# Microsphere Polymers in Molecular Imprinting: Current and Future Perspectives
## 3 Preparation of Molecularly Imprinted Microspheres
### Suspension Polymerisation  
Suspension polymerisation can be used to replace bulk polymerisation with easy operation and avoiding the grinding and sieving steps [118]. MIMs are mostly prepared in organic solvents. It is hard to recognise the template on MIMs in a polar solvent with aqueous suspension polymerisation. Water or a polar solvent can weaken the non-covalent interaction between the monomer and the template. Based on research by [PERSON] [110], the components needed to make MIMs using the suspension polymerisation method are molecular templates that are drugs or compounds that will be targeted for research, and monomers to be formed as polymers. Several studies have separated drug compounds using methacrylic acid (MAA) as a monomer (see Table 1) [80,120], and non-polar solvents as porogens, crosslinkers and initiators. Substances that play a major role in this method are suspension agents that are mixed with the disperse phase. Certain compounds, such as retinoic acid, were tried by [PERSON] [140], but require catalysts such as triethylamine. Suspension polymerisation uses a two-phase system, i.e., a disperse phase and a continuous phase or dispersing phase (Figure 6). The disperse phase contains the template molecule, monomer, crosslinker, initiator and porogen, while the dispersing phase contains water and the dispersing agent. The template molecule and functional monomer are dissolved in the porogen. The crosslinker and the initiator are added to this mixture and sonicated to dissolve. For the dispersing phase, the dispersing agent is dissolved in water at 60 \({}^{\circ}\)C under a nitrogen atmosphere in a flanged reactor flask with a mechanical stirrer, reflux condenser, nitrogen inlet and dropping funnel. The mixture has to reach room temperature before being admitted to the flask at 400-600 rpm under a gentle stream (about 60 bubbles per minute) of nitrogen (99.99%). The temperature is then raised to 60-70 \({}^{\circ}\)C for the polymerisation process for 24 h. The microspheres have to be washed with a combination of distilled water, methanol and acetic acid [110,120], ethanol [80] or acetone [140]. The ratio between monomers and molecule template must be adjusted--too many or too few monomers will affect the affinity and selectivity of the polymer formed; this was examined by [PERSON] [120] with five different monomer to molecule target ratios of 2:1, 3:1, 4:1, 6:1 and 8:1. The optimum ratio that showed specific binding of chloramphenicol as the molecule template was 4:1, with the highest recovery of 81.6%, while the MIMs with the same ratio were lower at 28.5%. The specific adsorption recovery for each ratio, i.e., 2:1, 3:1, 4:1, 6:1 and 8:1, was 33.5%, 25.9%, 53.1%, 20.1% and 17.6%, respectively. The reason for the adjustment ratio for the monomer to molecule template has been explained [81]. A lower monomer to template molecule ratio means that the absorption capacity is worse because the insufficient amount of monomers leads to less complexation to the binding site, whereas a higher monomer to template molecule ratio leads to worse imprinting performance, because excessive binding sites cause more non-selective binding with the template molecule. Figure 6: The mechanism of suspension polymerisation. The effect of the amount of surfactant or suspending agent on the size and distribution of particles has also been assessed. A higher amount of surfactant produces smaller average particle diameters and a narrower size distribution [140]. Gluten and PEG 4000 at 4%, and PVA1788 at 2%, 4% and 6% as a dispersant or suspending agent, were tested by [PERSON] [120]. Different types of dispersants could be seen from the formation of agglomerates. With the use of PVE, a few agglomerates formed, while the use of gluten led to agglomerate blocks, and PEG 4000 formed many agglomerates. Different concentrations of dispersants result in different particle sizes. PVA1788 at 2%, 4% and 8% formed particles with diameters of 120, 80 and 40 \(\upmu\)m, respectively.