# Microsphere Polymers in Molecular Imprinting: Current and Future Perspectives
## 3 Preparation of Molecularly Imprinted Microspheres  
### Precipitation Polymerisation  
Precipitation polymerisation is one of the polymerisation methods that are widely used in the preparation of MIMs. [PERSON] et al. [40, 41] stated that uniform MIP microspheres could be produced using precipitation polymerisation under a very dilute conditions, but this requires a greater amount of solvent than that used in the conventional method (approximately \(<\)5% [6] of the total volume of monomer loading relative to the solvent), producing an average particle diameter around 0.2-0.3 \(\upmu\)m.  
The mechanism of precipitation polymerisation was proposed by [PERSON] and [PERSON] [121], where initiation takes place in a homogenous solution containing monomers, crosslinkers, initiators and solvent. In conventional precipitation polymerisation, polymer formation starts by forming nuclei and oligomers. As these oligomers grow, they will begin to crosslink. Instead of overlapping with one another, the polymer chains grow individually by adding newly formed oligomers and monomers from the solvent. The chain length grows until it exceeds its solubility and eventually precipitates from the solution with uniformly spherical morphologies [41] (Figure 1). As reported previously [40], conventional MIPs have an irregular shape and size, while MIMs produce spherical microgels with a narrow size distribution. The formation of MIMs is dependent on various factors. For example, to produce larger particles, adjusting the solubility parameter of the growing polymer is critical, notably when the goal is simultaneously the control of polymer morphology. The monomer concentration, solvent mixture and method of agitation also play roles in the development of MIMs [6, 51]. Polymerisation of divinylbenzene (DVB) or trimethylolpropane trimethacrylate (TRIM) as the crosslinker in acetonitrile together with azobisisobutyronitrile (AlBN, initiator) is known as the basic procedure of precipitation polymerisation, and thus has been widely studied with various templates [40, 41, 70, 121]. Polymerisation is induced by either thermal or UV irradiation. The microspheres can be readily obtained through centrifugation [40]. In precipitation polymerisation, the morphology of the polymer is not significantly affected by the presence or absence of a template [6, 52, 122], but this has an impact on particle growth [70]. As mentioned above, in this case, the precipitation of the chains is caused by crosslinking. This approach requires no stabiliser or surfactant, which is beneficial in molecular imprinting, since these compounds can disrupt template-monomer complex formation. However, the fact that microsphere formation occurs in very dilute solutions (using solvent \(>\)95 wt.%) makes this technique economically unfavourable and leads to environmental pollution [51, 52]. This problem led [PERSON] et al. [52] to develop modified precipitation polymerisation (MPP), which needs a smaller amount of solvent (about 50 wt.%). This method uses a mixture of toluene and alkane (mineral oil) instead of the normal non-proton solvents (e.g., acetonitrile, chloroform, toluene). The morphology of particles was controlled by the volume ratio of mineral oil to toluene. MIMs obtained via conventional precipitation polymerisation and MPP had similar selectivity, in terms of imprinting and separation factors. However, the concentration of the crosslinker affects the binding capacity and selectivity. Some researchers [88, 109, 121] have stated that MIMs prepared using the minimum concentration of crosslinker have the highest binding capacity and selectivity, but excess could reduce stability due to the increased solvency of the continuous phase and particle swelling. A study conducted by [PERSON] et al. [88] showed that agitation has an influence on MIM production by MPP. MIMs were achieved only without agitation, which is different from earlier precipitation polymerisation methods in which MIMs could be produced with or without agitation.