# Methacrylate Polymer Monoliths for Separation Applications
## 3 Applications of Methacrylate Polymer Monoliths in Separation Science
### Biological Separations and DNA Purification
#### 3.2.1 Amplification and Pre-Concentration of DNA, Plasmids, Viruses and Peptides  
In this section the use of methacrylate monoliths for the enrichment, purification and separation of DNA and peptides are presented. The DNA is generally in the form of plasmid or genomic DNA in these applications. The separation and purification of plasmids has significant usage within the pharmaceutical industry (in the production of plasmid vaccines and plasmid based therapies) and therefore on the improvement of patient health. Consequently, there has been a significant amount of investigation into the purification of plasmid DNA carried out by many research groups, as discussed below [141; 142; 143; 144; 145]. Some Good Manufacturing Practice (GMP) validated methods have also been reported [140; 146]. A comprehensive review of the extraction and purification applications of polymer monoliths with an emphasis on biomolecular species was published by [PERSON] and [PERSON] [147] and the reader is directed to this article and the references contained therein for further detailed information on this area, as well as a more recent review published in 2012 [148]. The plasmid nucleotides are negatively charged; therefore, it follows that anion exchange chromatography has been a popular technique in the enrichment of plasmid DNA. As discussed previously, DEAE and amine groups can be used as effective anion exchange functionalities on methacrylate monoliths. Plasmids are generally much larger and exhibit much lower diffusion efficiencies than proteins do. Therefore, they bind to more sites than proteins, meaning lower binding efficiencies for polymer monoliths. Initially such amplification work was focused on small (<10 kb) plasmid samples [141]. More recently, the process has been seen to be scalable and reproducible [142],. In this latter work, of the cell genetic material 99% of the genomic DNA, proteins and RNA were removed from the mixture, leaving the highly purified plasmid DNA behind [149]. This was achieved using Hydrophobic Interaction Chromatography (HIC) and anion exchange functionalities on the same monolith, indicating the usefulness of multi-modal chromatography (MMC) for this application. Larger plasmids have also been investigated as in the case of a 62 kb plasmid, which was purified using a commercial CIM(tm) monolith with DEAE functional groups [150]. Enrichment factors of ~100 were obtained, and the authors noted that the column loading was much lower than the maximum possible loading, indicating scope for further improvement of the purification factor. The effect of plasmid size on the binding efficiency of the monolith was studied by [PERSON] _et al._[151] using a glycidyl methacrylate-\(\alpha\)-ethylene di-methacrylate monoliths which in some cases were grafted with carbonyl di-imidazole functional groups. A number of different plasmid sizes were investigated in this work, of around 2, 6, 10 and 14 kbps. All of these plasmids were resistance or R plasmids, conferring antibiotic resistance to a bacterial cell should they be present in it. They found that all the plasmids could be separated using the same procedure, and that the flow rates used in the study did not have a strong effect on the dynamic binding capacity, a feature previously noted for polymer monoliths. The monoliths were seen to have lower binding capacities for the larger plasmids. In addition, pH was seen to be an important factor, at lower pH, interactions between the phosphate groups in the plasmid DNA and the carbonyl di-imidazole groups. Critically, the structure of the plasmid DNA was not affected by the separation procedure, at lower pH values. [PERSON] _et al._ also investigated the purification of small plasmids with the intention of possible scaling up of the process. [146]. The DEAE modified CIM(tm) monoliths showed better binding capacities, larger pore sizes and better recovery of the pDNA. The 800 mL monolith exhibited 2000 mL/min flow rates under nominal back pressures and showed high throughput for DNA purification.