# Methacrylate Polymer Monoliths for Separation Applications
## 3 Applications of Methacrylate Polymer Monoliths in Separation Science
### Biological Separations and DNA Purification
#### 3.2.2 Chromatographic Separation of Proteins, Peptides and Amino Acids  
[PERSON] _et al._[53] fabricated a polyHIPE type methacrylate monolith to separate a mixture of proteins (myoglobin, conA and soybean trypsin inhibitor) and found that this type of monolith yielded separations under the applied conditions and also showed a similar performance to the commercial CIM(tm) monolith, but showed a slightly higher dispersion and lower surface loading which may explain the poor resolution and broad peaks, particularly for the conalbumin and soybean trypsin analyte peaks. However the authors noted that neither the polymerization reaction nor the modification of the monolith with DEAE was fully optimized. [PERSON] _et al._[154] used a methacrylate column and inter-particle methacrylate monoliths to separate a protein mixture composed of insulin, trypsin, BSA and lactoferrin, and compared the performance of the prepared monoliths for this mixture, against the commercial Chromolith(tm) Silica-C\({}_{18}\) columns which are often used as the standard monolithic media for performance comparisons. The prepared monoliths showed better separations than the commercial ones and exhibited shorter retention times. A number of different monolith columns were used, and the hybrid inter-particle monoliths showed improved performance compared with the \"whole-volume\" monoliths, yet in general, most exhibited relatively poor performance by modern standards. However, a markedly different picture is reported some six years later using a more conventional monolith. [PERSON] and co-workers [85] prepared lauryl methacrylate based monoliths modified with either C\({}_{12}\) or sulfonate groups. These were then used to facilitate the reverse phase separation of a mixture of proteins, as shown in Figure 11. The resolution achieved was reasonable, almost baseline however difficulties with separating smaller molecules were noted. [PERSON] _et al._ examined the effect of polymerization time on the retention times and separation efficiencies of methacrylate polymers modified for cation exchange and found that increasing the polymerization time led to longer retention times but that the back pressures did not vary appreciably [138]. However, the morphologies of the monoliths did vary quite dramatically which would be expected to contribute to altered separation characteristics. Anion exchange mediated separation was reported by [PERSON] _et al._[44]. Strong and weak anion exchange separations of acidic proteins were achieved, with reasonable resolution, in times of 20-30 min. Amine functional groups were used to affect the anion exchange. Cation exchange via vinyl sulfonic acid moieties on a poly ethylene glycol di-acrylate monolith were reported by [PERSON] and co-workers [136]. Strong cation exchange was observed, with near--baseline resolution and separation times of 30-40 min reported. It was noted by the authors that tuning of the level of hydrophobicity in the polymer is crucial in order to minimize non-specific interactions. The linker molecule and the backbone polymer chain are both important factors in this regard. Proteins have also been separated using immuno-affinity [61], HIC [43], and Size Exclusion Chromatography (SEC) [155]. Most recently, [PERSON] _et al._[130] used methacrylate columns as stationary phases for Normal phase, reversed phase, cation exchange, HIC, and HILIC type chromatographic separations for a number of different types of analytes, including proteins. A protein mixture of BSA, Ovalbumin, Myoglobin, Lysozyme and Cytochrome C were well separated within 40 min, albeit using an acetonitrile/trifluoroacetic acid based linear gradient elution. [PERSON] _et al._[155] investigated the effect of column length and diameter of a Poly Ethylene glycol-Methyl Ether Acrylate based monolith on the size exclusion based separation of protein mixtures. They found that increasing column length improved separation and resolution, but also increased the separation time.