Carboxymethyl Chitosan and Its Hydrophobically Modified Derivative as pH-Switchable Emulsifiers

The emulsification properties of carboxymethyl chitosan (CMChi) and hydrophobically modified carboxymethyl chitosan (h-CMChi) were studied as a function of pH and dodecane/water ratio. The pH was varied between 6—10, and the oil/water ratio between 0.1—2.0. In CMChi solution, the emulsion stability increased as the pH was lowered from 10 to 7, and the phase inversion was shifted from oil/water ratio 1.0 to 1.8, respectively. The system behaved differently in pH 6 due to the aggregation of CMChi and the formation of nanoparticles (∼200—300 nm). No phase inversion was observed and the maximum amount of emulsified oil was reached at oil/water ratio 1.2. The h-CMChi showed similar behavior as a function of pH but, due to hydrophobic modification, the phase inversion was shifted to higher values in pH 7—10. In pH 6, the behavior was similar, but the maximum amount of emulsified oil was higher compared to CMChi. The amount of adsorbed particles correlated with the emulsified amount of oil. Reversible emulsification of dodecane was demonstrated by pH adjustment using CMChi and h-CMChi solutions. The formed emulsions were gel-like, suggesting particle–particle interaction.


Synthesis of carboxymethyl chitosan in sodium salt form (CMC-Na)
One gram of chitosan, 1.35 g of NaOH, 6 ml of water, and 24 ml of 2-propanol were mixed and stirred in a round bottom flask (100ml) at 50 °C for one hour. Then, 1.5 g of chloroacetic acid dissolved in 2 ml of 2-propanol was added dropwise into the mixture while stirring. After 4 hours, the solid product was separated by centrifuging and washed thoroughly with ethanol (70v-% and 100v-%) and dried in room temperature on a watch glass over-night. The dried CMC-Na was purified by dissolving in 50 ml of water and separating the insoluble fraction by centrifugation. The CMC-Na was precipitated by slowly pouring the liquid phase into 400 ml of ethanol (100v-%). The precipitate was washed thoroughly with ethanol (70v-% and 100v-%) and separated by centrifuging. The product was dried as previously described and ground into a fine powder. The product was in sodium salt form (CMC-Na) due to the alkaline reaction medium.

Synthesis of hydrophobically modified carboxymethyl chitosan in sodium salt form (h-CMC-Na)
Portion of the purified and dried CMC-Na (750 mg) was dissolved in 42 ml of water. After the dissolution, 30 ml of ethanol (100v-%) was added slowly into the polymer solution while stirring. Dodecanal (30 µl) was added corresponding to approximately 5% substitution degree. The mixture was stirred for 20 minutes, NaCNBH 3 (30 mg) was added, and the stirring was continued for 20 h in room temperature. The reaction mixture was poured slowly into 400 ml of ethanol (100v-%) to precipitate the h-CMC-Na. The precipitate was separated by centrifuging and washed thoroughly with ethanol (90v-% and 100v-%). The product (h-CMC-Na) was separated by centrifuging, dried, and ground as previously described.

S3
Characterizations of CMC-Na and h-CMC-Na

FTIR analysis
The FTIR spectra of native chitosan, CMC-Na, and h-CMC-Na are presented in Figure S1. Figure S1. FTIR spectrum of native chitosan, CMC-Na, and h-CMC-Na. Figure S1. shows the FTIR spectrum of the chitosan used in the synthesis of CMC-Na and h-CMC-Na, and their FTIR spectra, respectively. In the spectra of CMC-Na and h-CMC-Na, a broad band appears at 1600 cm -1 which is due to overlapping of -COONa (1598 cm -1 ) and -NH 2 (1592 cm -1 ) bands [1,2]. In CMC-Na and h-CMC-Na the carboxyl groups are in sodium salt form due to the alkaline pH during the synthesis. Therefore, the band of -COOH group is not detected at around 1720 cm -1 [3]. The spectra of CMC-Na and h-CMC-Na are almost identical due to the low substitution degree of hydrophobic modification, and show successful carboxymethylation of chitosan.

H 1 NMR analysis
The 1 H NMR spectra of CMC-Na and h-CMC-Na are presented in Figures S2 and S3, respectively. The spectrum of h-CMC-Na shows the similar characteristic proton signals as can be seen in CMC-Na spectrum which was assigned according to our previous studies [4]. In the spectrum of h-CMC-Na, a new triplet with a typical coupling constant (J = 7.0 Hz) can be found at 1.26 ppm. The peak is assigned to CH3-group of dodecanyl subtituent (marked with a in Fig. S3). Also, a clear signal can be seen at 1.68 S4 ppm which is assigned to nine CH2-groups of dodecanyl chain (marked with b in Fig. S3). The signals of rest two CH2-groups can be hardly observed at 2.13 and 3.32 ppm. These peaks are assigned to protons c and d next to the nitrogen atom. In summary, the performed 1H NMR analysis clearly indicates the successful attachment of dodecanyl substituent to CMC-Na.    Figure S3. 1 H NMR spectrum of h-CMC-Na.