2.3. Synthesis of CMC

Carboxymethyl chitosan (CMC) was prepared following the method reported previously [23]: chitosan (10 g) and sodium hydroxide (15 g) were suspended in isopropanol (100 ml) to swell and alkalize at room temperature for 1 h. The monochloro-acetic acid (10 g) was dissolved in 20 ml isopropanol, and added to the reaction mixture drop-wise within 30 min and reacted for 4 h at 55 °C. Then the reaction was stopped by removing the reaction mixture from heat and discarding the isopropanol (Fig. 1). Ethyl alcohol (80%) was added and the solid product was filtered and rinsed with 80%~90% ethyl alcohol to desalt and dewater, and vacuum-dried at 50 °C. The product yield of CMC from the synthetic scheme was calculated by the following formula:

Synthetic routes of CMC (1. O-CMC, 2. N-CMC).

The degree of substitution (DS) of CMC was determined by pH titration. The accurately weighed CMC was dissolved in water, and then the pH was adjusted to less than 2 with hydrochloric acid (12 mol/l). The standard sodium hydroxide solution (1 mol/l) was used for titration. The amount of aqueous sodium hydroxide was determined by the second order differential method [19]. The DS can be calculated by the following equation:

where m_CMC is the mass of CMC (g), 161 and 58 are the molecular weights of glucosamine (chitosan skeleton unit) and a carboxymethyl group respectively, V_NaOH and C_NaOH are the respective volume and molarity of the aqueous sodium hydroxide solution for the titration [19].

Fourier transform infrared spectroscopy (FT-IR) was used to verify the chemical structure of the synthesized CMC. The measurements were carried out on a FT-IR (Perkin Elmer, Model 100). Samples were analyzed in the atmospheric environment, and the FT-IR curves of chitosan and CMC were acquired [19]. The synthesized CMC was characterized by using a Perkin Elmer PE2400 Series II CHNS/O analyzer.

Nuclear magnetic resonance (NMR) analyses were carried out as described previously [24]: 15 mg of the CMC sample was dissolved in 1 ml of deuterated water before analyzed using an AC-400 Bruker spectrometer at 353 K. Elemental analysis data were obtained using a Carlo Erba elemental analyzer. Samples were tested in both ¹H NMR and ¹³C NMR.