Determination Chitosan Accurately By Spectrophotometric Methods And HPLC Method

ObjectiveTo develop the new quantitative analysis method to determinate the concentration of chitosan in functional food and other chitosan sample.MethodsChitosan protonized to NH4+ under the weak acid, then binding with anion dye to form a stable ion-association complex through electrostatic forces and hydrophobic interaction force. According to the change of the spectra of the ion-association complex, developed 5 resonance Rayleigh scattering method and a fluorescence quenching method to quantify chitosan, and optimize the experimental conditions; To explore the influence of molecular weight, and find the solution to solve the influence of chitosan molecular weight; To explore the influence of the multiple coexistence material and find the appropriate ionic masking agent; With the establishment of a new resonance Rayleigh scattering method and fluorescence quenching method for determining content of chitosan samples,and compared with the results determined by two method. Under nitrogen protection acid hydrolysis combined with UV-vis spectrophotometry, high performance liquid chromatography(HPLC) method for the determination of chitosan.Results1. Resonance Rayleigh-scattering method for the determination of chitosan with Ponceau 4R, Congo red as probeIn B-R buffer solution, chitosan combined with Ponceau 4R or Congo red that resulted in RRS significant enhancement. The optimum condition, the influence factor as well as relationship between the RRS intensity and chitosan were investigated. It found the RRS intensities were proportional to the certain range of chitosan.Therefore, we developed two new methods for determination the concentration of chitosan that has highly sensitive. The limit detection of chitosan- Ponceau 4R system is 0.019 μg/mL and chitosan-Congo Red system is 0.044 μg/mL. The chitosanPonceau 4R system cannot be effect by chitosan molecular weight on low concentration, and chitosan-Congo Red system totally cannot be effect by chitosan molecular weight. Applied these two methods in determination real sample got satisfy results.2. Resonance Rayleigh-scattering method for the determination of chitosan with Aniline Blue, Naphthol green B as probeIn weak acid condition, investigated chitosan react with Aniline Blue, Naphthol green B, developed two new Resonance Rayleigh-scattering methods that the intensities of chitosan-dyes ion association complexes enhanced significant under under water bath. The results showed that under optimum condition, Aniline Blue,Naphthol green B reacted with chitosan, formed ion association complexes and enhanced the RRS intensity significantly. In chitosan- Aniline Blue system, the intensities of RRS were linearly proportional to the concentration of CTS in the range of 0.01 to 3.5 μg/mL, and the limit of detection was 6.94 ng/mL; In chitosanNaphthol green B system, the intensities of RRS were linearly proportional to the concentration of CTS in the range of 0.01 to 5.5 μg/mL, and the limit of detection was8.87 ng/mL. Moreover, the RRS result of the different chitosan molecular weight had no statistical significance and that this method could accurately determine CTS when the molecular weight of the CTS sample was different than the CTS standard.Therefore, Aniline Blue, Naphthol green B as probe Resonance Rayleigh-scatteringmethod were simple, highly sensitive and accurate methods.3. Fluorescence quenching and Resonance Rayleigh scattering spectra of chitosan-Cibacron Brilliant Red 3B-A association system and its application for the determination of chitosanA novel method was presented for the determination of chitosan based on its quenching effect on the fluorescence of Cibacron Brilliant Red 3B-A in B-R buffer solutions. Under the condition of λEx/λEm=285/341 nm, the degree of fluorescence quenching of Cibacron Brilliant Red 3B-A was linearly proportional to the concentration of chitosan in the range of 0.050-2.00 μg/mL with the linear equation of the method ΔF= 68.78c+2.648(c: μg/mL)(R2=0.999 2)and detection limit of 0.039μg/mL. Moreover, chitosan-Cibacron Brilliant Red 3B-A ion-association complex showed significant Resonance Rayleigh scattering peak at the wavelength of 342 nm,and the intensity of Resonance Rayleigh scattering was directly proportional to the concentration of chitosan. In the range of 0.050~6.00 μg/mL, linearity equation of chitosan-Cibacron Brilliant Red 3B-A system was ΔI = 681.31c+114.95(c: μg/mL)(R2=0.999 1) with detection limit of 0.033 μg/mL. Therefore, two methods(fluorescence quenching and Resonance Rayleigh scattering) by using Cibacron Brilliant Red 3B-A as a probe had been established and validated for rapid determination of chitosan and its capsule. Meanwhile, it had been also investigated on the effect of the molecular weight of chitosan on its accurate determination. These two methods were applied in two real chitosan samples analysis, and there was no significant difference between the results from two methods with acceptable recoveries.4. Determination of chitosan with a modified acid hydrolysis under the atmosphere of nitrogen and HPLC methodThis paper investigated the optimum hydrolysis condition under the atmosphere of nitrogen, with glucosamine hydrochloride efficiency as indictor. TLC results were showed that the end product of hydrolyzed chitosan under the atmosphere of nitrogen was glucosamine hydrochloride, and the longer hydrolysis time, the higher efficiency.UV-vis results were stated that the optimum condition is 100 ℃ HCl solutionhydrolyzed 54 h. Compared the end productivity of Uv-vis method and HPLC method,the content of chitosan detected by HPLC method were significantly less than the results detected by UV-vis method, which might be related to the relative specificity of HPLC method and incompleteness of hydrolysis.ConclusionsCompared with five kinds of dyes combined with chitosan, found that five kinds of dyes are with multiple- SO3- groups and multiple rigid plane; Five new resonance Rayleigh scattering methods applied in determining the concentration of chitosan, the results have no significant difference; Resonance Rayleigh scattering method and fluorescence quenching method with Cibacron Brilliant Red 3B-A as probe applied in determining the concentration of chitosan, the results are basically identical; Put forward two methods of diminishing the effect of chitosan molecular weight;Compared the chitosan hydrolysis sample results determined by HPLC method and Uv-vis method, found that the hydrolysis efficient from HPLC method more lower,reflected the hydrolysis was not completely, and the hydrolysis method must be optimized further.