Study on Frequency Doubling Scattering and Second-Order Scattering Spectra of Heparin-Methylene Blue System

Binding of heparin with methylene blue (MB) in pH 5.7 Britton Robinson buffer can result in a significant enhancement of frequency doubling scattering (FDS) and second order scattering (SOS). Their maximum scattering wavelengths ( λ max ) appear at 350 nm for FDS and 700 nm for SOS, respectively. The optimum conditions of the reaction, the influencing factors and the relationship between the two scattering intensities and the concentration of heparin have been investigated. The new methods for the determination of trace amounts of heparin based on the FDS and SOS methods have been developed, which exhibit high sensitivities. The detection limits of heparin are 4.36 ng/mL for the FDS method and 3.55 ng/mL for the SOS method, respectively. Both of the methods have fairly good selectivity and were applied to the determination of heparin in sodium heparinate injection samples with satisfactory results. Moreover, the relative mechanisms have also been discussed.