# Preparation of Freeze-Dried Porous Chitosan Microspheres for the Removal of Hexavalent Chromium
## 3 Results
### The Morphology of Porous Chitosan Micospheres  
#### 3.2.1 Effect of the Freezing Drying Method  
Figure 2 show the surface and sectional morphologies of the porous chitosan microspheres prepared using microneedles with a diameter of 800 \(\upmu\)m, and supported by two types of freezing-drying methods, which were freezing temperatures being maintained at \(-20\)\({}^{\circ}\)C for 24 h followed by lyophilizing (called freezing and lyophilizing, Figure 2a,b) and direct lyophilization without being frozen (Figure 2c,d). As shown in Figure 2a, when the concentration of chitosan solution was 2% and drying method was freezing-lyophilizing, chitosan microspheres were obtained, but the formed material exhibited a flat ellipsoid structure. There was also a mountain-like fold tabular structure on the surface of the chitosan material with a sponge-like stacked structure with plentiful gaps and a thin-walled interior. When the chitosan concentration was 3%, stereosphere structures were formed and traditional round pore-structures were found from the sectional morphologies of those microspheres. The morphologies of the porous chitosan microspheres obtained by direct lyophilization were quite different. Figure 2c,d shows a smooth and compact surface with a few cracks and a porous internal structure. Specifically, a low-magnification SEM image of the cross section revealed a highly concentrated internal structure with a few round pores, while a high-magnification showed a few large pores scattered among a large number of small pores with excellent connectivity. It was noticed that forming a dense and smooth surface and small pores in the interior of the chitosan microspheres was easier with direct lyophilization, while forming chitosan microspheres with rough surface structures and large pores, which were more suitable for the adsorption of materials to treat wastewater was easier by first freezing then lyophilizing [25]. These phenomena could be due to the fact that = ice crystals were formed during the freezing-drying process [25]. The water was frozen at sub-zero temperatures, and ice nucleuses were produced and then grew into ice crystals, which squeezed the chitosan chains into ice-crystal gaps. After the sublimation of ice, specific sponge-like structures are formed. There was no accumulation of chitosan nor obvious pores, which was because the ice crystal nucleation rate and growth rate were functions of both the heat and mass transfer rates [24; 28]. In other word, when the chitosan content was constant, the lower the temperature was, the faster the heat transfer rate was, and the more ice nuclei there were. Porous chitosan-based microspheres had relatively larger pores when the freezing temperature was higher according to previous studies [21; 22; 23; 24; 25]. For example, gelatin/chitosan/Ag porous sponge compound prepared by freezing at \(-80\)\({}^{\circ}\)C for 24 h had a dense pore structure with a pore size of about 100-250 \(\upmu\)m [21]. The pore diameter of chitosan-poly(vinyl alcohol)-methylcellulose porous scaffolds fabricated by freezing at \(-20\)\({}^{\circ}\)C for 24 h, however, was in the range of 200-500 \(\upmu\)m [22]. In this work, the temperature of \(-20\)\({}^{\circ}\)C was not sufficiently cold to produce enough ice crystal nuclei, so the few ice crystals that formed did not interfere with other crystals' growth and had enough space to grow into flakes. Larger pores appeared when the chitosan concentration was 3% (\(w/v\)), as shown in Figure 3a, which was due to the increase in chitosan concentration, resulting in an increase of ice crystal nuclei. This finding was consistent with previous results [24; 25; 28; 29]. On the other hand, direct lyophilization without freezing resulted in a compact and cumulate structure, which was due to the fact that no ice crystals were separated by self-crosslinking or the aggregated chitosan chain. After the evaporation of water droplets, a large number of stacked structures with small pores remained.