# Less Is More: Influence of Cross-Linking Agent Concentration on PFOS Adsorption in Chitosan
## 3 Results and Discussion  
### Molecular Imprinting on Chitosan  
The synthesized chitosan polymer used in this work differs from many other published forms in polymer shape. Due to the lack of machines and tools to make small (<5 mm) uniform chitosan beads, we decided to alter the synthesis protocol and focus on string-shaped chitosan polymers, as documented in the following. After dissolving chitosan in acetic acid, the resulting viscous mixture was quickly injected into the NaOH solution using a short syringe cannula. Subsequently, the strings were cut to an appropriate length (-1 cm) and used in the adsorption experiments. To check if the adsorption kinetics of the polymer strings were comparable to the work described by [PERSON] et al. [23], the adsorption at two different PFOS concentrations (0.1 & 0.01 mmol/L) at neutral pH was monitored at different times. The results showed a similar behavior regarding the adsorption kinetics as described by [PERSON] et al., with negligible adsorption happening after 48 h (see Figure S1). Therefore, for the following experiments, an adsorption time of 72 h was chosen to ensure no kinetic influence on the results.  
After confirming that the shape change did not affect the adsorption kinetics negatively, the next step was testing whether the molecular imprinting method used in an older work by [PERSON] et al. [23] improves the PFOS adsorption even more combined with their updated cross-linking protocol [24]. However, no significant difference in adsorption capacity between molecular imprinted and non-imprinted chitosan could be observed in the experiment (see Figure 2). As the difference between the two polymers was better visible at the higher concentration, a concentration of 0.1 mmol/L PFOS was chosen in the following experiments.  
In addition to this adsorption experiment, we also investigated the microscopic structure of the polymers via a scanning electron microscope (SEM) to investigate the influence of the cross-linking with and without molecular imprinting on the internal structure of the chitosan polymer (see Figure 3).  
Consistent with the adsorption experiment, there was no difference observable in the internal structures between fresh fracture edges of non-imprinted and imprinted polymers (Figure 3b,c). However, the pre-cross-linked polymer showed a noticeably different structure from those two in Figure 3a. While all three materials showed a homogenous micropore scaffold structure on the micrometer level, the cavities for pre-cross-linked chitosan were bigger on average. Additionally, there are membranes partly filling the pores of the scaffold structure. In the SEM images published by the [PERSON] group, the same structural motive as in Figure 3b,c can be seen below the collapsed outer surface layers as they likely did not create fresh fracture edges but looked at the outside layers of the chitosan gel after drying (which as shown later influences the morphology) [23]. Other reports on chitosan focused on the external surface of the particles rather than the internal structure, making a comparison with the internal structure found in our study difficult (e.g., [30; 31; 32; 33; 34; 35; 36]). There is, however, one work that showed an inner structure for ammonium hydroxide cross-linked chitosan. The results from this study were consistent with the inner structures shown in Figure 3b,c [37].  
Figure 3: Scanning electron microscopy (SEM) image of chitosan polymer: (**a**) before ECH cross-linking, (**b**) after cross-linking but without PFOS-based molecular imprinting (MIP), and (**c**) after cross-linking and with MIP. The Supplementary Materials contains the pictures with higher resolution.  
Figure 2: Comparison of PFOS sorption capacity between molecular imprinted and non-imprinted chitosan at two different PFOS concentrations. The inset Figure Shows the error bars for the 0.01 mmol/L PFOS experiment. The error bars show the standard deviation (\(n=3\)). 72 h adsorption time, 100 mg adsorption material, 100 mL solution.