An investigation of glycidyl methacrylate terpolymers for mixed mode LC separations

Enhancing efficient separation techniques for use in proteomics or metabolomics is a growing area in recent years. Monolithic columns, as innovative separation materials have gained more attention and acceptance for proteomic and metabolic separation as alternatives to conventional packed columns. Moreover, monolithic columns have advantages over conventional columns such as fast and easy modifications, no requirement for retaining frits, ability to improve analyte mass transfer, lower back pressure due to high porosity, and broad selectivity. Five monomers were used to prepare the monolithic columns alongside glycidyl methacrylate, styrene, 2-(diethylamino)ethyl methacrylate, butyl methacrylate, lauryl methacrylate, and stearyl methacrylate. Ethylene glycol dimethacrylate (EDMA) was used as cross-linker, while 2,2-dimethoxy-2-phenylacetophenone was used as initiator. The v/v ratio between the two monomers has been investigated, while the ratio between the monomer to cross-linker was not changed. Three ratios (90:10, 50:50, and 10:90)% between the two monomers were used to prepare monoliths with appropriate surface area and pore size that can be used to separate small and macro molecules. The results indicated that the glycidyl methacrylate-co-stearyl methacrylate-co-ethylene glycol dimethacrylate monolith was formed at all the ratios tested, yet (30:70, 20:80, and 10:90) ratios that gave higher average surface area compared to other ratios. The average surface area was between 19.5938-21.0283 m2g-1, and the average pores size was 5.12, 4.80, and 4.2 nm for (30:70, 20:80, and 10:90)% of GMA:SMA respectively. Correct formation of the monolith was proven using different techniques such as FTIR, and 1HNMR.Mixed mode properties were investigated by ring opening the epoxy group of glycidyl methacrylate to form free hydroxyl functions to give HILIC/revers phase functionality and by converting the epoxy groups to sulphonate groups to give cation exchange/revers phase properties. The effect of several porogenic solvents and the irradiation time on the monolith formation were investigated to obtain even higher surface areas. The best results were obtained using a50:50 v/v ratio of 1-propanol to methanol with an irradiation time of 23 minutes. The monolithic column produced gave average surface areas of 73m2 g-1and was tested for the separation of proteins, however, base line separation could only be achieved with the strong cation exchange/revers phase monolith. Base line separation was obtained with all proteins samples tested, depending on the pI value of each protein, also, with peptides, hydrophobic compounds, and a mixture of hydrophilic and hydrophobic compounds, which was a better result compared to the HILIC/RP monolithic columns. The strong cationic exchange/ reversed phase monolithic columns were also prepared inside a glass microchip device and used for LC separation. The result showed base line separation was obtained forpharmaceutical and hydrophobic compounds