The powder of 88% deacetylated chitosan (ACROS, ORGANICS) was dissolved into 1% acetic acid to form 3% chitosan colloid. In a 2 cm × 2 cm square container, 2 mL of 3% chitosan colloid was poured and was placed into an oven (OVEN 453, HSIANGTAI CO., Taiwan) to dehydrate for 24 hours to form a chitosan film. The chitosan film was solidified by slowly stirring in 1% sodium hydroxide for 30 minutes by using a magnetic stirrer (SP18425, Apogent Technologies). After drying at 30˚C, the chitosan film with a thickness of about 0.3 mm can be stored at 25˚C and 65% RH in a programmed temperature and humidity chamber.

By using a syringe, 2 mL of 3% chitosan colloid was dropped slowly into 1% sodium hydroxide and was solidified for 30 minutes. The solidified chitosan microsphere was then washed with deionized water and was dried at 30˚C in an oven. The dried chitosan microsphere was stored at 25˚C and 65% RH in a programmed temperature and humidity chamber. Measured by the MASTERSIZER, a Laser Diameter Analyzer (Hydro 2000SM, Malvern Instrument, UK), the size of each chitosan microsphere was about 1.2 ± 0.3 mm.

The 3% chitosan colloid was pumped with air to form bubbles and was homogenized at 30,000 rpm by using a high speed homogenizer (HG-300D, HSIANGTAI CO., Taiwan). In a container with a diameter of 1.5 cm and a height of 2 cm, 2 mL of the 3% chitosan colloid was dropped to fix the shape at low temperature, followed by solidifying in 1% sodium hydroxide for 30 minutes. The solidified chitosan scaffold absorbent was washed with deionized water and was dried by using a freeze dryer (SMR-100, FUYUAN CO., Taiwan). The dried chitosan scaffold was stored at 25˚C and 65% RH in a programmed temperature and humidity chamber. A SEM picture of the chitosan scaffold was shown in Figure 1 (6335F, JEOl, Japan).

In this experiment, five different dye samples (i.e., direct, reactive, acidic, disperse, azo dyes) were tested. In

Table 1, the chemical structures of the dyes were shown. Except for the azo dye, which was synthesized in the lab, all the other dyes were provided by the Plastoform Industries Ltd (Taiwan). About 10 mg of each dye was used to form a corresponding dye solution with a concentration of 0.1 mg/mL. About 2 mL of each dye solution was poured into a quartz test tube and was placed into a photospectrometer (V-670, JASCO). The wavelength was increased by 1 nm each time from 350 to 760 nm. The light absorbances of each dye solution at different wavelengths were recorded. A standard curve was formed for each dye solution according to the greatest absorbance and the corresponding wavelength. For a single dye, five different concentrations were prepared and 2 mL of each concentration was then poured into a quartz test tube to measure the absorbencies. According to the absorbance of different concentrations, a formula of the standard curve of each dye solution was formed. The greatest wavelength, the formula of the standard curve, and the R² value of each dye solution were shown in Table2

Three different forms of chitosan samples (i.e. film, microsphere and scaffold) were put into 0.05 g of each dye solution, respectively. The adsorptive properties of the samples were recorded daily and the time for saturated adsorption was noted for each sample. The chitosan sample with saturated adsorption was dissolved into 5% acetic acid. Using 5% acetic acid as the standard solution, the adsorbencies of the samples were recorded. The adsorptive rate of each sample can be calculated by using the formula:

C₁ is the adsorbance difference between the chitosan sample before dyeing and the chitosan sample dissolved into 5% acetic acid after dyeing.

C₂ is the adsorbance difference between the purified water and the dye solution.