepared is a mesoporous methacrylate- based polymer monolith. In some embodiments, the mesoporous methacrylate-based polymer monolith displays a mesopore volume of up to about 0.620 mL / g in dry state, which is at least approximately 25 %v / v with respect to the total volume of the monolith. In some embodiments, the mesoporous methacrylate-based polymer monolith displays a BET surface area of about 137 m2 / g, for example up to about 155 m2 / g, for pores having a diameter in the range of 2-250 nm. In some embodiments, the mesopores in the monolith is up to about 35% by volume, for example about 25% by volume, with respect to the total volume of the monolith. In some embodiments, a non-solvating solvent selected from dodecanol, toluene and cyclohexanol and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture. In some further embodiments, the non-solvating solvent and the solvating solvent are used in a weight ratio of about 50:50. In even further embodiments, the porogenic mixture consists of dodecanol and dioxane, for example in a weight ratio of about 50:50. In even further embodiments, the porogenic mixture consists of dodecanol and tetrahydrofuran, for example in a weight ratio of about 50:50. In some embodiments, an inert polymer selected from poly(ethylene glycol) (PEG), poly(acrylic acid) (PAA) and polymethacrylates such as poly(tert-butyl methacrylate) (P(t-BMA)) and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture. In some further embodiments, the inert polymer and the solvating solvent are used in a weight a ratio of about 50:50. In even further embodiments, the porogenic mixture consists of a PEG having a molecular weight of about 6K g / mol and dioxane, for example in a weight ratio of about 50:50. In some embodiments, the RAFT polymerisation is conducted at a temperature of about 60 °C.

[0025] In certain embodiments of the first aspect, the monolith prepared is a hierarchically porous methacrylate-based polymer monolith. In some embodiments, the hierarchically porous methacrylate- based polymer monolith exhibits a dual pore size distribution in dry state. In some embodiments, the hierarchically porous methacrylate-based polymer monolith exhibits a dual pore size distribution in macropore region in dry state, for example with about 60 nm and about 550 nm. In further embodiments, the pores in a size range of about 100 nm to about 1000 nm and the pores in a size range of about 20 nm to about 100 nm are in a volume ratio of about 50:50. In some further embodiments, the pore volume of the hierarchically porous methacrylate-based polymer monolith is about 0.167 mL / g in a pore size rangeof about 20-100 nm and about 0.584 ml / g in a pore size range of about 100-1,000 nm. In even further embodiments, the hierarchically porous methacrylate-based polymer monolith has about 22 %v / v pores in a size range of about 20 nm to about 100 nm and about 78 %v / v pores in a size range of about 100 nm to about 1000 nm with respect to the total pore volume. In even further embodiments, the hierarchically porous methacrylate-based polymer monolith has about 12 %v / v pores in a size range of about 20 nm to about 100 nm and about 43 %v / v pores in a size range of about 100 nm to about 1000 nm with respect to the total monolith volume. In some embodiments, a non-solvating solvent such as dodecanol, an inert polymer and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture. In some further embodiment, an inert polymer, a non-solvating solvent such as dodecanol, and a solvating solvent selected from dioxane and tetrahydrofuran are in a weight ratio of about 12.5:50:37.5. In some further embodiments, PEG, dodecanol, and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture. In some further embodiments, the porogenic mixture consists of a PEG having a molecular weight of about 6K g / mol to about 20K g / mol, dodecanol and dioxane, for example in a weight ratio of about 12.5:50:37.5. In some embodiments, the RAFT polymerisation is conducted at a temperature of about 50 °C.

[0026] In certain embodiments of the first aspect, the monolith prepared is a macroporous methacrylate-based polymer monolith, particularly a fully macroporous methacrylate-based polymer monolith developed via spinodal decomposition. In some embodiments, a solvating solvent such as dioxane and tetrahydrofuran together with an inert polymer such as polymethacrylate are used to form the porogenic mixture. In some embodiments, the inert polymer and the solvating solvent is in a weight ratio of about 22:78 to about 25:75, for example about 23:77. In some further embodiments, the polymethacrylate is poly(tert-butyl methacrylate) (P(t-BMA)), for example having a molecular weight of about 4.7K g / mol, 10K g / mol or 21K g / mol. In some embodiments, the porogenic mixture consists of poly(tert-butyl methacrylate) (P(t-BMA)) having a molecular weight of about 4.7K g / mol to about 21K g / mol and dioxane. In some further embodiments, the P(t-BMA) having a molecular weight of about 4.7K g / mol o about 21K g / mol and the dioxane is in a weight ratio of about 22:78 to about 25:75.

[0027] In a second aspect, provided herein is a porous methacrylate-based polymer monolith obtained through the method according to the first aspect. In some embodiments, the porous methacrylate-based polymer monolith is a mesoporous methacrylate-based polymer monolith. In some embodiments, the porous methacrylate-based polymer monolith is a hierarchically porous methacrylate-based polymer monolith, particularly a hierarchically porous methacrylate-based polymer monolith which exhibits a dual pore size distribution in dry state. In some further embodiments, the hierarchically porous methacrylate- based polymer monolith exhibits a dual pore size distribution in macropore region, for example with about 60 nm and about 550 nm. In some embodiments, the porous methacrylate-based polymer monolith is a macroporous methacrylate-based polymer monolith, particularly a fully macroporous methacrylate- based polymer monolith developed via spinodal decomposition.

[0028] In a third aspect, provided herein is a separation medium that comprises the porous methacrylate-based polymer monolith obtained through the method according to the first aspect. In some embodiments, the medium is used for a liquid chromatography application selected from normal phase chromatography, hydrophilic interaction liquid chromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography and ion exchange chromatography.

[0029] In a fourth aspect, provided herein is use of the porous methacrylate-based polymer monolith obtained through the method according to the first aspect in a liquid chromatography application. In some embodiments, the liquid chromatography application is selected from normal phase chromatography, hydrophilic interaction liquid chromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography, and ion exchange chromatography.BRIEF DESCRIPTION OF THE FIGURES

[0030] Non-limiting embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein:

[0031] Figure 1 shows (I) scanning electron microscopy (SEM) images and (II) pore size distributions ((a) from nitrogen adsorption isotherms or (b) and (c) from mercury intrusion porosimetry) for poly(GMA-co-EDMA) monoliths with different porous structures, i.e. (a) fully mesoporous, (b) hierarchically porous, and (c) fully macroporous by spinodal decomposition. In all cases, the reaction compositions contained 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture, 1 %w azobisisobutyronitrile (AIBN) with respect to the monomer and the crosslinker and CDSTS / AIBN=2 (molar ratio). The porogenic mixtures contained (a) PEG(6K):dioxane = 50:50 by weight, (b) PEG(20K): dodecanol: dioxane=12.5:50:37.5 by weight and (c) P(t-BMA)(10K): dioxane = 22:78 by weight.

[0032] Figure 2 shows nitrogen adsorption-desorption isotherms for monoliths obtained by free radical or RAFT polymerization using CDSTS, CETCPA or CTBCOOH as the chain transfer agent. The reaction compositions contained 20 %w GMA, 20 %w EDMA, 30 %w PEG(6K), 30 %w dioxane, 1 %w AIBN with respect to the monomer and the crosslinker, and CTA / AIBN=2 (molar ratio).

[0033] Figure 3 shows pore size distributions for monoliths obtained by free radical or RAFT polymerization using CDSTS, CETCPA or CTBCOOH as the chain transfer agent. The reaction compositions contained 20 %w GMA, 20 %w EDMA, 30 %w PEG(6K), 30 %w dioxane, 1 %w AIBN with respect to the monomer and the crosslinker, and CTA / AIBN=2 (molar ratio).

[0034] Figure 4 shows SEM images for monoliths obtained by RAFT polymerization using CDSTS, CETCPA or CTBCOOH as the chain transfer agent or obtained by free radical polymerisation. Thereaction compositions contained 20 %w GMA, 20 %w EDMA, 30 %w PEG(6K), 30 %w dioxane, 1 %w AIBN with respect to the monomer and the crosslinker, and CTA / AIBN=2 (molar ratio).

[0035] Figure 5 shows X-ray photoelectron spectroscopy (XPS) spectra indicating the elemental composition of monoliths synthesized by (a) free radical polymerisation or RAFT polymerisation using (b) CDSTS, (c) CTBCOOH or (d) CETCPA as the chain transfer agent. The reaction compositions contained 20 %w GMA, 20 %w EDMA, 30 %w PEG(6K), 30 %w dioxane, 1 %w AIBN with respect to the monomer and the crosslinker, and CTA / AIBN=2 (molar ratio).

[0036] Figure 6 shows first order plots for the monolith synthesis by (a) free radical or RAFT polymerisation with (b) CDSTS, (c) CTBCOOH or (d) CETCPA as the chain transfer agent. In all cases the reaction compositions contained 20 %w GMA, 20 %w EDMA, 30 %w PEG(6K), 30 %w dioxane, 1 %w AIBN with respect to the monomer and the crosslinker, and CTA / AIBN=2 (molar ratio).

[0037] Figure 7 shows apparent molar mass (M) obtained by SEC vs. conversion for linear GMA in dioxane and poly(GMA-co-EDMA) monoliths in 100 % dioxane as the solvent or dodecanol / dioxane 50:50 as the solvents. For all reactions 40 %w monomer / crosslinker (either GMA or GMA / EDMA 50:50 weight ratio) and 60 %w porogenic mixture were used, with 1 %w AIBN with respect to the monomer and the crosslinker and a CDSTS / AIBN =2 (molar ratio).

[0038] Figure 8 shows (I) pore volume (2-100 nm) from nitrogen adsorption-desorption isotherms and (II) SEM images for monoliths with variable compositions of a porogenic mixture. Monoliths were synthesised with 20 %w GMA, 20 %w EDMA, 60 %w porogenic mixture (dodecanol, dioxane and PEG either (a) (6K), (b) (10 K) or (c) (20 K)), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). Polymerisations were performed at 50 °C.

[0039] Figure 9 shows pore size distributions from nitrogen adsorption isotherms and mercury intrusion porosimetry (MIP) for monoliths with different porogenic mixtures. The reaction compositions contained 40 %w monomer / crosslinker (GMA / EDMA 50:50), 60 %w porogenic mixture (PEG, dodecanol and dioxane), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). The porogenic mixtures were PEG(6K) / dodecanol / dioxane (a) 0:50:50, (b) 12.5:50:37.5, (c) 25:50:25, (d) 37.5:50:12.5, (e) PEG(10K) / dodecanol / dioxane=12.5:50:37.5, (f) PEG(20K) / dodecanol / dioxane =12.5:50:37.5 and (g) PEG(20K) / dodecanol / dioxane =12.5:50:37.5 by free radical polymerisation.

[0040] Figure 10 shows SEM images of some hierarchically porous methacrylate-based polymer monoliths prepared by the method disclosed herein with (a) PEG(10K) / dodecanol / dioxane =12.5:50:37.5 and (b) PEG(20K) / dodecanol / dioxane=12.5:50:37.5. The reaction compositions contained 40 %wmonomer / crosslinker (GMA / EDMA 50:50), 60 %w porogenic mixture (PEG, dodecanol and dioxane), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio).

[0041] Figure 11 shows procedure for column filling: (a) pouring the reaction composition in a column attached to a vial, (b) sealing the column top end and (c) inverting the column, as well as the set up filled with reaction composition before and after polymerisation (c) and (d).

[0042] Figure 12 shows elugrams of PMMA standards in a 4.6x150 mm column obtained from 20 %w GMA, 20 %w EDMA, 60 %w porogenic mixture (PEG(20K) / dodecanol / dioxane 12.5:50:37.5), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 molar ratio (column 3 in Table 3). Chromatographic conditions: mobile phase: THF, F=0.2 mL / min, Vinj=5pl, Tcoi=40°C, 7=230 nm.

[0043] Figure 13 shows elugrams for PMMA standards in the range of 2.58K g / mol to 4,810K g / mol in 4.6x150mm monolithic columns synthesised by (a) RAFT polymerisation (CDSTS / AIBN=2 molar ratio) or (b) free radical polymerisation. The reaction compositions for the monoliths contained 20 %w GMA, 20 %w EDMA, 60 %w porogenic mixture (PEG(20K) / dodecanol / dioxane 12.5:50:37.5), 1 %w AIBN with respect to the monomer and the crosslinker. Chromatographic conditions: mobile phase: THF, F=0.2 mE / min, Vinj=5pl, Tcoi=40°C, 7=230 nm.

[0044] Figure 14 shows calibration curves from (a) columns 1-5 (see Table 3) or (b) columns 3 and 5 in series for PMMA analytes with molar masses from 2.58K g / mol to 4,810K g / mol.

[0045] Figure 15 depicts phase separation processes by (a) nucleation and growth and (b) spinodal decomposition35.

[0046] Figure 16 shows SEM images of monoliths prepared from 20 %w GMA, 20 %w EDMA, 60 %w porogenic mixture, 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). The porogenic mixtures contained P(t-BMA) / dioxane mixtures in various weight proportions. Polymerisations were performed at 60 °C.

[0047] Figure 17 shows SEM images of monoliths prepared with either P(t-BMA)(10K) or PEG (10K) by conventional free radical or RAFT polymerisations. Reaction mixtures contained 20 %w GMA, 20 %w EDMA, 60 %w porogenic mixtures consisting of P(t-BMA) or PEG and dioxane in various compositions, 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio) for the monoliths by RAFT polymerisation. Percentages recited in the figures indicate the weight percentage of polymer in the porogenic mixture.

[0048] Figure 18 shows pore size distributions obtained from MIP analysis for monoliths with (a) different porogenic mixtures or (b) different polymerisation mechanisms. Monoliths contained 20 %w GMA, 20 %w EDMA, 60 %w porogenic mixtures consisting of P(t-BMA) and dioxane, 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio) for all RAFT polymerisations.

[0049] Figure 19 shows elugrams for PMMA standards in the range of 520K g / mol to 4, 81 OK g / mol in 4.6x150mm monolithic columns synthesised by (a) RAFT polymerisation (column 1 in Table 4) or (b) conventional free radical polymerisation. The reaction compositions for the monoliths contained 20 %w GMA, 20 %w EDMA, 60 %w porogens (P(t-BMA) / dioxane=23:77 by weight), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio) for the column by RAFT polymerisation. Chromatographic conditions: mobile phase: THF, F=0.33 mE / min, Vinj=5 pl, Tcoi=40 °C, TRBD=35 °C.

[0050] Figure 20 shows calibration curves from (a) individual columns (see Table 4) or (b) columns in series for PMMA analytes with molar masses from 520K g / mol to 4, 81 OK g / mol. For the x axis t corresponds to the elution time of the analyte and to the elution time of toluene.DESCRIPTION OF EMBODIMENTS

[0051] Details of terms and methods are given below to provide greater clarity concerning materials, compositions, methods and use(s) thereof for the purpose of guiding those of ordinary skill in the art in the practice of the present disclosure. The terminology in this disclosure is understood to be useful for the purpose of providing a better description of particular embodiments and should not be considered limiting.

[0052] The term “monolith” used herein refers to a single discrete piece of porous material, which may be used in, for example, the separation or pre-concentration of an analyte.

[0053] The term “reversible addition-fragmentation chain-transfer (RAFT) polymerization” used herein refers to a controlled radical polymerization that can be achieved by introducing in the reaction mixture a chain transfer agent. The chain transfer agent is a molecule with the ability to react with the propagating radical in a reversible reaction. Because of this equilibrium between the active propagating radical and the dormant or inactive specie, the polymerization kinetic changes (with respect to a conventional free radical polymerization) and the product has a predictable molar mass (depending on the chain transfer agent / monomer ratio), low poly dispersity and a linear dependency of molar mass relative to conversion35. Therefore, a RAFT polymerization becomes an option to design polymers with enhanced properties.

[0054] The term “hierarchically porous” used herein suggests that the polymer monolith has permanent porosity and exhibits porosity on two or more distinct length scales. In some circumstances, the hierarchically porous methacrylate polymer monolith exhibits a dual pore size distribution in dry state, for example in the range of about 20 nm to about 100 nm and in the range of about 100 nm to about 1000 nm. In some circumstances, the hierarchically porous polymer monolith with the dual pore size distribution includes interconnected macropores as well as accessible mesopores in structure.

[0055] The phrase “mesoporous methacrylate -based polymer monolith” used herein suggests that the polymer monolith has permanent porosity and the maxima of the pore size distribution is in the mesopore range when measured on the basis of nitrogen adsorption-desorption isotherm. The phrase “macroporous methacrylate-based polymer monolith” used herein suggests that the polymer monolith has permanent porosity and the maxima of the pore size distribution is in the macropore range when measured on the basis of mercury intrusion porosimetry. The phrase “fully macroporous methacrylate-based polymer monolith” used herein suggests that macroporosity is about 100% of the pore volume.

[0056] The term “permanent porosity” used herein suggests that the structural integrity of the porosity can be retained in dry or wet state, during use, for example by means of a high degree of crosslinking, which is different from the non-crosslinked porous methacrylate materials that have been reported. Also, in order to achieve permanent porosity, phase separation due to insolubility of the growing polymer in the reaction media needs to occur.

[0057] The term “methacrylate-based” used herein suggests that the monolith has a methacrylate monomer and a methacrylate crosslinker. The term “methacrylate” used herein refers to any derivative of methacrylic acid, such as glycidyl methacrylate and methyl methacrylate.

[0058] The term “liquid chromatography” used herein includes within its scope any known liquid chromatography technique or mode and includes normal phase chromatography, hydrophilic interaction liquid chromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography and / or ion exchange chromatography.

[0059] According to the International Union of Pure and Applied Chemists (IUPAC) classification, pores with a size smaller than about 2 nm are called micropores, pores with a size larger than about 50 nm are called macropores, and pores with an intermediate size of about 2 nm to about 50 nm are called mesopores.

[0060] The disclosure arises from the inventors’ surprising finding that the porosity of a porous methacrylate-based polymer monolith may be advantageously tuned through a reversible additionfragmentation chain-transfer (RAFT) polymerization mechanism compared to through a conventional free radical polymerization. The first-order kinetic plot in Figure 6 shows slower kinetics and a delay in theonset of phase separation, from which RAFT conditions can be assumed. In this regard, the addition of a small molecular chain transfer agent with a relatively high reactivity to a typical polymerisation mixture containing a methacrylate monomer, a methacrylate crosslinker, a porogenic mixture of solvents (including a solvating solvent together with a non-solvating solvent and / or an inert polymer) and a radical initiator allowed a controlled free radical polymerization by the RAFT mechanism. The porogenic mixture also contributed to RAFT control for the porous methacrylate-based polymer monoliths.

[0061] Accordingly, a method for preparing a porous methacrylate-based polymer monolith through reversible addition-fragmentation chain-transfer (RAFT) polymerization is provided. The method comprises: introducing a reaction composition into a mold, the reaction composition comprising at least one methacrylate monomer, at least one methacrylate crosslinker, a radical initiator, a chain transfer agent (CTA), and a porogenic mixture of a solvating solvent with a non-solvating solvent and / or an inert polymer; conducting the RAFT polymerisation to form a polymer monolith; and optionally, separating the polymer monolith from unreacted reagents.

[0062] In fabricating the porous methacrylate-based polymer monolith, a reaction composition comprising the initiator, the CTA, the monomer, the crosslinker and the porogenic mixture may be introduced into a mold and purged with N2. In some circumstances, it is suggested to form a monomer mixture comprising the initiator, the CTA, the monomer and the crosslinker in a container (for example a vial) and to form a porogenic mixture of a solvating solvent with a non-solvating solvent and / or an inert polymer in a mold (for example a vial), and then add the monomer mixture to the porogenic mixture within the mold. Before being used for polymerisation, the monomer and the crosslinker may be purified, for example by flushing the monomer and crosslinker through an alumina column. If desirable, the monomer units of the polymer monolith may be functionalised prior to the polymerisation or functionalised in situ after the polymerisation. The mold may be, for example, a capillary, a column and a vial. The mold may be preheated before the reaction composition is transferred into it. After the reaction composition is introduced, the mold may be sealed at both ends.

[0063] The porous methacrylate-based polymer monolith is made from a methacrylate monomer which is generally of moderate polarity due to the presence of the ester bond. It is considered that this type of monomer has some ease of preparation and functionalisation. The methacrylate monomer to be used herein may comprise a monovinyl monomer having a methacrylate unit. In some embodiments, the methacrylate monomer is a monovinyl monomer having a methacrylate unit. Non-limiting examples of the mono vinyl monomer having a methacrylate unit include glycidyl methacrylate (GM A), methyl methacrylate, ethyl methacrylate, butyl methacrylate, octadecyl methacrylate, lauryl methacrylate andpoly(ethylene glycol) methacrylate. Glycidyl methacrylate is a commonly used monomer as it contains an epoxy group which allows a wide variety of functionalisations and is also used as a grafting agent. Optionally, other monomers such as methacrylic acid and hydroxyethyl acrylates may be used in the polymer mixture to tailor the hydrophilicity of the resulting stationary phase.

[0064] The crosslinker is responsible for the degree of crosslinking and the amount of swelling of the monolith during the polymerisation step. The crosslinker to be used herein may comprise a multi-vinyl monomer having a methacrylate unit, for example di vinyl monomer having a methacrylate unit. In some embodiments, the crosslinker is a multi-vinyl monomer having a methacrylate unit, for example divinyl monomer having a methacrylate unit. Non-limiting examples of the crosslinker include ethylene glycol dimethacrylate (EDMA), triethylene glycol dimethacrylate (TEGDMA), trimethylol-propane trimethacrylate (TRIM), poly(ethylene glycol) dimethacrylate. In some circumstances where glycidyl methacrylate (GMA) is used as the monomer, it may be preferable to use ethylene glycol dimethacrylate (EDMA) as the crosslinker.

[0065] Structural rigidity of the polymer monolith produced could be secured through extensive crosslinking. However, too much crosslinker is likely to cause a decrease in the pore volume of the monolith. For the purpose of illustration, the crosslinker and the monomer for the monolith may be in a molar ratio of about 1:0.9 to about 1:3.3, for example about 1.4 to about 2.1. In some embodiments, the crosslinker (for example EDMA) and the monomer (for example GMA) for the monolith may be in a molar ratio of about 1:1.4.

[0066] A chain transfer agent (CTA) is used to react with the propagating radical in a reversible reaction in order to control the molecular weight (or the degree of polymerisation (DP)) of a polymer to be produced. The addition of a CTA in a polymerisation mixture does not generate more radicals, but the equilibrium between the CTA and the propagating radicals usually causes a period of inhibition and / or a rate retardation because of the additional steps in the propagation process. It is suggested that a small molecular chain transfer agent with a relatively high reactivity be used for the RAFT polymerization disclosed herein. For instance, the small molecular chain transfer agent may have the following formula (I):wherein Z is selected from an aryl such as phenyl, an alkylthio group, and a carboxyalkylthio group, R is a substituent having a tertiary carbon and optionally a nitrile group connected to the tertiary carbon. Thealkyl may have 1 to 20 carbon atoms, for example 2 to 12 carbon atoms, that is, the alkyl may be ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl. The aryl and the alkyl may be substituted with a group selected from H, Ci-6 alkyl, halogen, -OH, -SH, -O-Ci 6 alkyl, and -S- Ci-6 alkyl. Z groups such as aromatic groups and alkylthio substituents increase the reactivity of the thiocarbonyl group while tertiary R substituents (specially involving a nitrile group) may be good leaving groups. Accordingly, the small molecular chain transfer agent may be selected from 4-cyano-4- ((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDSTS), 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CETCPA), and 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTBCOOH), 2-cyano-2-propyl benzodithioate, 4-cyano-4- (phenylcarbonothioylthio) pentanoic acid, 2-phenyl-2- propyl benzoditioate, 2-cyano-2-propyl dodecyl trithiocarbonate.4-cyano-4-((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDSTS)4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTBCOOH)4-((((2-carboxyethyl)thio)carbonothioyl)thio) -4-cyanopentanoic acid (CETCPA)

[0067] The chain transfer agent and the monomer / the crosslinker may be used in a molar ratio of about 0.5:100 to about 3:100. In some circumstances, the molar ratio between the chain transfer agent and the monomer / the crosslinker is about 0.75:100 to about 1.25: 100, for example about 1:100. If the amount of the chain transfer agent is too high, the molecular weight of the polymer produced may be too low and this could cause a larger number of dormant sites across the structure, which may in turn lead to smaller pores within the monolith. If the amount of the chain transfer agent is too low, the molecular weight ofthe polymer produced may not be well controlled and lead to an unwanted high poly dispersity, and also larger pores would be created by free radical polymerisation.

[0068] For the synthesis of a monolith by RAFT polymerisation, a solvent or solvents is or are desirable to enable precipitation of the polymer formed from the reaction system. The solvent(s) and the inert polymer should not react with the monomer or the crosslinker. It is also required that the solvent(s) and the inert polymer be compatible with the initiation method. If a thermal initiator is to be used, it is normally desirable for the solvent(s) to have a boiling point higher than the decomposition temperature of the initiator. A solvating solvent that favours the dissolution of reagents as well as a non-solvating solvent and / or an inert polymer that promote(s) the precipitation of the polymer may be used in combination. The non-solvating solvent and / or the inert polymer is / are chosen to allow RAFT polymerization for the monomer and crosslinker as well as to promote phase separation of the growing chains during monolith synthesis. The phase separation happens when the solubility parameter or polarity of the inert polymer and the crosslinked polymer that is forming are sufficiently different to induce phase separation. For the purpose of the present disclosure, the solvating solvent may be selected from dioxane and tetrahydrofuran, and examples of the non-solvating solvent include, but are not limited to, toluene, cyclohexanol and dodecanol. Examples of the inert polymer include, but are not limited to, poly(ethylene glycol) (PEG), poly(acrylic acid) (PAA) and polymethacrylates such as poy(tert-butyl methacrylate) (P(t- BMA)). When an inert polymer is used, the pore volume is very similar to the inert polymer volume added in the reaction composition and the pore size may be proportional to the molar mass of the inert polymer. If both an inert polymer and a non-solvating solvent are used, they must be miscible with each other. Only for the purpose of illustration, PEG used herein may have a molecular weight of about 6K g / mol to about 35K g / mol, for example about 6K g / mol, 10K g / mol, 20K g / mol and 35K g / mol, which are commercially available from Sigma Aldrich. If the molecular weight is too high, macropores might be formed at the expense of mesopores, that is, the volume of mesopores decreases significantly. The poly(tert-butyl methacrylate) (P(t-BMA)) used herein may have a molecular weight of about 4.7K g / mol to 21K g / mol, for example about 4.7K g / mol, 10K g / mol and 21K g / mol. As an example, a combination of dioxane and dodecanol may be used to form the porogenic mixture. If needed, more than one nonsolvating solvent is used. In some embodiments, the weight ratio between the solvating solvent and the non-solvating solvent and / or the inert polymer is in a range of about 20:80 to about 80:20.

[0069] A non-solvating solvent and / or an inert polymer causes no chemical change to the monolith and is used to induce pores in the resulting polymer monolith. A minimum amount of the solvating solvents and / or the inert polymer may be required to obtain a polymer monolith. To achieve a desirable pore volume, there might be a maximum amount of the solvating solvents. For the purpose of illustration, the solvating solvent may be in the range of 12-48 %wt, and the non-solvating solvent and / or the inert polymer may be in the range of 12-48 %wt, based on the reaction composition. The amount of the porogenic mixture of the solvating solvent with the non-solvating solvent and / or the inert polymer mayvary depending on a desirable monolith structure, and may be about 60 %wt based on the reaction composition.

[0070] A radical initiator is used to start the reaction between the monomer, the crosslinker and the chain transfer agent. The radical initiator used herein may be thermally or photolytically activated. Suitable thermal radical initiators may include azo compounds such as azobisisobutyronitrile (AIBN) and 4,4'-azobis(4-cyanovaleric acid) (ACVA; also called 4,4'-azobis(4-cyanopentanoic acid)). Other suitable radical initiators include peroxide compounds such as benzoyl peroxide (BPO)and di-t-butyl peroxide (DTBP). Radical photo-initiators may include 1 -hydroxy cyclohexyl phenyl ketone (also known as Irgacure). The initiator may be present in an amount up to about 5 wt% (for example about 1 wt%) with respect to the amount of the monomer(s) and the crosslinker(s).

[0071] The porous methacrylate-based polymer monoliths are continuous rigid structures and they can be fabricated in situ in a range of formats, shapes or sizes. They can be fabricated within the confines of a mold having a shape and / or a dimension that is adapted for the application of the monolith, for example, liquid chromatography applications. In some circumstances, it may take a form of a capillary, column or vial which is made of, for example, silica, stainless steel and polyimide. The mold may also be in the form of a microfluidic device or a channel. It is also possible to fabricate monoliths in the format of flat sheets. Flat monolithic sheets provide a particularly suitable medium for the storage of whole blood which allows for ease in both storage and transportation of blood samples. For a photoinitiator to work, the mold must be transparent to the chosen wavelength, for example a UV lamp with 320-500 nm or a LED source with 600 nm.

[0072] The RAFT polymerisation may be carried out at a temperature at about 50 °C to about 70 °C, for example, at about 50 °C, 55 °C, 60 °C, 65 °C and 70 °C. It is possible to use a water bath or an oil bath to provide the temperature for reaction. Alternatively, the mold together with the reaction composition may be placed in an oven set at a suitable temperature. If needed, a chain termination agent such as catechol may be added to stop the reaction. The polymerisation starts in solution and as the polymer grows, it becomes insoluble and the reaction is completed in a heterogeneous system. Phase separation can be promoted by crosslinking or difference in polarity between the growing polymer and the solvent(s) or the inert polymer, but in order to obtain a continuous porous structure, precipitation due to some degree of insolubility is needed. The polymerization is then allowed to proceed until the polymer monolith of appropriate porosity and surface area is obtained. Following that, the unreacted reagents including the non-solvating solvent(s) may be thoroughly flushed from the mold with a suitable solvent.

[0073] When an inert polymer is used, a step of removing the inert polymer from the monolith produced is recommended as presence of the inert polymer may alter the porosity as well as thesuperficial chemistry of the monolith. This can be done through extraction, for example with a Soxhlet extractor.

[0074] It has also been surprisingly found by the present inventors that a mesoporous methacrylate- based polymer monolith can be obtained through the method disclosed herein. It is believed that the change in polymerization mechanism from free radical to RAFT polymerization could drastically change the porous properties of the obtained monoliths from macroporous to mesoporous materials. These structural differences may be attributed to the following characteristics of monoliths prepared by the RAFT process: a reduced degree of polymerisation, delays in the onset of phase separation and the preference of inter-crosslinking over intra-crosslinking during the polymerization reaction. Formation of a mesoporous methacrylate-based polymer monolith may be supported by a type IV shape for nitrogen adsorption-desorption isotherms (see for example Figure 2), which is characteristic of mesoporous materials. It is possible to adjust the mesopore size by changing the CTA / initiator ratio and / or changing the chemical nature of the non-solvating solvents or the inert polymer. Furthermore, the mesopore volume can be tuned by changing the composition of the porogenic mixture, for example the amount of a nonsolvating solvent or an inert polymer.

[0075] It is advantageous that synthesis of the mesoporous methacrylate-based polymer monolith disclosed herein may be achieved in a one-step process using a RAFT polymerization, wherein removal of the chain transfer agent is not necessary. In other words, the mesoporous methacrylate-based polymer monolith disclosed herein may be achieved in a one-step process without removal of the chain transfer agent. This contrasts with some previously reported methods wherein preparing a polymer monolith by a RAFT polymerisation with the aid of a macro-CTA (wherein a polymer is covalently bonded to the CTA) needed to etch the macro-CTA and the etching took a few days. The mesoporous methacrylate-based polymer monolith disclosed herein has permanent mesoporosity and low pore polydispersity in the mesopore range. As shown by the Nitrogen adsorption-desorption isotherms in Figure l(a.II), the pore width of the pore distribution is about 50 nm.

[0076] In order to prepare a mesoporous methacrylate-based polymer monolith, a non-solvating solvent such as dodecanol, toluene and cyclohexanol and a solvating solvent such as dioxane and tetrahydrofuran may be used to form the porogenic mixture. It is possible for the non-solvating solvent and the solvating solvent to be used in a weight ratio of about 50:50. In some embodiments, a porogenic mixture of dodecanol and dioxane can be used. For example, the dodecanol and the dioxane is used in a weight ratio of about 50:50. In some embodiments, a porogenic mixture of dodecanol and tetrahydrofuran can be used. For example, the dodecanol and the tetrahydrofuran is used in a weight ratio of about 50:50. Alternatively, an inert polymer such as poly(ethylene glycol) (PEG), poly( acrylic acid) (PAA) and polymethacrylates (for example poly(tert-butyl methacrylate)) and a solvating solvent such as dioxane and tetrahydrofuran may be used to form the porogenic mixture, for example in a weight ratio of about50:50. In some embodiments, a porogenic mixture of a PEG having a molecular weight of about 6K g / mol and dioxane can be used, wherein the PEG and the dioxane may be in a weight ratio of about 50:50. For the purpose of illustration, the RAFT polymerisation can be conducted at a temperature of about 60 °C.

[0077] Higher surface areas and greater numbers of smaller pores could contribute to an increase in potential interaction sites for an analyte in the mobile phase, therefore, in theory increase separation efficiencies. The mesoporous methacrylate-based polymer monolith disclosed herein may display a mesopore volume of up to about 0.620 mL / g in dry state, which is at least approximately 25 %v / v with respect to the total volume of the monolith. In comparison, a monolith obtained through free radical polymerisation mechanism only demonstrates a pore volume for pores in the range of 2-250 nm of about 0.013 mL / g with respect to the same pore sizes. Alternatively or in addition, the mesoporous methacrylate-based polymer monolith may display a BET surface area of about 137 m2 / g, for example up to about 155 m2 / g, for pores having a diameter in the range of 2-250 nm. This is significantly higher than a 5.4 m2 / g surface area of the one obtained through free radical polymerisation mechanism. Alternatively or in addition, the mesopores in the monolith may be up to about 35% by volume, for example about 25% by volume, with respect to the total volume of the monolith. The surface area can be determined from nitrogen adsorption-desorption isotherms with a BET model. The pore volume can be calculated from nitrogen adsorption-desorption isotherms with a DFT model.

[0078] It has also been surprisingly found by the present inventors that a hierarchically porous methacrylate-based polymer monolith, particularly a hierarchically porous methacrylate-based polymer monolith which exhibits a dual pore size distribution in dry state, can be obtained through the RAFT polymerisation disclosed herein. In some embodiments, the hierarchically porous methacrylate-based polymer monolith exhibits a dual pore size distribution in macropore region in dry state, for example with about 60 nm and about 550 nm. The hierarchically porous methacrylate-based polymer monolith includes interconnected macropores as well as accessible mesopores in structure.

[0079] In order to prepare a hierarchically porous methacrylate-based polymer monolith, an inert polymer, a non-solvating solvent such as dodecanol, and a solvating solvent such as dioxane and tetrahydrofuran may be used to form the porogenic mixture, for example in a weight ratio of about 12.5:50:37.5. In some embodiments, the porogenic mixture consists of a PEG, dodecanol and dioxane. For example, the PEG has a molecular weight of about 6K g / mol to about 20K g / mol. It is possible for dodecanol, the inert polymer (for example a PEG) and the solvating solvent (for example dioxane) are used in a weight ratio of about 12.5:50:37.5. When the inert polymer is a PEG, the melting point for PEG having a molecular weight of about 10K g / mol to about 20K g / mol ranges from 60-64 °C. Thus, if PEG is precipitated during the monolith synthesis, a solid template will be formed. However, reducing thepolymerisation temperature could affect the monolith conversion. Accordingly, it may be suggested that the RAFT polymerisation is conducted at about 50 °C.

[0080] The effect of the polymerisation mechanism on the porous properties of the monoliths is shown in Figure 9. When the monolith was synthesised by RAFT polymerisation, a dual pore size distribution was obtained, while for a monolith synthesised by free radical polymerisation a single pore size distribution was observed. In free radical polymerisations, a large degree of polymerisation is reached at low conversions, while for controlled radical polymerisations, a linear increase of molar mass with respect to conversion is observed. Moreover, the reaction rate is higher for free radical polymerisations with respect to RAFT polymerisations. Both effects contribute to an early onset of phase separation for monoliths synthesised by free radical polymerisation. Such long chains of the inert polymer contained in the reaction composition promote monoliths with large macropores. For monoliths obtained by RAFT polymerisation, not only is there a delay in the onset of phase separation, which promotes mesoporosity, but also the slower polymerisation kinetics allow a better control over the phase separation mechanism before gelation. It was observed that the effect of the porogenic mixture comprising an inert polymer on the phase separation mechanism was only observed in the synthesis of monoliths by RAFT polymerisation, suggesting that the polymerisation kinetics play an important role in allowing a certain type of phase separation before gelation.

[0081] For the hierarchically porous methacrylate-based polymer monolith disclosed herein, the pores in a size range of about 100 nm to about 1000 nm and the pores in a size range of about 20 nm to about 100 nm can be in a volume ratio of about 50:50, and this is beneficial for size-based separations. In some embodiments, the pore volume of the hierarchically porous methacrylate-based polymer monolith is about 0.167 mL / g in a pore size range of about 20-100 nm and about 0.584 ml / g in a pore size range of about 100-1,000 nm. Alternatively, or in addition, the hierarchically porous methacrylate-based polymer monolith has about 22 %v / v pores in a size range of about 20 nm to about 100 nm and about 78 %v / v pores in a size range of about 100 nm to about 1000 nm with respect to the total pore volume. Alternatively, or in addition, the hierarchically porous methacrylate-based polymer monolith has about 12 %v / v pores in a size range of about 20 nm to about 100 nm and about 43 %v / v pores in a size range of about 100 nm to about 1000 nm with respect to the total monolith volume.

[0082] It has also been surprisingly found by the present inventors that a macroporous methacrylate- based polymer monolith, particularly a fully macroporous methacrylate-based polymer monolith by spinodal decomposition can be obtained through the method disclosed herein. For the synthesis of a polymer monolith, the reaction system moves from a stable region to either a metastable or unstable region. When phase separation occurs in the metastable region, the mechanism for phase separation is nucleation and growth, however, when phase separation occurs in the unstable region, the mechanism for demixing is spinodal decomposition. Each mechanism results in a distinctive morphology for the two-phase system. Specifically, a nucleation process involves the formation of a discontinued second phase, or nuclei, that increase its size over time, spinodal decomposition allows the obtention of two continuous phases. Figure 15 depicts the schematic evolution of phase separation due to nucleation and growth and spinodal decomposition mechanisms. Those morphological differences can result in differences in the chromatographic performance when monoliths are used as stationary phases.

[0083] In order to prepare a fully macroporous methacrylate -based polymer monolith by a spinodal decomposition mechanism, an inert polymer and a solvating solvent such as dioxane and tetrahydrofuran may be used to form the porogenic mixture. Furthermore, the porogenic mixture may be composed of the inert polymer and the solvating solvent in a weight ratio of about 22:78 to about 25:75, for example about 23:77. The inert polymer may be selected from polymethacrylates, such as poly(tert-butyl methacrylate) (P(t-BMA)). The P(t-BMA) may have a molecular weight of about 4.7K g / mol to about 21K g / mol, for example 10K g / mol. In some embodiments, the porogenic mixture consists of poly(tert-butyl methacrylate) (P(t-BMA)) (for example with a molecular weight of about 10K g / mol) and dioxane. In some further embodiments, the P(t-BMA) (for example with a molecular weight of about 10K g / mol) and the dioxane is in a weight ratio of about 22:78 to about 25:75.

[0084] It is believed that the monoliths by spinodal decomposition would present a lower tortuosity compared to the nucleation monoliths and have increased percentage of overall convective flow. A lower tortuosity could be beneficial for the efficiency of chromatographic analysis, and may make the macroporous methacrylate-based polymer monolith well-suited for applications requiring high rates of mass transfer.

[0085] Also disclosed herein is a separation medium that comprises the porous methacrylate-based polymer monolith disclosed herein or prepared by the method disclosed herein. The medium may be useful in a liquid chromatography application.

[0086] Also disclosed herein is a use of the porous methacrylate-based polymer monolith disclosed herein or prepared by the method disclosed herein in a liquid chromatography application.

[0087] The liquid chromatography application mentioned herein includes, but is not limited to, normal phase chromatography, hydrophilic interaction liquid chromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography, and ion exchange chromatography. For example, it can be used as a stationary phase for chromatographic separations of macromolecules, such as peptides and proteins, DNA fragments, plasmid DNA, oligonucleotide, oligodeoxyethymidylic acids.

[0088] EXAMPLESMaterials

[0089] Glycidyl methacrylate (GMA, 97 %), ethylene glycol dimethacrylate (EDMA, 98 %), azobisisobutyronitrile (AIBN, 12 %w in acetone), 4-cyano-4- ((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDSTS, 97 %), 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CETCPA, 95 %), 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTBCOOH), toluene (>99.9 %), cyclohexanol (>99 %), dodecanol (>98 %), tetrahydrofuran (THF, anhydrous, >99.9 %), poly(ethylene glycol) (PEG, 6K g / mol; 10K g / mol; 20K g / mol and 35K g / mol), poly(acrylic acid) (PAA, 1.8K g / mol), 1 ,2-dihydroxybenzene (catechol, >99 %), and CDCE (>99.8 %) were purchased from Sigma Aldrich. 1,4-dioxane (>99.5 %) and methanol (HPLC grade) were purchased from Merck. Poly(methyl methacrylate) (PMMA 2.58K, 10.1K, 31.6K, 54.5K, 93.3K, 158.3K, 267K, 520K, 701K, 981K, l,280K, 2,650K, 3,220K and 4,810K g / mol) and poly(tert-butyl methacrylate) (P(t-BMA), 4.7K g / mol, 10K g / mol and 21K g / mol) were obtained from Polymer Standards Service GmbH, Mainz, Germany.

[0090] Both GMA and EDMA were purified by flushing the monomers through an alumina column.Instrumentation

[0091] Size Exclusion Chromatography (SEC) determinations were performed using an Agilent 1260 Infinity II with a Refractive Index Detector. Calibration curve was obtained with poly(styrene) standards (ReadyCal Kit, PSS) with molar masses ranging from 162 to 2,520,000 g / mol. All samples were filtered (PTFE, 0.22 pm) prior to injection. Two sets of columns were used: a PSS SDV Linear S (8x300 mm; Mainz, Germany) with 3 pm particle diameter for polymers obtained by RAFT polymerization and a set of three PSS SDV Lux columns with pores of 1,000 A, 100,000 A and 1,000,000 A (8x300 mm with 5 pm particle diameter; Mainz, Germany) were used to characterize polymers synthesized by free radical polymerizations. Mobile phase was THF at a flow rate of 1 mL / min. Injection volume was 10 pL, the column temperature was 40 °C and the RI detector was set at 35 °C. Data was processed with a PSS WinGPC UniChrom software.

[0092] A Bruker Advance III 600 NMR equipment was used for kinetic and conversion studies. For single runs, aliquots of 200 pL of the sample were diluted in 600 pL of CDCI3 and for polymerisations performed inside the NMR no deuterated solvent was used and the temperature was set at 60 °C. In all cases trioxane was used as internal standard and the signals corresponding to the protons from the vinyl groups of GMA (5 6.16 and 6.66 ppm) or EDMA (5 6.14 and 6.60 ppm) were used to calculate conversion.

[0093] For scanning electron microscopy (SEM) imaging, vacuum dried samples were coated with Platinum (approx. 5 nm thickness) using an Agar High Resolution Sputter Coater. Images were taken with a Zeiss Merlin Scanning Electron Microscope, using secondary electrons for the measurements.

[0094] Nitrogen adsorption-desorption isotherms were measured with a Micrometrics ASAP 2420 analyser. Samples were degassed at 60 °C for 10 hours prior to analysis. Brunauer-Emmett-Teller (BET) model was used to obtain the surface area of the porous materials and pore size distribution and pore volume were obtained from the density functional theory (DFT) model. Pore size was estimated from the maxima in the DFT incremental pore volume vs. pore size distribution and pore volume was obtained from the DFT cumulative pore volume vs. pore size distribution curves for a pore size range of 2-100 nm.

[0095] Differential Scanning Calorimetry (DSC) analysis was performed in a TA Instruments Discovery using aluminium T-zero pans with hermetic lids. Measurements were run under a N2 current from 0 °C to 100 °C using a 10 °C / min ramp.

[0096] Surface elemental analysis was performed in a Kratos Axis Ultra X-ray Photoelectron Spectroscopy (XPS) system. XPS measurements were performed by Dr. Alex Cavallaro at the Future Industries Institute at the University of South Australia.

[0097] Mercury intrusion porosimetry (MIP) analysis were performed in a Micromeritics Pore Sizer 9310. Dr. Petr Smejkal kindly performed MIP analysis at the University of Tasmania. Pore size was approximated as the maxima in the pore size distribution curve (incremental intrusion vs. pore diameter).

[0098] Example 1 - Mesoporous methacrylate-based polymer monoliths using different chain transfer agents

[0099] A reaction composition containing 20 wt% GMA, 20 wt% EDMA, 30 wt% PEG (6K), 30 wt% dioxane, 1 wt% AIBN (with respect to the monomer and the crosslinker) and a chain transfer agent with a molar ratio of CTA / AIBN=2 (molar ratio) was prepared. The chain transfer agent used was CDSTS, CTBCOOH or CETCPA. The reaction composition was introduced into a glass vial and purged with N2 for 5 min. The glass vial containing the reaction composition was kept in a water bath at 60 °C for 24 hours. The resulting monolith was extracted by a Soxhlet extractor with methanol for 48 hours to remove PEG and other unreacted reagents.

[0100] Figures 2-4 show the comparative structures of the obtained monoliths and their respective nitrogen adsorption-desorption isotherms and pore size distributions obtained with a DFT model. A dramatical morphological difference was observed between the monoliths synthesised in the presence of CTA and the one polymerized by free radical mechanism. The N2 isotherms obtained for the monoliths synthesized by RAFT polymerization show a type IV shape, characteristic of mesoporous materials, whilethe isotherm from the free radical polymerization monolith is type II, which is typical of macroporous structures52. The pore volume of pores with diameters in the range of 2-250 nm was 0.013 mL / g for the monolith obtained by free radical polymerization, while the addition of CDSTS, CTBCOOH or CETCPA increased the pore volume in the meso- and small macropore range to 0.673 mL / g, 0.623 mL / g and 0.628 mL / g respectively. The surface areas obtained by a BET model for these monoliths were 5.4 m2 / g for the free radical polymerization synthesis, 137.8 m2 / g for the reaction with CDSTS, 138.6 m2 / g for CTBCOOH and 155.7 m2 / g for CETCPA. The pore size distribution plots show a narrow pore distribution for all monoliths obtained by RAFT polymerization, with maxima between 30 nm and 35 nm. As the monoliths obtained with all three CTAs have a similar structure, CDSTS was arbitrarily chosen as the CT A for further experiments.

[0101] Surface analysis of the monoliths obtained by RAFT polymerization shows the presence of sulphur. Figure 5 depicts the XPS spectra of monoliths synthesised by free radical polymerization or with either CDSTS, CTBCOOH or CETCPA as the chain transfer agent. The presence of sulphur in the monoliths synthesized in the presence of a CT A suggests the polymerization by a RAFT mechanism. Moreover, the exposure of the CTA in the surface suggests the possibility of performing a posterior grafting step by RAFT polymerization as previous reported36,37. However, if the CTA end group needs to be removed due to undesired interactions or reactions, multiple removal approaches has been reported, such as radical or thermal induced, as well as specific reactions according to the chemical nature of the end group38.

[0102] The mesoporous structure in the monoliths obtained by RAFT polymerization could be explained by three phenomena. First, DP is lower for linear polymers obtained under RAFT control with respect to free radical polymerizations. In the monolith, even when the polymer is crosslinked, the trend is expected to be the same. It was found that the minimum CDSTS / monomers molar ratio necessary to have RAFT control in the monolith was 1:100, thus every 100 monomer units (or less), the polymer growth is interrupted by the presence of the CTA. This results in a structure with ‘shorter chains’ with respect to a monolith obtained by free radical polymerization. Moreover, Table 1 shows that the DP of poly(GMA) obtained by free radical polymerization is 647, while it ranges from 41 to 47 for RAFT polymerization (with either CDSTS, CTBCOOH or CETCPA). This similarities in the chain length for linear poly(GMA)s with the three CTAs tested could be extrapolated to the crosslinked polymer and could explain the similarities in the pore sizes for the monoliths obtained by RAFT polymerization with those CTAs.

[0103] Table 1. Conversion, DP, PDI and Mnobtained for poly(GMA) synthesised in solution with different RAFT agents in various solvents. Reaction conditions: molar ratios of GMA / CTA=100 and CTA / AIBN=2 were used and polymerisation was performed at 60 °C for 24 hours.Solvent CTA Mn* DP* PDI* C% dioxane - 92,200 647 2.83 82.5 dioxane CTBCOOH 6,970 47 1.35 84.0 dioxane CDSTS 6,200 41 1.47 74.3 dioxane CETCPA 6,390 43 1.40 97.1 toluene - - 68.9 toluene CTBCOOH 6,190 42 1.44 74.2 toluene CDSTS 6,280 41 1.55 71.7 toluene CETCPA 6,220 42 1.80 66.4 cyclohexanol - - 99.7 cyclohexanol CTBCOOH - - - 99.1 cyclohexanol CDSTS 5,610 37 1.97 99.5 cyclohexanol CETCPA - - - 98.610 %v / v cyclohexanol in CDSTS 7,080 47 1.43 79.9 dioxane10 %v / v dodecanol in CDSTS 7,100 47 1.39 79.9 dioxane10 %v / v toluene in dioxane CDSTS 7,230 48 1.40 84.3*Conditions with no available data for Mnand PDI correspond to insoluble polymers.

[0104] Second, with RAFT polymerisation the onset of phase separation is delayed with respect to its free radical counterpart. Figure 6 shows a delay in the onset of phase separation. This delay in phase separation is caused by both a shorter length of the polymer chains, as discussed before, and by a lower reaction rate. An early onset of phase separation promotes large monolith globules as the precipitation occurs while the number of polymer nuclei is still low and reaction continues over those nuclei in a heterogeneous system, while a late onset of phase separation allows the formation of a larger number of nuclei in solution, resulting in smaller globules.

[0105] Third, it was found for the poly(GMA-co-EDMA) monolith that with RAFT polymerization inter-crosslinking is favoured over intra-crosslinking. A linear polymer in solution will adopt a conformation of a random coil, however, with a crosslinked polymer, both inter- and intra-crosslinking reactions would occur. When intra-crosslinking is favoured, the polymer chains will adopt a compact conformation, smaller than the ideal random coil. On the other hand, when inter-crosslinking dominates, multiple growing polymer chains will be bonded together, resulting in a much larger structure39. In order to evaluate the inter- or intra-crosslinking processes before phase separation, samples were taken from the early stages of a monolith polymerization every 15 minutes and the size of the growing polymer was estimated by its apparent molar mass (M) by SEC. Figure 7 shows the apparent M of monoliths polymerized in two different solvent mixtures (namely, 100% dioxane and dodecanol / dioxane 50:50) and the M of poly(GMA) obtained in the same experimental conditions. It can be seen that the sizes of the crosslinked polymers are larger than the linear poly(GMA), showing that inter-crosslinking reactions are favoured over intra-crosslinking processes. As inter-crosslinking dominates, a highly interconnected network structure is formed from the early stages of polymerization reaction, resulting in a uniform structure all over the reaction volume in the monolith.

[0106] Example 2 - Hierarchically porous methacrylate-based polymer monoliths

[0107] For the synthesis, the porogenic mixture and the monomer mixture were first prepared in separate vials. PEG (either 6K, 10K or 20K g / mol), dodecanol and either dioxane or THF were weighted in a vial and kept at 60 °C while preparing the monomers mixture to allow complete dissolution of the polymer. The monomer mixture contained AIBN, CDSTS, GMA and EDMA and the overall composition of the reaction mixture was 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture, 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). Once the mixtures in both the vials were homogeneous, the monomer mixture was poured into the porogenic mixture, shaken and purged with N2 for 5 min in a warm water (approx. 55 °C) bath to avoid PEG precipitation. The synthesis was then performed by leaving the vials in a water bath at 50 °C for 24 hours. Monoliths were then washed with methanol in a Soxhlet apparatus for 48 hours.

[0108] To promote PEG precipitation, ternary porgenic mixtures were tested, namely, PEG (6, 10 and 20K g / mol), dodecanol and either dioxane or THF because both dioxane and THF are solvating solvents for PEG, while dodecanol is a non-solvating solvent for PEG as well as for the monolith. Moreover, the polymerisation temperature was reduced to 50 °C because the melting point of PEG ranges from 60-64 °C for molar masses between 6K g / mol and 20K g / mol (measured by DSC). Thus, if PEG is precipitated during monolith synthesis, a solid template will be formed. However, reducing the polymerisation temperature could affect the monolith conversion, therefore, the conversion of monoliths at 50 °C was evaluated by following the consumption of monomers over time with1H NMR as well as measuring the mass difference between the initial monomers in a polymerisation mixture and the resulting monolith. Aconversion of 98 % was observed by mass difference after 24 hours of polymerisation at 50 °C for a monolith with 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture (dodecanol / THF 50:50), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). Conversion studies performed by 'H NMR showed total consumption of monomers after 12 hours of polymerisation at 50 °C.

[0109] The effect of the composition of the ternary porogenic mixture was evaluated for monoliths containing 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture, 1 %w AIBN and CDSTS / AIBN=2 (molar ratio). Figure 8 shows the SEM pictures and pore volume (in the range of 2-100 nm, measured by nitrogen adsorption-desorption isotherms) for monoliths with variable compositions of PEG (either 6K, 10K or 20K g / mol), dodecanol and dioxane. Even when the SEM images do not provide quantitative data for the porosity of the materials, it gives an idea of the presence of macropores in the structure. It can be observed that as the mesopore volume decreases, macropores are observed by SEM in the monoliths. Overall, the larger the amount of PEG (or the larger its molar mass), the larger the pore size. Both dioxane and THF have been tested as solvating solvents for ternary porogenic mixtures and similar trends have been observed.

[0110] There are some conditions where the mesopore volume exceeds 0.2 mL / g and the SEM image suggest the presence of macropores, like the monolith with a porogenic mixture of PEG(6K) / dodecanol / dioxane 12.5:50:37.5 which presented an average mesopore volume of 0.257 mL / g (measured by nitrogen adsorption-desorption isotherms) as well as an opened structure, based on SEM (highlighted with a circle in Figure 8(a)). The total pore volume found for mesopore monoliths prepared with PEG / dioxane mixtures was up to around 0.6 mL / g, and macropores are formed at the expense of mesopores, thus, the aim was to keep at least one third of those pores in the mesopore range (0.2 mL / g mesopore volume) while introducing macropores in the structure.

[0111] Pore size distribution was evaluated by both nitrogen adsorption-desorption isotherms and mercury intrusion porosimetry (MIP), as both techniques are complementary and provide information about the porous properties of a material for different pore size ranges. Figure 9 and Table 2 summarise the pore size distribution, pore volume and surface area obtained by nitrogen isotherms and MIP. Panels a-d in Figure 9 show the effect of the amount of PEG (6K) in the porogenic mixture (corresponding to the four conditions highlighted in a line in Figure 8(a)) and it can be seen that the pore size increases as the concentration of PEG(6K) increases in the polymerisation mixture, at constant dodecanol concentration. This is what is expected for a polymerisation induced phase separation mechanism, as a larger concentration of a non-solvating solvent would promote an earlier onset of phase separation. Panels b, e and f of Figure 9 show the effect of the molar mass of PEG (corresponding to the individual conditions highlighted in a circle in Figure 8 (a), (b) and (c) and it can be seen that for PEG(10K and 20K) the monoliths present dual pore size distributions in the dry state. Interestingly, the pores sizes at the peakmaxima of the curves are 60 nm and 550 nm for both monoliths, but the volume of smaller pores decreased from 0.310 to 0.167 mL / g when the molar mass of PEG increases, while the volume of the larger pores increased from 0.457 to 0.584 mL / g.

[0112] It was found that both RAFT polymerisation as well as the right composition of the porogenic mixture was required to achieve dual porosity. Panels f and g of Figure 9 show the effect of the polymerisation mechanism on the porous properties of the material. When the monolith was synthesised by RAFT polymerisation, a dual pore size distribution was obtained, while for a monolith synthesised by free radical polymerisation a single pore size distribution was observed. This difference might be due to the differences in kinetics between RAFT and free radical polymerisations. In free radical polymerisations, a large degree of polymerisation is reached at low conversions, while for controlled radical polymerisations, a linear increase of molar mass with respect to conversion is observed. Moreover, the reaction rate is higher for free radical polymerisations with respect to RAFT polymerisations. Both effects contribute to an early onset of phase separation for monoliths synthesised by free radical polymerisation. Such long polymer chains precipitating in the reaction mixture promote monoliths with large macropores. On the other hand, for monoliths obtained by RAFT polymerisation, not only is there a delay in the onset of phase separation, which promotes mesoporosity, but the slower polymerisation kinetics allows a better control over the phase separation mechanism before gelation. It was observed that the effect of the polymeric non-solvating solvent on the phase separation mechanism was only observed in the synthesis of monoliths by RAFT polymerisation, suggesting that the polymerisation kinetics play an important role in allowing a certain type of phase separation before gelation.

[0113] Table 2 summarises the porous properties of the selected monoliths and it can be seen that their total porosity is close to the volume percentage of the solvents in the polymerisation mixture, which is expected for the synthesis of organic monoliths. Total pore volume values lower than 60 %v (which is the theoretical value) could have been obtained due to shrinkage of the material in the dry state. As the total pore volume is approximately constant, but pore size distributions differ for all conditions tested, it is evident that macro- and mesopores are obtained at the expense of each other.

[0114] Figure 10 shows the SEM images of the monoliths with hierarchical porosity, which are prepared from the porogenic mixture consisting of PEG / dodecanol / dioxane.

[0115] Table 2. Surface area, pore volume for various pore size ranges and total pore volume from nitrogen adsorption isotherms and MIP for monoliths with 40 %w monomers (GMA / EDMA 50:50), 60 %w the porogenic mixture, 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio) (conditions highlighted in Figure 8). Polymerisations were performed at 50 °C.*pore range: 100-2000 nm

[0116] Example 3 - Macroporous methacrylate-based polymer monoliths

[0117] Monoliths were prepared in vials for material characterization. For the synthesis, the porogenic mixture and the monomer mixture were first prepared in separate vials. P(t-BMA) and dioxane were weighted in a vial and kept at 60 °C while preparing the monomer mixture to allow complete dissolution of the polymer. The monomer mixture contained AIBN, CDSTS (or either CETCPA or CTBCOOH), GMA and EDMA and the overall composition of the reaction mixture was 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture, 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). Once the mixtures in both the vials were homogeneous, the monomer mixture was poured into the porogenic mixture, stirred until obtaining a homogeneous solution and purged with N2 for 5 min in a warm water (approx. 55 °C) bath to avoid polymer precipitation. The synthesis was then performed by leaving the vials in a water bath at 60 °C for 24 hours. Monoliths werethen washed with methanol in a Soxhlet apparatus for 48 hours and dried in a vacuum oven before SEM, MIP or nitrogen adsorption-desorption analysis.

[0118] In order to obtain monoliths by spinodal decomposition, it is suggested that the interaction parameter between the methacrylate polymer network and the non-solvating solvent should be as large as possible as the (the change in Gibbs free energy) of mixing would be larger, thus demixing would be thermodynamically favoured and it will be easier to reach the unstable region in the phase diagram of a binary system. It is also suggested that the difference between 6 (Hildebrand solubility parameters) of the non-solvating solvent and the monomers should be as large as possible in order to consider such a solvent as a porogen for spinodal decomposition. Moreover, as previous publications found in literature about spinodal decomposition for organic monoliths were reported for polymeric non-solvating solvents, an inert polymer was chosen as a potential non-solvating solvent to promote phase separation by spinodal decomposition. From all polymeric non-solvating solvent tested for a P(GMA-co-EDMA) monolith, P(t- BMA) presented the larger difference in 6 value with respect to the monolith ( ®WA=14.7 MPa1 / 2and A / 'V / V / .4= 18.6 MPa1 / 2,40PMMA was used as an approximation of the monolith), thus, it was chosen as a non-solvating solvent for the synthesis of monoliths.

[0119] Figure 16 shows SEM images of P(GMA-co-EDMA) monoliths synthesised with increasing amounts of P(t-BMA) in the porogenic mixture. The evolution of the morphology of the obtained monoliths follows the expected trend for a phase separation process induced by a spinodal decomposition mechanism. There are two processes taking place during the synthesis of a monolith, phase separation and gelation. While the crosslinked polymerisation reaction promotes gelation, phase separation is affected by the difference in polarity between the growing monolith and the non-solvating solvent, the volume ratio of the two phases, the mobility of the participating species and the temperature. Therefore, as the amount of monomer, crosslinker, initiator, CTA and reaction temperature were constant for all conditions, the polymerisation kinetics and thus the gelation time is expected to be the same for all monoliths shown in the figure. However, if the composition of the porogenic mixture changes, the onset of phase separation will change as well. As the amount of P(t-BMA) increases, the onset of phase separation decreases, thus, gelation takes place at a later stage of phase separation. Therefore, by changing the amount of the polymeric non-solvating solvent in the reaction mixture, we can observe different stages of phase separation, which suggest that the mechanism is spinodal decomposition. It is interesting to note that very small increments of P(t-BMA) in the porogenic mixture causes significant changes in porosity. Similar behaviours, where small changes in the amount of polymeric non-solvating solvent promoted important changes in the pore size of monoliths obtained by spinodal decomposition, can also be found in literature.

[0120] The effect of the polymerisation mechanism to achieve spinodal decomposition was evaluated by performing monolith synthesis by either RAFT or conventional free radical polymerisations with various mixtures of P(t-BMA) and dioxane as the porogenic mixture. The first two rows in Figure 17show the SEM images of the obtained monoliths, and it was observed that while the morphologies of the monoliths obtained by RAFT polymerisation (using CDSTS as a CTA) resemble the structures expected for a spinodal decomposition mechanism, however, the monoliths obtained by free radical polymerisation showed a completely different morphology, with a globular structure, typically observed for monoliths by nucleation. Thus, a RAFT polymerisation mechanism is necessary to achieve spinodal decomposition for a P(GMA-co-EDMA) monolith with P(t-BMA) and dioxane for the porogenic mixture.

[0121] MIP analysis was performed for monoliths with variable porogenic mixtures obtained by spinodal decomposition. It was observed that the pore size of monoliths obtained in the presence of P(t- BMA) as polymeric non-solvating solvent by RAFT polymerisation increased as the amount of P(t-BMA) increased (Figure 18 (a)). The pore size shifted from the mesopore range (pore size was 40 nm for a monolith with 19 %w P(t-BMA) in the porogenic mixture) to the macropore range (reaching a pore size of 1,170 nm for a monolith with 24 %w P(t-BMA) in the porogenic mixture), but the pore size distribution was unimodal for all conditions tested. It is interesting to note that for monoliths synthesised with 23 %w P(t-BMA) in the porogenic mixture, the pore size was around 500 nm regardless of the polymerisation mechanism as observed in Figure 18(b) (pore size was 500 nm for the monolith synthesised by RAFT polymerisation and 550 nm when the monolith was obtained by free radical polymerisation), even when the structures look different on SEM images (Figure 17).

[0122] When a polymerisation is performed by a RAFT mechanism, the reaction rate is lower with respect to a free radical polymerisation. Moreover, the crosslinking density is higher and both the DP and PDI of a linear polymer are lower when it is obtained by RAFT polymerisation as compared to a polymer synthesised by free radical polymerisation. Even when it was observed that RAFT polymerisation is needed in order to obtain a phase separation by spinodal decomposition for the co-polymerisation of GM A and EDMA in a porogenic mixture of P(t-BMA) and dioxane, the inventors do not have enough evidence to attribute the phase separation mechanism to the polymerisation kinetic or the characteristics of the crosslinked network. Nonetheless, the inventors have studied the effect of the polymerisation temperature, which affects the kinetics, as well as the effect of the chemical structure of the CTA.

[0123] Monoliths were also prepared with PEG 10K as a polymeric non-solvating solvent ( SPEG=20.2 MPa1 / 2and 8PMMA= 18.6 MPa1 / 2) with and without CDSTS. The two bottom rows in Figure 17 depict the SEM images of poly(GMA-co-EDMA) monoliths synthesised with various mixtures of PEG and dioxane for the porogenic mixture by either RAFT or free radical polymerisations and it can be observed that irrespective of the polymerisation mechanism, monoliths presented a globular structure typical of a nucleation and growth phase separation mechanism. However, while the pore size noticeable changed when the composition of the porogenic mixture changed for the monoliths by free radical polymerisation, it remains apparently constant for the monoliths obtained by RAFT polymerisation (the mesopore size obtained by nitrogen isotherms of monoliths by RAFT polymerisation with various amounts of PEG(6K)and it was observed that once the polymeric non-solvating solvent reached a minimum value, the pore size was not affected). Besides the effect of the polymerisation mechanism on the pore size of the resulting monolith, when PEG is used as a polymeric non-solvating solvent, the phase separation mechanism is nucleation and growth, which is different to the behaviour observed for P(t-BMA) as the non-solvating solvent. Thus, these results suggest that the difference in solubility parameter between the polymeric non-solvating solvent and the crosslinked network is a defining factor to allow phase separation by spinodal decomposition.

[0124] Use of the porous polymer monoliths in chromatography applications

[0125] Example 4 - Use of monoliths with hierarchical porosity in chromatography applications: size exclusion chromatography

[0126] 4.1. Materials

[0127] Glycidyl methacrylate (GMA, 97 %), ethylene glycol dimethacrylate (EDMA, 98 %), azobisisobutyronitrile (AIBN, 12 %w in acetone), 4-cyano-4- ((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDSTS, 97 %), toluene (>99.9 %), dodecanol (>98 %), tetrahydrofuran (THF, anhydrous, >99.9 %), poly(ethylene glycol) (PEG 6K, 10K and 20K g / mol) were purchased from Sigma Aldrich. 1,4-dioxane (>99.5 %) was purchased from Merck and poly(methyl methacrylate) (PMMA 2.58K, 10.1K, 31.6K, 54.5K, 93.3K, 158.3K, 267K, 520K, 701K, 981K, l,280K, 2,650K, 3,220K and 4,810K g / mol) were obtained from Polymer Standards Service GmbH, Mainz, Germany.

[0128] Both GMA and EDMA were purified by flushing the monomers through an alumina column.

[0129] 4.2. Instrumentation

[0130] Size Exclusion Chromatography (SEC) determinations were performed using an Agilent 1260 Infinity II system equipped with a G7110B isocratic pump, a G7129A vial sampler, an Extrema A071561685 column oven and a with a G1314F UV Detector with a semi-micro flow cell (5 pl volume and 6 mm path length). Mobile phase was THF at a flow rate of 0.2 mL / min. Injection volume was 5 pl, the column temperature was 40 °C and the UV detector was set at 254 nm for the injection of toluene 1 %v (void volume marker) and 230 nm for PMMA standards (1 mg / mL). Data was processed with a PSS WinGPC UniChrom software for SEC runs and PoroCheck for ISEC.

[0131] 4.3. Column synthesis

[0132] 4.6x30 mm columns were prepared by introducing the column tube in a 3 mL vial and filling the vial with polymerisation mixture in order to have extra volume of the reaction mixture and avoid shrinking. Synthesis was performed in a water bath at 50 °C for 24 hours and after the polymerisations were completed, the vials were broken and the extra monolith around the column was removed.

[0133] For the synthesis of monoliths in 4.6x150 mm columns, 5 g of polymerisation mixture were prepared in order to fill the entire volume of the column and have some spare reaction mixture in an extra volume to avoid shrinkage in the column. The extra volume was achieved by attaching a 3 mL vial to the top end of the column through a hole in the rubber from the vial cap (see Figure 11(a)). In order to allow complete dissolution of the polymeric non-solvating solvent, the porogenic mixture was prepared the day before the column synthesis. Thus, 0.375 g PEG(20K), 1.5 g dodecanol and 1.125 g dioxane were weighted in a vial and left overnight at 60 °C. The following day, the monomers mixture was prepared by weighting 20 mg AIBN, 99 mg CDSTS, 1 g GMA and 1 g EDMA. The homogenised monomer mixture was then added to the porogenic mixture and purged with N2 for 5 min in a warm water bath (approx. 55 °C). The column was then filled with the polymerisation mixture by pouring the reaction mixture in the column with the vial attached at the bottom (Figure 11(a)), closing the top end (Figure 11(b)) and then turning the column upside down (Figure 11(c)). The column was then sonicated to avoid bubbles and placed in a water bath at 50 °C for 24h. After polymerisation (Figure 11(d)), the vial was broken and the extra monolith removed from the top of the column. The monolith was then washed in the HPLC instrument with methanol at 60 °C to reproduce the Soxhlet conditions. After washing the columns with methanol overnight, the mobile phase was changed to THF for chromatographic evaluation.

[0134] 4.4. Chromatographic characterization

[0135] Permeability was calculated according to Equation 4.1, where Bo is the permeability, rj the viscosity of the mobile phase (0.455 cP for THF), F the volumetric flow rate, P the back pressure and L and A the length and cross-sectional area of the column. The column was flushed with THF at increasing flowrates in the range of 0.05-0.20 mL / min and Bo was obtained by linear regression of AP vs. F curves.

[0137] Plate count and asymmetry were obtained from the integration of the peak from toluene 1 %v in THF, which was used as a void volume marker. For these measurements, THF was used as a mobile phase at a flowrate of 0.2 mL / min and the UV detector was set at 254 nm. The calculation of the plate count and asymmetry factor were done using a WinGPC software. Asymmetry was calculated according to ISO13885 at 10 % height of the peak maxima and plate count according to Equation 4.2, where N isthe plate count, L the length of the column in cm, vpthe elution volume and W1 / 2 the peak width at half its height.

[0139] Calibration curves were obtained from the injection of PMMA standards with molar masses ranging from 2.58K to 4,810K g / mol. THF was used as a mobile phase at 0.2 mL / min and the column temperature was 40 °C. Detection of the analytes was performed with a UV detector at 230 nm and a polynomial regression (grade 3) was used for the calibration curves.

[0140] The total exclusion pore volume (Vp) of the columns was obtained by the difference in elution volume between the toluene peak (void volume marker, Vo) and the first PMMA standard that elutes at the exclusion limit based on the calibration curve (interstitial volume marker, Vi). ISEC was used to obtain the exclusion pores size in the wet state using PoroCheck as software.

[0141] 4.5. Size exclusion chromatography results

[0142] Monoliths containing 20 %w GMA, 20 %w EDMA, 60 %w porogens (PEG(20K) / dodecanol / dioxane 12.5:50:37.5 weight ratio), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio) were prepared in 4.6x150 mm columns for chromatographic evaluation. PMMA standards were injected (ranging from 2.58K to 4,810K g / mol) and it was observed that the analytes were successfully separated and their elution order followed the expected order for an SEC mechanism. Figure 12 shows the elugrams for the poly (methyl methacrylate) (PMMA) standards, and it can be observed that the analytes with molar masses larger than 2,650K g / mol are in the exclusion limit of the column and cannot be separated.

[0143] The presence of exclusion pores in the stationary phase is a requirement for SEC separations and that is possible due to a RAFT mechanism during the monolith synthesis. It was shown in Figure 9 that a monolith with a mixture of PEG(20K) / dodecanol / dioxane 12.5:50:37.5 obtained by RAFT polymerisation showed a dual pore size distribution, while a monolith with the same polymerisation mixture obtained by free radical polymerisation showed a monomodal pore size distribution. When stationary phases were prepared by RAFT polymerisation, separation of PMMA standards according to its size was achieved, while a control column obtained by free radical polymerisation showed coelution of all PMMA standards as well as non-gaussian analyte profiles. Figure 13 shows the elugrams for PMMA standards in the range of 2.58K to 4,810K g / mol for the columns synthesised by either RAFT or free radical polymerisation, and it can be seen not only that with the column by free radical polymerisation is not possible to separate the analytes, but also that the peak width, shape and asymmetry significantly differs between the two columns. Those differences are due to the absence of exclusion pores in thecolumn obtained by free radical polymerisation and a gel-like structure for the monolith by free radical polymerisation.

[0144] 4.6. Column reproducibility

[0145] The stationary phase reproducibility was evaluated by the synthesis and chromatographic evaluation of five columns, where column replicates were obtained by preparing a new polymerisation mixture each time. Table 3 summarises the permeability, plate count, asymmetry factor and exclusion pore volume obtained for all column replicates. The variations in permeability seem to have a correlation with the differences observed in efficiency, thus, for the columns with higher permeability (1.1-1.9x1015m2, columns 1, 2 and 4), the plate count ranged from 2,600 to 6,600 plates / m, while for the columns with lower permeabilities (7.1-7.6x1016m2, columns 3 and 5), the efficiencies were 12,400 and 14,300 plates / m respectively. These results suggest that there might be some voids or cracks in the monoliths with higher permeabilities, which affected the efficiency of analysis. The asymmetry factor ranged from 0.57 to 0.78 for columns 1,3,4 and 5, while for column 2 the asymmetry factor was 1.57, which again suggest the presence of an internal crack or void. Figure 14(a) shows the calibration curves obtained for the injection of PMMA standards, and it can be observed that the selectivity, or slope of the calibration curve, was very similar for all columns, which goes in hand with the similarities observed for the exclusion pore volume in Table 3.

[0146] Table 3. Permeability, plate count, asymmetry factor and exclusion pore volume for 4.6x150 mm columns. Monoliths were synthesised with 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture (PEG(20K) / dodecanol / dioxane 12.5:50:37.5), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). Plate count and asymmetry factor were calculated from a toluene 1 %v peak and exclusion pore volume was obtained from the difference in elution volume between the largest PMMA standard from the linear range of the calibration curve and the toluene peak.Column Bo (m2) N (plates / m) As VP(mL)1 l.lxlO156,000 0.57 0.682 1.3xl0152,600 1.57 0.653 7.1xl01612,400 0.70 0.674 1.9xl0156,600 0.73 0.565 7.6xl01614,300 0.78 0.67 free radical 4.5x 10148 0.04 0

[0147] The possibility to couple columns to increase the total exclusion pore volume was evaluated, thus, columns 3 and 5 have been used in series. Figure 14(b) shows the calibration curves of columns 3and 5 individually as well as both used together, and it can be observed that the slope of the calibration curve decreased for the two-column system. However, this increase in selectivity was not accompanied by an increase in resolution, as the flow rate needed to be decreased from 0.2 to 0.1 mL / min in order to maintain a back pressure below 200 bar, which resulted in a decrease in efficiency.

[0148] Example 5 - Use of monoliths with macroporosity in chromatography applications: hydrodynamic chromatography

[0149] 5.1. Materials

[0150] Glycidyl methacrylate (GMA, 97 %), ethylene glycol dimethacrylate (EDMA, 98 %), azobisisobutyronitrile (AIBN, 12 %w in acetone), 4-cyano-4- ((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDSTS, 97 %), 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CETCPA, 95 %), 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTBCOOH), toluene (>99.9 %), dodecanol (>98 %) and tetrahydrofuran (THF, anhydrous, >99.9 %) were purchased from Sigma Aldrich. 1,4-dioxane (>99.5 %) and methanol (HPLC grade) were purchased from Merck. Poly(tert-butyl methacrylate) (P(t-BMA), 4.7K,10K and 21K g / mol) and poly(methyl methacrylate) standards (PMMA 520K, 701K, 981K, l,280K, 2,650K, 3,220K and 4,810K g / mol) were obtained from Polymer Standards Service GmbH, Mainz, Germany.

[0151] Both GMA and EDMA were purified by flushing the monomers through an alumina column before using for any reaction.

[0152] 5.2. Instrumentation

[0153] Size Exclusion Chromatography (SEC) determinations were performed in an Agilent 1260 Infinity II system equipped with a G7110B isocratic pump, a G7129A vial sampler, an Extrema A071561685 column oven and two detectors, a G7162A refractive index detector (RID) and a G1314F UV Detector with a semi-micro flow cell (5 pl volume and 6 mm path length). For the injection of PMMA standards, the mobile phase was THF and the flow rate 0.33 mL / min. Injection volume was 5 pl, the column temperature was 40 °C and RI was used for detection of all analytes. The void volume was measured by injection of a toluene 1 %v solution in THF and the concentration of PMMA standards wasl mg / mL.

[0154] 5.3 Column synthesis

[0155] 4.6x30 mm columns were prepared by introducing the column tube in a 3 mL vial and filling the vial with polymerisation mixture in order to have extra volume of the reaction mixture and avoidshrinking inside the column. Synthesis was performed in a water bath at 60 °C for 24 hours and after the polymerisations were completed, the vials were broken and the extra monolith around the column was removed.

[0156] For the synthesis of monoliths in 4.6x150 mm columns, 5 g of polymerisation mixture were prepared in order to fill the entire volume of the column and have some spare reaction mixture in an extra volume to avoid shrinkage in the column. The extra volume was achieved by attaching a 3 mL vial to the top end of the column through a hole in the rubber from the vial cap (see Figure 11(a)). The porogen mixture was first prepared in a vial, which contained 690 mg P(t-BMA) and 2.310 g dioxane, and it was kept at 60 °C to allow the polymer dissolution while the monomers mixture was prepared. The monomer mixture contained 20 mg AIBN, 99 mg CDSTS, 1 g GMA and 1 g EDMA. The homogenized monomers mixture was added to the porogens mixture and purged with N2 for 5 min in a warm water bath (around 55 °C). The column was then filled with the polymerisation mixture by pouring the reaction mixture in the column with the vial attached at the bottom (Figure 11(a)), closing the top end (Figure 11(b)) and then turning the column upside down (Figure 11(c)). The column was then sonicated to avoid bubbles and placed in a water bath at 60 °C for 24h. After polymerisation (Figure 11(d)), the vial was broken and the extra monolith removed from the top of the column, the monolith was then washed in the HPLC instrument with methanol at 60 °C to reproduce the Soxhlet conditions. After washing the columns (at 0.1 mL / min) with methanol overnight, the mobile phase was changed to either THF or an aqueous mobile phase for chromatographic evaluation.

[0157] 5.4. Chromatographic evaluation

[0158] Permeability was calculated according to Equation 5.1, where Bo is the permeability, rj the viscosity of the mobile phase (0.455 cP for THF), F the volumetric flow rate, P the back pressure and L and A the length and cross-sectional area of the column. The column was flushed with THF at increasing flowrates in the range of 0.05-0.33 mL / min and Bo was obtained by linear regression of the AP vs. F curves.

[0160] Plate count and asymmetry were obtained from the integration of the peak from toluene 1 %v in THF, which was used as a void volume marker. For these measurements, a flowrate of 0.33 mL / min was used and UV detection was set at 254 nm. The calculation of the plate count and asymmetry factor were done using a WinGPC software. Asymmetry was calculated according to ISO13885 at 10 % height of the peak maxima and plate count according to Equation 5.2, where N is the plate count, L the length of the column in cm, vpthe elution volume and W1 / 2 the peak width at half its height.

[0162] Calibration curves were obtained for the linear polymeric standards from the injection of PMMA standards with molar masses ranging from 2.58K to 4,810K g / mol. THF was used as a mobile phase at 0.33 mL / min and the column temperature was 40 °C. Detection of the analytes was performed with the RI detector and a polynomial regression (grade 3) was used for the calibration curves

[0163] 5.5. Hydrodynamic chromatography results

[0164] A set of 5 columns (4.6x150 mm) with monoliths with 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture (P(t-BMA) / dioxane 23:77 weight ratio), 1 %w AIBN with respect to monomers and CDSTS / AIBN=2 molar ratio were synthesised by RAFT polymerisation and another column with the same polymerisation mixture was synthesised by conventional free radical polymerisation as a control. Table 4 summarises the permeability, plate count and asymmetry factor (measured from the injection of toluene as a void volume marker) for all columns. It was found that the permeability of the column prepared by free radical polymerisation was an order of magnitude lower than the columns obtained by RAFT polymerisation. This result is surprising as the pore size distribution from both monoliths was very similar in the dry state as measured by MIP and it suggests that the monoliths swelling structure in THF are different. This difference in swelling could be due to a more heterogeneous crosslinking density as well as lower crosslinking density in the monolith obtained by free radical polymerisation with respect to the monolith synthesised by RAFT polymerisation. Regardless of the good reproducibility of the permeability values obtained for the columns synthesised by RAFT polymerisation, the dispersity of the plate count values and asymmetry factors was considerable large, suggesting that the monolith filling in columns 3, 4 and 5 is not homogeneous, which could be due to the presence of bubbles or cracks inside the column.

[0165] Table 4. Permeability, plate count and asymmetry factor for polymer monoiths prepared within 4.6x150 mm columns with THF as mobile phase. Monoliths were synthesised with 20 %w GMA, 20 %w EDMA, 60 %w the porogenic mixture (P(t-BMA) / dioxane 23:77 weight ratio), 1 %w AIBN with respect to the monomer and the crosslinker, and CDSTS / AIBN=2 (molar ratio). Plate count and asymmetry factor were calculated from a toluene 1 %v peak at 7=254 nm.

[0166] Poly(methyl methacrylate) (PMMA) standards with molar masses in the range of 520K-4,810K g / mol were injected in all columns using THF as the mobile phase. Figure 19 depicts the elugrams obtained for the PMMA standards in a column obtained by RAFT polymerisation as well as the column synthesised by conventional free radical polymerisation. Size-based separations were achieved with both types of stationary phases and analytes with larger molar masses eluted earlier than those of lower molar masses. As the columns tested as stationary phases lack of mesopores, the only separation mechanism possible in the macropores is hydrodynamic chromatography (HDC). Figure 20(a) shows the calibration curves for all columns tested and it can be observed that the slope of the curves for the columns obtained by RAFT polymerisation are similar with the exemption of column 4, where there is almost no separation of the analytes. Because the larger analytes from the calibration curves are still in the quasi-linear range of the curve, it is evident that the upper limit of the separation range has not been reached for the columns synthesised by RAFT polymerisation. On the other hand, the larger analytes coeluted in the column obtained by free radical polymerisation and the elugrams in Figure 19 show tailing for the analytes from 2,250K to 4,810K g / mol, indicating that the limit of the separation range was reached.

[0167] In order to increase selectivity, multiple columns synthesised by RAFT polymerisation have been coupled in series. Figure 20(b) shows the calibration curves for the columns in series. The main difference in the calibration curves can be found at the lower molar masses, where analytes coelute when one column is used, but they are separated when two or more columns are used in series. However, besides the improvement in selectivity with two columns, further coupling of columns did not seem to improve separations. Thus, by using two columns the best compromise between higher selectivity and lower analysis time can be achieved.

[0168] It will be evident from the foregoing that disclosed herein is the following.

[0169] A method for preparing a porous methacrylate-based polymer monolith through reversible addition-fragmentation chain-transfer (RAFT) polymerization, wherein the method comprises: introducing a reaction composition into a mold, the reaction composition comprising at least one methacrylate monomer, at least one methacrylate crosslinker, a radical initiator, a chain transfer agent (CT A), and a porogenic mixture of a solvating solvent with a non-solvating solvent and / or an inert polymer;conducting the RAFT polymerization to form a polymer monolith; and optionally, separating the polymer monolith from unreacted reagents.

[0170] A method as above, wherein the methacrylate monomer comprises a monovinyl monomer having a methacrylate unit.

[0171] A method as above, wherein the methacrylate monomer is a mono vinyl monomer having a methacrylate unit.

[0172] A method as above, wherein the monovinyl monomer having a methacrylate unit is selected from the group consisting of glycidyl methacrylate (GMA), methyl methacrylate, ethyl methacrylate, butyl methacrylate, octadecyl methacrylate, lauryl methacrylate and poly (ethylene glycol) methacrylate.

[0173] A method as above, wherein the mono vinyl monomer having a methacrylate unit is glycidyl methacrylate (GMA).

[0174] A method as above, wherein the methacrylate crosslinker comprises a multi-vinyl monomer having a methacrylate unit.

[0175] A method as above, wherein the methacrylate crosslinker is a multi-vinyl monomer having a methacrylate unit.

[0176] A method as above, wherein the multi-vinyl monomer having a methacrylate unit is a divinyl monomer having a methacrylate unit.

[0177] A method as above, wherein the multi-vinyl monomer having a methacrylate unit is selected from the group consisting of ethylene glycol dimethacrylate (EDMA), triethylene glycol dimethacrylate (TEGDMA), trimethylol-propane trimethacrylate (TRIM), poly(ethylene glycol) dimethacrylate.

[0178] A method as above, wherein the multi-vinyl monomer having a methacrylate unit is ethylene glycol dimethacrylate (EDMA).

[0179] A method as above, wherein the crosslinker and the monomer for the monolith are in a molar ratio of about 1:0.9 to about 1:3.3.

[0180] A method as above, wherein the crosslinker and the monomer for the monolith are in a molar ratio of about 1:1.4 to about 1:2.1.

[0181] A method as above, wherein the molecular chain transfer agent has the following formula (I):wherein Z is selected from an aryl, an alkylthio group, and a carboxyalkylthio group, R is a substituent having a tertiary carbon and optionally a nitrile group connected to the tertiary carbon, and the alkyl has 1 to 20 carbon atoms, each of the aryl and the alkyl is optionally substituted with a group selected from H, Ci-6 alkyl, halogen, -OH, -SH, -O-Ci 6 alkyl, and -S-Ci-6 alkyl.

[0182] A method as above, wherein the alkyl has 2 to 12 carbon atoms.

[0183] A method as above, wherein the chain transfer agent is selected from 4-cyano-4-((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid (CDSTS), 4-((((2- carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CETCPA), and 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTBCOOH), 2-cyano-2-propyl benzodithioate, 4-cyano-4- (phenylcarbonothioylthio) pentanoic acid, 2-phenyl-2- propyl benzoditioate, 2-cyano-2-propyl dodecyl trithiocarbonate.

[0184] A method as above, wherein the chain transfer agent and the monomer / the crosslinker is in a molar ratio of about 0.5:100 to about 3:100.

[0185] A method as above, wherein the chain transfer agent and the monomer / the crosslinker is in a molar ratio of about 0.75:100 to about 1.25:100.

[0186] A method as above, wherein the chain transfer agent and the monomer / the crosslinker is in a molar ratio of about 1:100.

[0187] A method as above, wherein the solvating solvent within the porogenic mixture is selected from the group consisting of dioxane and tetrahydrofuran.

[0188] A method as above, wherein the non-solvating solvent within the porogenic mixture is selected from the group consisting of toluene, cyclohexanol, and dodecanol.

[0189] A method as above, wherein the inert polymer within the porogenic mixture is selected from poly(ethylene glycol) (PEG), poly(acrylic acid) (PAA) and polymethacrylates such as poly(tert-butyl methacrylate) (P(t-BMA)).

[0190] A method as above, wherein the PEG has a molecular weight of about 6K g / mol to about 35K g / mol.

[0191] A method as above, wherein the PEG has a molecular weight of about 10K g / mol to about 20K g / mol.

[0192] A method as above, wherein the poly( tert-butyl methacrylate) (P(t-BMA)) has a molecular weight of about 4.7K g / mol to about 21 K g / mol.

[0193] A method as above, wherein the solvating solvent is in the range of 12-48 %wt, and the nonsolvating solvent and / or the inert polymer is(are) in the range of 12-48 %wt, based on the reaction composition.

[0194] A method as above, wherein the porogenic mixture of the solvating solvent with the nonsolvating solvent and / or the inert polymer is about 60 %wt based on the reaction composition.

[0195] A method as above, wherein the weight ratio between the solvating solvent and the nonsolvating solvent and / or the inert polymer is in a range of about 20:80 to about 80:20.

[0196] A method as above, wherein the RAFT polymerisation is conducted at a temperature of about 50 °C to about 70 °C.

[0197] A method as above, wherein the RAFT polymerisation is conducted at a temperature of about 50 °C to about 60 °C.

[0198] A method as above, wherein the monolith prepared is a mesoporous methacrylate-based polymer monolith.

[0199] A method as above, wherein the mesoporous methacrylate-based polymer monolith displays a mesopore volume of up to about 0.620 mL / g in dry state, which is at least approximately 25 %v / v with respect to the total volume of the monolith.

[0200] A method as above, wherein the mesoporous methacrylate-based polymer monolith displays a BET surface area of about 137 m2 / g, for example up to about 155 m2 / g, for pores having a diameter in the range of 2-250 nm.

[0201] A method as above, wherein the mesopores in the monolith is up to about 35% by volume, for example about 25% by volume, with respect to the total volume of the monolith.

[0202] A method as above, wherein a non-solvating solvent selected from dodecanol, toluene and cyclohexanol and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture.

[0203] A method as above, wherein the non-solvating solvent and the solvating solvent are used in a weight ratio of about 50:50.

[0204] A method as above, wherein the porogenic mixture consists of dodecanol and dioxane.

[0205] A method as above, wherein the porogenic mixture consists of dodecanol and tetrahydrofuran.

[0206] A method as above, wherein an inert polymer selected from poly(ethylene glycol) (PEG), poly(acrylic acid) (PAA) and polymethacrylates such as poly(tert-butyl methacrylate) (P(t-BMA)) and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture.

[0207] A method as above, wherein the inert polymer and the solvating solvent are used in a weight ratio of about 50:50.

[0208] A method as above, wherein the porogenic mixture consists of a PEG having a molecular weight of about 6K g / mol and dioxane.

[0209] A method as above, wherein the RAFT polymerisation is conducted at a temperature of about 60 °C.

[0210] A method as above, wherein the monolith prepared is a hierarchically porous methacrylate- based polymer monolith.

[0211] A method as above, wherein the hierarchically porous methacrylate-based polymer monolith exhibits a dual pore size distribution in dry state.

[0212] A method as above, wherein the hierarchically porous methacrylate-based polymer monolith exhibits a dual pore size distribution in macropore region in dry state.

[0213] A method as above, wherein the hierarchically porous methacrylate-based polymer monolith exhibits a dual pore size distribution with about 60 nm and about 550 nm in dry state.

[0214] A method as above, wherein the hierarchically porous methacrylate-based polymer monolith has the pores in a size range of about 100 nm to about 1000 nm and the pores in a size range of about 20 nm to about 100 nm in a volume ratio of about 50:50.

[0215] A method as above, wherein the pore volume of the hierarchically porous methacrylate-based polymer monolith is about 0.167 mL / g in a pore size range of about 20-100 nm and about 0.584 ml / g in a pore size range of about 100-1,000 nm.

[0216] A method as above, wherein the hierarchically porous methacrylate-based polymer monolith has about 22 %v / v pores in a size range of about 20 nm to about 100 nm and about 78 %v / v pores in a size range of about 100 nm to about 1000 nm with respect to the total pore volume.

[0217] A method as above, wherein the hierarchically porous methacrylate-based polymer monolith has about 12 %v / v pores in a size range of about 20 nm to about 100 nm and about 43 %v / v pores in a size range of about 100 nm to about 1000 nm with respect to the total monolith volume.

[0218] A method as above, wherein an inert polymer, a non-solvating solvent such as dodecanol, and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture.

[0219] A method as above, wherein an inert polymer, a non-solvating solvent such as dodecanol, and a solvating solvent selected from dioxane and tetrahydrofuran are in a weight ratio of about 12.5:50:37.5.

[0220] A method as above, wherein the porogenic mixture consists of PEG, dodecanol, and a solvating solvent selected from dioxane and tetrahydrofuran.

[0221] A method as above, wherein the porogenic mixture consists of a PEG having a molecular weight of about 6K g / mol to about 20K g / mol, dodecanol and dioxane.

[0222] A method as above, wherein the RAFT polymerisation is conducted at a temperature of about 50 °C.

[0223] A method as above, wherein the monolith prepared is a macroporous methacrylate-based polymer monolith.

[0224] A method as above, wherein the macroporous methacrylate-based polymer monolith is a fully macroporous methacrylate-based polymer monolith developed via spinodal decomposition.

[0225] A method as above, wherein an inert polymer and a solvating solvent selected from dioxane and tetrahydrofuran are used to form the porogenic mixture.

[0226] A method as above, wherein the inert polymer and the solvating solvent is in a weight ratio of about 22:78 to about 25:75.

[0227] A method as above, wherein the inert polymer is a polymethacrylate.

[0228] A method as above, wherein the polymethacrylate is poly(tert-butyl methacrylate) (P(t-BMA)).

[0229] A method as above, wherein the porogenic mixture consists of poly(tert-butyl methacrylate)(P(t-BMA)) having a molecular weight of about 4.7K g / mol to about 21K g / mol and dioxane.

[0230] A method as above, wherein the RAFT polymerisation is conducted at a temperature of about 60 °C.

[0231] A porous methacrylate-based polymer monolith obtained through the method as described above.

[0232] A porous methacrylate-based polymer monolith as above, wherein the porous methacrylate- based polymer monolith is a mesoporous methacrylate-based polymer monolith.

[0233] A porous methacrylate-based polymer monolith as above, wherein the porous methacrylate- based polymer monolith is a hierarchically porous methacrylate -based polymer monolith.

[0234] A porous methacrylate-based polymer monolith as above, wherein the porous methacrylate- based polymer monolith is a hierarchically porous methacrylate -based polymer monolith which exhibits a dual pore size distribution in macropore region in dry state.

[0235] A porous methacrylate-based polymer monolith as above, wherein the hierarchically porous methacrylate-based polymer monolith exhibits a dual pore size distribution with about 60 nm and about 550 nm in dry state.

[0236] A porous methacrylate-based polymer monolith as above, wherein the porous methacrylate- based polymer monolith is a macroporous methacrylate-based polymer monolith.

[0237] A porous methacrylate-based polymer monolith as above, wherein the porous methacrylate- based polymer monolith is a fully macroporous methacrylate-based polymer monolith developed via spinodal decomposition.

[0238] A separation medium that comprises the porous methacrylate-based polymer monolith obtained through the method as above.

[0239] A separation medium as above, wherein the medium is used for a liquid chromatography application selected from normal phase chromatography, hydrophilic interaction chromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography and ion exchange chromatography.

[0240] Use of the porous methacrylate-based polymer monolith obtained through the method as above in a liquid chromatography application.

[0241] A use as above, wherein the liquid chromatography application is selected from normal phase chromatography, hydrophilic interaction chromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography, and ion exchange chromatography.

[0242] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge.

[0243] It will be understood that the terms “comprise” and “include” and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

[0244] In some cases, a single embodiment may, for succinctness and / or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to “at least one of’ a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c.

[0245] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and / or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.REFERENCES

[0246] [1] Wu, J.; Xu, F.; Li, S.; Ma, P.; Zhang, X.; Liu, Q.; Fu, R.; Wu, D. Porous Polymers asMultifunctional Material Platforms toward Task-Specific Applications. Adv. Mater. 2019, 31 (4), el802922, DOI: 10.1002 / adma.201802922.

[0247] [2] Melde, B. J.; Johnson, B. J. Mesoporous Materials in Sensing: Morphology andFunctionality at the Meso-Interface. Anal. Bioanal. Chem. 2010, 398 (4), 1565-1573, DOI: 10.1007 / s00216-010-3688-6.

[0248] [3] Da'na, E. Adsorption of Heavy Metals on Functionalized-Mesoporous Silica: A Review.Micropor. Mesopor. Mat. 2017, 247, 145-157, DOI: 10.1016 / j.micromeso.2017.03.050.

[0249] [4] Brady, R.; Woonton, B.; Gee, M. L.; O'Connor, A. J. Hierarchical Mesoporous SilicaMaterials for Separation of Functional Food Ingredients — A Review. Innov. Food. Sci. Emerg. Technol. 2008, 9 (2), 243-248, DOI: 10.1016 / j.ifset.2007.10.002.

[0250] [5] Zhang, Y.; Ang, C. Y.; Li, M.; Tan, S. Y.; Qu, Q.; Luo, Z.; Zhao, Y. Polymer-CoatedHollow Mesoporous Silica Nanoparticles for Triple -Responsive Drug Delivery. ACS Appl. Mater. Interfaces 2015 , 7 (32), 18179-18187, DOI: 10.1021 / acsami.5b05893.

[0251] [6] Lim, E.; Jo, C.; Lee, J. A mini Review of Designed Mesoporous Materials for Energy-Storage Applications: From Electric Double-Layer Capacitors to Hybrid Supercapacitors. Nanoscale 2016, 8 (15), 7827-7833, DOI: 10.1039 / c6nr00796a.

[0252] [7] Santoyo-Gonzalez, F.; Hernandez-Mateo, F. Silica-Based Clicked Hybrid Glyco Materials.Chem. Soc. Rev. 2009, 38 (12), 3449-3462, DOI: 10.1039 / b909363j.

[0253] [8] Ali, U.; Karim, K. J. B. A.; Buang, N. A. A Review of the Properties and Applications ofPoly (Methyl Methacrylate) (PMMA). Polymer Reviews 2015, 55 (4), 678-705, DOI: 10.1080 / 15583724.2015.1031377.

[0254] [9] Vlakh, E. G.; Tennikova, T. B. Preparation of Methacrylate Monoliths. J. Sep. Sci. 2007, 30(17), 2801-2813, DOI: 10.1002 / jssc.200700284.

[0255]

[0010] Wu, D.; Xu, F.; Sun, B.; Fu, R.; He, H.; Matyjaszewski, K. Design and Preparation ofPorous Polymers. Chem. Rev. 2012, 112 (7), 3959-4015, DOI: 10.1021 / cr200440z.

[0256]

[0011] Liu, Q.; Tang, Z.; Ou, B.; Liu, L.; Zhou, Z.; Shen, S.; Duan, Y. Design, Preparation, andApplication of Ordered Porous Polymer Materials. Mater. Chem. Phys. 2014, 144 (3), 213-225, DOI: 10.1016 / j.matchemphys.2014.01.013.

[0257]

[0012] Yang, M.; Ma, J.; Zhang, C.; Yang, Z.; Lu, Y. General Synthetic Route toward FunctionalHollow Spheres with Double-Shelled Structures. Angew. Chem. Int. Ed. Engl. 2005, 44 (41), 6727-6730, DOI: 10.1002 / anie.200501556.

[0258]

[0013] Johnson, S. A.; Olliver, P. J.; Mallouk, T. E. Ordered Mesoporous Polymers of TunablePore Size from Colloidal Silica Templates. Science 1999, 283, 963-965.

[0259]

[0014] Desire, C. T.; Khodabandeh, A.; Schiller, T. L.; Wilson, R.; Arrua, R. D.; Bon, S. A. F.;Hilder, E. F. Preparation of Highly Interconnected Hydrophilic Polymers from Emulsion Templates with Improved Mechanical Properties. Eur. Polym. J. 2018, 102, 56-67, DOI:10.1016 / j .eurpolymj .2018.02.002.

[0260]

[0015] Mezhoud, S.; Le Droumaguet, B.; Aimedieu, P.; Monchiet, V.; Bornert, M.; Grande, D.Investigation of Morphology Associated with Biporous Polymeric Materials Obtained by the Double Porogen Templating Approach. Colloid Polym. Sci. 2020, DOI: 10.1007 / s00396-020-04747-9.

[0261]

[0016] Turiel, E.; Martin-Esteban, A. Molecularly Imprinted Polymers for Sample Preparation: AReview. Anal. Chim. Acta 2010, 668 (2), 87-99, DOI: 10.1016 / j.aca.2010.04.019.

[0262]

[0017] Li, C.; Li, Q.; Kaneti, Y. V.; Hou, D.; Yamauchi, Y.; Mai, Y. Self-Assembly of BlockCopolymers Towards Mesoporous Materials for Energy Storage and Conversion Systems. Chem. Soc. Rev. 2020, 49 (14), 4681-4736, DOI: 10.1039 / d0cs00021c.

[0263]

[0018] Peters, E. C.; Svec, F.; Frechet, J. M. J. Rigid Macroporous Polymer Monoliths. Adv.Mater. 1999, 11, 1169-1181.

[0264]

[0019] Svec, F. Porous Polymer Monoliths: Amazingly Wide Variety of Techniques EnablingTheir Preparation. J. Chromatogr. A 2010, 1217 (6), 902-924, DOI: 10.1016 / j.chroma.2009.09.073.

[0265]

[0020] Aggarwal, P.; Tolley, H. D.; Lee, M. L. Monolithic Bed Structure for Capillary LiquidChromatography. J. Chromatogr. A 2012, 1219, 1-14, DOI: 10.1016 / j.chroma.201 L 10.083.

[0266]

[0021] Svec, Frantisek. Quest for Organic Polymer-based Monolithic Columns AffordingEnhanced Efficiency in High Performance Liquid Chromatography Separations of Small Molecules in Isocratic Mode. J. Chromatogr. A 2012, 1228, 250-262, DOI: 10.1016 / j.chroma.2011.07.019.

[0267]

[0022] Jiang, J.-X.; Cooper, A. I., Microporous Organic Polymers: Design, Synthesis andFunction. In Functional Metal-Organic Frameworks: Gas Storage, Separation and Catalysis, Schroder, M., Ed. Springer Berlin Heidelberg: Berlin, Heidelberg, 2010; pp 1-33.

[0268]

[0023] Xu, Z.; Yang, L.; Wang, Q. Different Alkyl Dimethacrylate Mediated Stearyl MethacrylateMonoliths for Improving Separation Efficiency of Typical Alkylbenzenes and Proteins. J. Chromatogr. A 2009, 1216 (15), 3098-106, DOI: 10.1016 / j.chroma.2009.01.089.

[0269]

[0024] Wang, H.-S.; Song, M.; Hang, T.-J. Functional Interfaces Constructed byControlled / Living Radical Polymerization for Analytical Chemistry. ACS Appl. Mater. Interfaces 2016, 8 (5), 2881-2898, DOI: 10.1021 / acsami.5bl0465.

[0270]

[0025] Peters, E. C.; Svec, F.; Frechet, J. M. J. Control of Porous Properties and SurfaceChemistry in "Molded" Porous Polymer Monoliths Prepared by Polymerization in the Presence of TEMPO. Macromolecules 1999, 32, 6377-6379.

[0271]

[0026] Nischang, I.; Causon, T. J. Porous Polymer Monoliths: From their Fundamental Structure to Analytical Engineering Applications. Trends Anal. Chem. 2016, 75, 108-117, DOI: 10.1016 / j.trac.2015.05.013.

[0272]

[0027] Gama, M. R.; Aggarwal, P.; Liu, K.; Lee, M. L.; Bottoli, C. B. G. Improvement in LiquidChromatographic Performance of Organic Polymer Monolithic Capillary Columns with Controlled Free- Radical Polymerization. J. Chromatogr. Sci. 2017, 55 (4), 398-404, DOI: 10.1093 / chromsci / bmwl93.

[0273]

[0028] Goldfeld, D. J.; Silver, E. S.; Valdez, J. M.; Hillmyer, M. A. Bicontinuous Ion-ExchangeMaterials through Polymerization-Induced Microphase Separation. ACS Macro Letters 2020, 10 (1), 60- 64, DOI: 10.1021 / acsmacrolett.0c00684.

[0274]

[0029] Oh, J.; Seo, M. Photoinitiated Polymerization-Induced Microphase Separation for thePreparation of Nanoporous Polymer Films. ACS Macro. Lett. 2015, 4 (11), 1244-1248, DOI: 10.1021 / acsmacrolett.5b00734.

[0275]

[0030] Shirahase, T.; Akasaka, S.; Asai, S. Organic Solvent-Free Fabrication of MesoporousPolymer Monolith From Miscible PLLA / PMMA Blend. Polymer 2020, 203, DOI:10.1016 / j .polymer.2020.122742.

[0276]

[0031] Saba, S. A.; Mousavi, M. P.; Buhlmann, P.; Hillmyer, M. A. Hierarchically PorousPolymer Monoliths by Combining Controlled Macro- and Microphase Separation. J. Am. Chem. Soc. 2015, 137 (28), 8896-8899, DOI: 10.1021 / jacs.5b04992.

[0277]

[0032] Weller, A.; Carrasco-Correa, E. J.; Belenguer-Sapina, C.; de los Reyes Mauri- Aucejo, A.;Amoros, P.; Herrero-Martinez, J. M. Organo-Silica Hybrid Capillary Monolithic Column with Mesoporous Silica Particles for Separation of Small Aromatic Molecules. Microchim. Acta 2017, 184 (10), 3799-3808, DOI: 10.1007 / s00604-017-2404-z.

[0278]

[0033] Wickenheisser, M.; Janiak, C. Hierarchical Embedding of Micro-Mesoporous MIL-101 (Cr) in Macroporous poly(2-hydroxy ethyl methacrylate) High Internal Phase Emulsions with Monolithic Shape for Vapor Adsorption Applications. Micropor. Mesopor. Mat. 2015, 204, 242-250, DOI: 10.1016 / j.micromeso.2014.11.025.

[0279]

[0034] Kip, C.; Liu, S.; Fu, X.; Tuncel, A.; Lammerhofer, M. In-Situ Photopolymerized C4-Functionalized Organosilicon Monoliths for Reversed-Phase Protein Separation in nano-Liquid Chromatography. Taianta 2019, 198, 330-336, DOI: 10.1016 / j.talanta.2019.01.116.

[0280]

[0035] K. Kanamori, J. Hasegawa, K. Nakanishi, T. Hanada, Facile Synthesis of MacroporousCross-Linked Methacrylate Gels by Atom Transfer Radical Polymerization, Macromolecules, 41 (2008) 7186-7193.

[0281]

[0036] Barlow, K. J.; Bernabeu, V.; Hao, X.; Hughes, T. C.; Hutt, O. E.; Polyzos, A.; Turner, K.A.; Moad, G. Triphenylphosphine-Grafted, RAFT-Synthesised, Porous Monoliths as Catalysts for Michael Addition in Flow Synthesis. React. Funct. Polym. 2015, 96, 89-96, DOI:10.1016 / j .reactfunctpolym.2015.09.008.

[0282]

[0037] Koler, A.; Krajnc, P. Surface Modification of Hypercrosslinked Vinylbenzyl ChloridePolyHIPEs by Grafting via RAFT. Macromol. Chem. Phys. 2021, 222 (3), DOI: 10.1002 / macp.20200038L

[0283]

[0038] Willcock, H.; O'Reilly, R. K. End Group Removal and Modification of RAFT Polymers.Polym. Chem. 2010, 1 (2), 149-157, DOI: 10.1039 / b9py00340a.

[0284]

[0039] Yan, M.; Huang, Y.; Lu, M.; Lin, F.-Y.; Hernandez, N. B.; Cochran, E. W. Gel PointSuppression in RAFT Polymerization of Pure Acrylic Cross-Linker Derived from Soybean Oil. Biomacromolecules 2016, 17 (8), 2701-2709, DOI: 10.1021 / acs.biomac.6b00745.

[0285]

[0040] Physical Properties of polymers Handbook, Second Edition ed., Springer, New York,2007.

Claims

CLAIMS1. A method for preparing a porous methacrylate-based polymer monolith through reversible addition-fragmentation chain-transfer (RAFT) polymerization, wherein the method comprises: introducing a reaction composition into a mold, the reaction composition comprising at least one methacrylate monomer, at least one methacrylate crosslinker, a radical initiator, a chain transfer agent (CTA), and a porogenic mixture of a solvating solvent with a non-solvating solvent and / or an inert polymer; conducting the RAFT polymerization to form a polymer monolith; and optionally, separating the polymer monolith from unreacted reagents.

2. The method according to claim 1, wherein the mono vinyl monomer having a methacrylate unit is glycidyl methacrylate (GM A).

3. The method according to any one of claims 1 to 2, wherein the methacrylate crosslinker comprises a multi-vinyl monomer having a methacrylate unit.

4. The method according to claim 3, wherein the multi-vinyl monomer having a methacrylate unit is ethylene glycol dimethacrylate (EDMA).

5. The method according to any one of claims 1 to 4, wherein the methacrylate crosslinker and the methacrylate monomer are in a molar ratio of about 1:1.4 to about 1:2.1.

6. The method according to any one of claims 1 to 5, wherein the molecular chain transfer agent has the following formula (I):wherein Z is selected from an aryl, an alkylthio group, and a carboxyalkylthio group, R is a substituent having a tertiary carbon and optionally a nitrile group connected to the tertiary carbon, and the alkyl has 1 to 20 carbon atoms, each of the aryl and the alkyl is optionally substituted with a group selected from H, Ci-6 alkyl, halogen, -OH, -SH, -O-Ci 6 alkyl, and -S-Ci-6 alkyl.

7. The method according to any one of claims 1 to 6, wherein the solvating solvent within the porogenic mixture is selected from the group consisting of dioxane and tetrahydrofuran.

8. The method according to any one of claims 1 to 7, wherein the non-solvating solvent within the porogenic mixture is selected from the group consisting of toluene, cyclohexanol, and dodecanol.

9. The method according to any one of claims 1 to 8, wherein the inert polymer within the porogenic mixture is selected from poly(ethylene glycol) (PEG), poly(acrylic acid) (PAA) and polymethacrylates such as poly(tert-butyl methacrylate) (P(t-BMA)).

10. The method according to any one of claims 1 to 9, wherein the monolith is a hierarchically porous methacrylate-based polymer monolith.

11. The method according to claim 10, wherein the hierarchically porous methacrylate-based polymer monolith has pores in a size range of about 100 nm to about 1000 nm and pores in a size range of about 20 nm to about 100 nm in a volume ratio of about 50:50.

12. The method according to any one of claims 10 to 11, wherein the hierarchically porous methacrylate-based polymer monolith has about 22 %v / v pores in a size range of about 20 nm to about 100 nm and about 78 %v / v pores in a size range of about 100 nm to about 1000 nm with respect to the total pore volume.

13. A porous methacrylate-based polymer monolith obtained through the method according to any one of claims 1 to 12.

14. The porous methacrylate-based polymer monolith according to claim 13, wherein the porous methacrylate-based polymer monolith is a hierarchically porous methacrylate-based polymer monolith which exhibits a dual pore size distribution in macropore region in dry state.

15. The porous methacrylate-based polymer monolith according to claim 14, wherein the hierarchically porous methacrylate-based polymer monolith exhibits a dual pore size distribution with about 60 nm and about 550 nm in dry state.

16. A separation medium that comprises the porous methacrylate-based polymer monolith obtained through the method according to any one of claims 1 to 12.

17. The separation medium according to claim 16, wherein the medium is used for a liquid chromatography application selected from normal phase chromatography, hydrophilic interactionchromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography and ion exchange chromatography.

18. Use of the porous methacrylate-based polymer monolith obtained through the method according to any one of claims 1 to 12 in a liquid chromatography application.

19. The use according to claim 18, wherein the liquid chromatography application is selected from normal phase chromatography, hydrophilic interaction chromatography, reversed phase chromatography, size exclusion chromatography, hydrodynamic chromatography, and ion exchange chromatography.

Images