Synthesis of FCS

First step: Perfluoroheptanoic acid (PFCA) was dissolved in DMSO and then mixed with EDC and NHS under stirring for 0.5 hours in the dark at room temperature to obtain activated perfluoroheptanoic acid (the molar ratio of perfluoroheptanoic acid, EDC, and NHS was 1:2:1). 

Second step: CS was dissolved in 1% acetic acid. The feeding ratios between CS and PFCA were 100:0, 100:15, 100:20, 100:25, 100:30 and 100:35. For example, the feeding ratio between CS and PFCA was 100:25, and the reaction weight of the CS was 200 mg, with the molarity was 1.24 mmoL. Then, the relevant molarity of PFCA was 0.31 mmoL, and the activated per-fluoroheptanoic acid was dropwise added to the CS solution under stirring in the dark for 20 hours.

Third step: After stirring, 1 M HCl was directly added to the reaction system and stirred for another 0.5 hours. The excess anhydrous ethanol was added to precipitate the FCS-HCl product. Then, the product was washed and vacuum-dried (30°C, overnight).

Last step: The degree of substitution of conjugated fluoroalkyl substituents was characterized by ninhydrin colorimetry and fluorine element analysis.

Ninhydrin hydrate reacts with an amino group, loses dimolecular water, then decarboxylates, generates imine, hydrolyzes to aminopindandione, then loses water with a molecule of ninhydrin hydrate, and then tautomerism, that is, gets a purple compound. Here, amino-substitution rate was measured by ninhydrin method

Firstly, the solution of the ninhydrin was prepared (85 mg hydrindantin, 15 mg ninhydrin, 10 mL ethylene glycol monomethyl ether). And the acetate - sodium acetate buffer solution (0.2 mol/L, pH=5.4) was also prepared.

Secondly, the standard curve was prepared. The concentration of CS with the above buffer solution were 0, 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2 mg/mL. what’s more, the FCSs at 0.1 mg/mL were also prepared. The volume of samples was 200 μL, and then added 200 μL the solution of the ninhydrin with 200 μL H₂O.

Thirdly, the samples above were moved to metal bath at 100°C for 10 min. After samples cooling, we added the 600 μL ethyl alcohol solution (ethyl alcohol : H₂O = 6:4).

Last step, the samples above were tested the absorption at 570nm. Then the amino-substitution rate of FCSs was calculated with the standard curve of CS.