**2.2. Spectroscopic measurements**

Previous studies have shown that the best conditions to solubilize chitosan are: chitosan 1% (w/v) dissolved in aqueous solution of glacial acetic acid 1% (v/v) under stirring (Signini & Campana Filho, 1999; Rodrigues, 2005; Rodrigues et al., 2008), so measurements in presence of chitosan were conducted in these conditions.

Chitosan has no fluorescence emission or absorption in the experimental conditions. The absorption spectra of chemicals (fluoxetine, B2 and B12) were measured using quartz cuvettes with 1 cm of optical pathway. The fluorescence measurements were performed at excit= 275 nm and emis= 305 nm for vitamin B12 (Li and Chen, 2000); at excit= 440 nm and emis= 305 nm for vitamin B2 (United States Pharmacopeia, 2007; Association of Official Analytical Chemists, 2005); and at excit= 230 nm and emis= 290 nm for fluoxetine (United States Pharmacopeia, 2007; Association of Official Analytical Chemists, 2005).

Absorbance measurements were taken at maximum of the absorption spectra and performed at room temperature.

Initially, variations of both fluorescence and absorption spectra of the chemicals (fluoxetine, B12 and B2) were taken as a function of their concentration in acid aqueous solution and after in different concentrations of chitosan in aqueous acid solution, at the same range of chemicals concentration. The variation in the spectra of the chemicals (fluoxetine, vitamins B2 and B12) was also studied by keeping fixed the concentration of vitamin and varying the concentration of chitosan in acid aqueous solution).
