## 3 Result and Discussion
### Optimization of the Particle Size and Porous Structure of the Microspheres  
As mentioned above, the significant influence of the stirring rate to the particle size was revealed. Moreover, when the time of the swelling process reduced to half (10 to 5 min), the particle size grew from 25 to 350 \(\upmu\)m (Figure 6) and the aggregations were prevented. Therefore, both the stirring speed and swelling time had significantly influenced the particle size (Table 3). Comparing the small droplets with the high surface energy intended to aggregate, the big droplet with lower energy was hard to aggregate. However, the pore size of the particle was only around 100 nm (less than one in a thousand of the particle size), and some of the big particles were hollow. Therefore, emulsion with a proper droplet size was needed.  
In order to control the particle size, the membrane emulsification method was used. When using the 50-\(\upmu\)m membrane, a large number of small particles formed, almost all of the small microspheres (about 15 \(\upmu\)m) aggregated to form larger aggregations (Figure 7), and the pore size of the microspheres was about 100 nm. Considering that the largest pore size of the commercial membrane is 50 \(\upmu\)m, a uniform screen with a hole size of around 300 \(\upmu\)m was used in the first step of emulsification to increase the size of the oil droplets. After polymerization, the microspheres had a particle size of about 20 \(\upmu\)m, and the aggregation was effectively inhibited (Figure 8).  
The pore size of the microspheres was about 90 nm (Figure 8c). Because the pore size was positively correlated to the amount of surfactant, we further optimized the content of Span 80 in the oil phase to control the porous structure. The pore characters with a different Span 80 is shown in Table 4. According to the table, when the content of Span 80 was 55% (based on the total amount of ST and DVB), the pore diameter was more than 150 nm (Figure 9, Table 4), which could satisfy the requirement of the separation and purification of large molecules. From the results, it can be concluded that the aggregation was reduced by utilizing the AIPS when excluding the effect of the small particles.  
Figure 6: (**a**) The SEM photographs of the microspheres with stirring speed 70 rpm, swelling time 5 min, (\(\times\)100) and (**b**) size distribution.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Stirring rate** & **Swelling time** & **Average particle** & **Uniformity of** & **Average pore** & **Yield (\%)** \\
**(rpm)** & **(min)** & **size (\(\upmu\)m)** & **particle size** & **size (nm)** & **Yield (\%)** \\ \hline
150 & 10 & 10 & 0.52 & 80 & 87 \\
70 & 10 & 25 & 0.33 & 130 & 91 \\
70 & 5 & 350 & 0.45 & 110 & 93 \\ \hline \hline \end{tabular}
\end{table}
Table 3: The relationship between stirring rate/ swelling time and particle size.  
Figure 8: The photographs of the polymer microspheres by screen emulsification (**a**) SEM (\(\times\)500); (**b**) size distribution; and (**c**) mercury porosimetry measurements.  
Figure 7: The photographs of the polymer microspheres of the (**a**) membrane emulsification (SEM \(\times\)500) and (**b**) size distribution (dispersed).