# 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
Some data from the report is shown in Figure 12. However, the effect of internal diameter appears inconclusive. More systematic data is required in order to fully explain the effect or lack thereof, of the column diameter, whether this is a one off result or something altogether more interesting. If column diameter does not play an obvious role in such separations at these diameters  
Figure 11: Reverse Phase separation of protein mixture on two different Lauryl methacrylate monoliths, (**a**) fabricated using photo-initiated polymerization and (**b**) fabricated via thermally initiated polymerization. Peaks are: (1) impurity; (2) ribonuclease A; (3) Cytochrome C; and (4) Myglobin. Reproduced from [85] with permission from The Royal Society of Chemistry. then what is the range of values at which this continues to be the case? A design of experiments where the length is varied with diameter kept constant and vice-versa would have been a useful exercise in order to better understand this result. The monolith was reported to exhibit better separations than a packed column of the same diameter, the interesting point to note is that the monolith was significantly shorter than the column (17 cm _vs._ 57 cm). He _et al._[70] investigated the separation of protein mixtures via electrophoresis, using hydrophilic and hydrophobic monomers (hydroxyl-ethyl methacrylate and butyl methacrylate respectively), in the polymer mixture. Baseline separations were reported, using the hydrophilic monomer-containing monolith. Yeast [156] and, more recently, whey [157] proteins have also been separated. In the case of the yeast protein mixture, a multi-dimensional separation setup was employed, involving strong cation exchange, followed by reverse phase chromatography. The separation took a relatively long time (12 h) to complete. Amino acids have also been investigated on methacrylate monoliths. Two particular examples are discussed here. The first is the separation of a series of dansylated amino acids on an ethyl  
Figure 12: SEC separation on methyl ether acrylate monolith using different column lengths (**A**) and diameter (**B**). Mobile phase was 20 mM phosphate with NaCl. Key: TG: Thyroglobulin; BSA: Albumin; STR: Trypsin Inhibitor; ANG1: Angiotensin 1; LE; Leucine encephalitis. Reprinted with permission from [155], Copyright (2009), American Chemical Society. methacrylate based monolith which was cured using electron beam (e-beam) curing [34]. Electron beam curing involved a linear accelerator, and irradiation with electrons to a dose of 22 kGy (kiloGrays) was performed which initiated the formation of the free radicals necessary for the polymerization step. Again, the comparison was made with commercial monoliths by using Chromolith(tm) (Silica-C18, Merck, Darmstadt, Germany) as the standard. The e-beam curing method was found to be an easy procedure, and amenable to large diameter monoliths. It would be interesting to investigate the uniformity of the porosity of monoliths fabricated using this method, and in particular to see if they exhibit the same variation at the wall as observed in methacrylate monoliths produced from thermally induced polymerizations. The second example of amino acid separation is a separation carried out on a microfluidic device. In recent years, the integration of separation and sensor media into microfluidic devices has garnered interest. The reasons for the increased interest are the lower analyte and solvent volumes required, the potential for autonomous, remote sensing, testing at point of analysis, instead of having to remove the sample to the lab, and the development of rapid, cheap, and disposable devices. In this second example [158] an acrylate monolith was fabricated in a silica capillary which was then integrated into a Poly Di-Methyl Siloxane (PDMS)/Glass chip.