| Some simple, rapid and sensitive techniques of molecular spectrum analysis for the direct determination of chondroitin sulfate have been developed by investigating the interactions between chondroitin sulfate and cationic surfactants or cationic dyes using resonance Rayleigh scattering (RRS), frequency-doubling scattering (FDS), second-order scattering (SOS), fluorophotometry, and spectrophotometry in this paper. This work is a supplement of the methods for the determination of chondroitin sulfate and an expansion of the application scope of the RRS, FDS, and SOS technology.The main contents are as follows:1. Resonance Rayleigh scattering method for the determination of the sodium chondroitin sulfate with cetyldimethyl benzylammonium chlorideThe resonance Rayleigh scattering (RRS) intensity of chondroitin sulfate (CS) or cetyldimethyl benzylammonium chloride (CDBAC) is weak while it will enhance greatly when CDBAC and CS form an ion-association complex in pH 6.50 Britton-Robinson (BR) buffer solution. The RRS spectrum of the CDBAC-CS system appears with the maximum RRS peak at 315 nm. The RRS spectral characteristics, the optimum conditions and the relation between RRS intensity and concentration of CS have been investigated and a simple, accurate and rapid method for the determination of CS was developed. The linear regression equation wasΔI= 20.08+ 291.1 C(μg/mL ). The correlation coefficient ( r ) was 0.9992. The linear range was 0.10～10.00μg/mL. The detection limits (3σ, n=10) was 0.045μg/mL. The RSD was 4.5%. This is a simple, accurate and rapid method used in the determination of CS in chondroitin sulfate injection samples.2. Resonance Rayleigh scattering spectral characteristic of the interaction of the interaction of sodium chondroitin sulfate with methyl violet and its analytical application A simple, accurate and rapid method for the determination of CS was developed. The intensity of RRS resulting from the ion-association complex formed between CS and methyl violet (MV), a basic triphenylmethane dye, in pH 9.37 BR buffer can be greatly enhanced. The RRS spectrum of the MV-CS system appears with two large RRS peaks at 505 nm and 661nm, respectively. The optimum conditions for the MV-CS interaction and the relationship between the relative intensity of RRS and the concentration of CS have been investigated in detail. The linear ranges of the calibration curves for CS at 505 nm and 661 nm were both 0.15～0.90μg/mL. The detection limits were 0.019μg/mL at 505 nm and 0.043μg/mL at 661 nm, respectively. It has been applied for the determination of CS in chondroitin sulfate injection samples with satisfactory results.3. Resonance Rayleigh scattering study on the interaction of sodium chondroitin sulfate with acridine yellow and its analytical applicationAcridine yellow (AY), an acridine fluorescence dye, exists as a HR+ cation which can form an ion-association complex with CS anion in pH 7.96 BR buffer by virtue of electrostatic and hydrophobic interaction force. This interaction resulted in absorption of light decrease and fluorescence quenching, whereas RRS intensity enhancement of the system. The spectral characteristics of AY-CS system, the optimum conditions of the reaction, the influencing factors and the relationship between the relative intensity of RRS and the concentration of CS have been investigated in detail and a new method for the determination CS by RRS technology has been proposed. The linear ranges of the calibration curves for CS were 0.05～1.00μg/mL (300 nm) , 0.015～0.10μg/mL (490 nm), 0.15～1.50μg/mL (508 nm) and the detection limits were 0.034, 0.008, 0.019μg/mL respectively.4. Frequency doubling scattering and second-order scattering technology as new methods for the determination of the sodium chondroitin sulfate with cetyldimethyl benzylammonium chlorideThe combination reaction of CS with CDBAC in the near neutral medium has been studied using the FDS and SOS technology. The results show that the binding of CS with CDBAC in the near neutral media can result in a significant enhancement of FDS and SOS intensities. Their maximum scattering wavelengths,λex/λem, appear at 785nm/398nm for FDS and 240nm/485nm for SOS, respectively. The linear range was 0.30～12.00μg/mL and the detection limit(3σ) was 0.10μg/mL for FDS. While for SOS technology, the linear range was 0.30～7.50μg/mL, the detection limit (3σ) was 0.11μg/mL. The FDS technology has been applied to the determination of CS in chondroitin sulfate injection samples with satisfactory results.5. Study on frequency doubling scattering and second-order scattering spectra of chondroitin sulfate-cetyltrimethylammonium bromide systemThe binding of CS with cetyltrimethylammonium bromide (CTAB) in pH 7.00 BR buffer can result in significant enhancement of FDS and SOS intensities. Their maximum scattering wavelengths,λex/λem, appear at 780 nm/395 nm for FDS and 240 nm/485 nm for SOS, respectively. The linear ranges was 0.40～20.00μg/mL and the detection limit (3σ) was 0.14μg/mL for FDS. The linear range was 10.00～24.00μg/mL and the detection limit (3σ) was 0.33μg/mL for SOS. FDS was better than the SOS technology. The FDS method was developed and applied to the determination of CS in chondroitin sulfate injection samples with satisfactory results.6. Fluorescence quenching method for the determination of sodium chondroitin sulfate with acridine orangeIn pH 6.00 BR buffer solution, CS will dissociate to become a macro anion, which can react with acridine orange (AO), an acridine fluorescence dye, to form an ion-association complex resulting in fluorescence quenching of AO. Fluorescence quenching method was developed based on the fluorescence quenching character. The maximum fluorescence quenching wavelength was 547 nm (λex = 502 nm). The fluorescence quenching value was directly proportional to the concentration of CS in the range of 0.20～1.00μg/mL and the detection limit: (3σ) was 0.19μg/mL. The method can be applied to the determination of CS in chondroitin sulfate injection samples. |
