# Less Is More: Influence of Cross-Linking Agent Concentration on PFOS Adsorption in Chitosan
## 3 Results and Discussion
### Varying the ECH Concentration During Synthesis  
Those differences in the polymer structure revealed by SEM between the cross-linked and non-cross-linked chitosan then led us to investigate the influence of the cross-linker concentration on the PFOS adsorption. Therefore, four different chitosan polymers were synthesized using 0, 1, 2, or 4% (v/v) epichlorohydrin (**ECH0**, **ECH1**, **ECH2**, **ECH4**) solutions in the cross-linking step, respectively. The results of those adsorption experiments are presented in Figure 4.  
While the non-cross-linked chitosan **ECH0** showed the highest PFOS adsorption in the batch experiments after 72 h, this material is not acid-stable in contrast to the three other chitosan gels (see Supplementary Materials) and thus of limited use in real-life applications. The data further showed that a decrease in the used ECH concentration in the cross-linking step resulted in higher adsorption of PFOS. The Student's \(t\)-test showed significant differences between **ECH0** and **ECH2** as well as **ECH4** (\(p<0.05\), see Table S8). As there is no further decrease from **ECH2** to **ECH4**, it seems that already at 2% ECH concentration, all possible cross-linking connections are made. Thus, no further PFOS adsorption decrease occurs by adding more ECH during the cross-linking step It is known that higher cross-linking of chitosan can negatively impact the adsorption capacity of metal ions due to decreased availability of free amine and hydroxyl groups [21,38]. A similar correlation between ECH concentration and PFOS adsorption has not yet been investigated so far. The internal structure of the polymer **ECH1** observed by SEM (see Figure S29) is indistinguishable from the ones seen before in Figure 3b for the polymers produced by the 2% ECH solution. Considering that the internal structures are not visibly different,  
Figure 4: Influence of epichlorohydrin (ECH) concentration in the cross-linking step on the loading q of PFOS on the adsorber (dry mass). The data is also shown in Table S7. The error bars represent the standard deviation from three adsorption experiments. The asterisk above the line linking the two data sets shows significant differences between those according to Student’s \(t\)-test (\(p<0.05\)). 72 h adsorption time, 100 mg adsorption material, 100 mL solution.  
the decrease of PFOS adsorption with higher ECH concentration is likely explainable by a lower number of free amine and hydroxyl groups instead of a change in structure, as seen in Figure 3a.  
The chitosan samples synthesized for this series of experiments showed lower adsorption capacities than those used for the molecular imprinting experiments (see Figure 2). The reason for this could not be completely revealed. However, the first set of experiments (Molecular imprinting) was done with a different batch of chitosan, while all other experiments afterward were done with a newly delivered lot of chitosan after the first was completely used. Therefore, the later ones are all consistent with each other, but as is visible in the difference in Figure 2, not with the first one. In this set of experiments, only the difference between imprinted and non-imprinted was important, not the absolute value. Moreover, we also measured the BET (Brunauer Emmet Teller) surface areas of all four materials, but these findings were inconclusive, as no clear correlation between ECH concentration and surface area could be determined (see Table S5). [PERSON] et al. reported lower BET surface areas for non-cross-linked chitosan than for ECH cross-linked chitosan. However, instead of applying freeze-drying, their polymer samples were desiccated at elevated temperatures under vacuum, resulting in surface area sizes an order of magnitude lower than in this work [33]. It has been shown by [PERSON] et al. that the drying method determines the measured BET surface area [34].