Studies On The New Methods For The Determination Of Colchicine, Resveratrol And Pepsin By Absorption, Fluorescence And Resonance Rayleigh Scattering Spectrometry

This dissertation mainly studied the effects of the interactions of colchicine and resveratrol with some dyes,inorganic acid,some amino acid and pepsin on the absorption,fluorescence and resonance Rayleigh scattering(RRS) spectra.The optimum reaction conditions,influencing factors and analytical properties were investigated.Some new spectrophotometric,fluorescence and resonance Rayleigh scattering methods for the determination of colchicines,resveratrol and pepsin were established.Belows are the specific research contents:1.Colchicine- acid sulfonephthalein dyes systemsIn pH 2.9～4.6 BR buffer solution,the hydrolyzed product of colchicine reacted with some acid sulfonephthalein dyes such as bromophenol blue(BPB),bromocresol green(BCG),bromothymol blue(BTB) and thymol blue(TB) etc.to form ion-association complexes.As a result,the resonance Rayleigh scattering(RRS) was enhanced greatly,and a new RRS spectrum appeared.The maximum RRS signals of four ion-association complexes were located at 327,311,305 and 306 nm,respectively. The intensities of RRS were proportional to the concentration of colchicine.And the detection limits for colchicine were 12.3,15.1,16.4 and 20.0 ng/mL,respectively.The optimum conditions and the influencing factors of the reaction were investigated.A sensitive,simple and fast method for the determination of colchicine by the RRS technique was developed.It was applied to the determination of colchicine in tablet, hemerocallis,serum and urine samples with satisfactory results. 2.Colchicine- some amino acids systemsIn pH 1.2～3.6 NaAc- HC1 acidic medium,Colchicine(COL) reacted with Tyrosine(Tyr),Tryptophan(Trp) and Phenylalanine(Phe) to form complexes resulting in the fluorescence quenching of Tyr,Trp and Phe.The maximum fluorescence quenching wavelength were at 350 nm,304nm and 284nm.Fluorescence quenching intensities(â–³F) were proportional to the concentration of colchicine in the ranges of 0.05～5.0,0.06～5.5 and 0.3-10μg/mL,and the detection limit for colchicine were 15.1,19.8 and 101.7 ng/mL, respectively.Among three systems,the sensitivity of Tyr-COL system was the highest, and that of the Phe-COL system was the lowest.The optimum conditions and the influencing factors of the Tyr-COL reaction system were investigated.A sensitive, simple and fast fluorescence quenching method for the determination of colchicine was developed.3.Colchicine-tungstophosphoric acid systemIn H₂SO₄ medium,colchicine and tungstophosphoric acidwhich could react to result in the enhancement of the resonance Rayleigh scattering(RRS),second-order scattering(SOS) and frequency doubling scattering(FDS) and appearance of new scattering spectra.The maximum scattering peaks were at 381 nm for RRS,750nm for SOS and 390 nm for FDS,respectively.The enhanced RRS,SOS and FDS intensities were directly proportional to the concentrations of colchicine.The detection limits for colchicine of the RRS,SOS and FDS methods were 8ng/mL,19ng/mL and 28ng/mL.These methods could be used for the determination of trace amounts of colchicine.In this work,the effects of the reaction of colchicine with tungstophosphoric acid on RRS,SOS and FDS spectral characteristics,intensities and the optimum conditions were investigated. Meanwhile,the influences of coexisting substances were tested.Based on the above researches,a sensitive,simple and rapid method for the determination of trace amounts of colchicine by resonance light scattering technique was developed.It could be applied to the determination of Colchicine in tablet,serum and urine with satisfactory results. 4.Colchicine-pepsin systemIn pH 1.9-3.8 acid solution,the hydrolyzed product of colchicine(H-COL) reacted with pepsin to form a binding product leading to the great enhancement of Resonance Rayleigh Scattering(RRS) and appearance of a new RRS spectrum.The maximum RRS peak was located at 406nm,and a smaller scattering peak was at 282nm. The intensities of RRS(â–³/) were proportional to the concentration of pepsin in the range of 0.018-3.5μg/mL.The reaction had a high sensitivity,and the detection limits for pepsin was 5.4ng/mL.The method also had good selectivity.Therefore,a sensitive, simple and fast method for the determination of pepsin by RRS technique was developed.In addition,the reaction mechanism was discussed.5.Resveratrol-HAuCl₄ systemIn pH 12-14 BR buffer medium,resveratrol(Res) and HAuCl₄ could react with each other to result in the great enhancement of the resonance Rayleigh scattering(RRS),second-order scattering(SOS) and frequency doubling scattering(FDS) intensities and appearance of new scattering spectra.The maximum RRS,SOS and FDS scattering peaks were at 396 nm,541nm and 393 nm.The enhanced RRS,SOS and FDS intensities were directly proportional to the concentrations of resveratrol.The detection limits for resveratrol of RRS,SOS and FDS methods were 8.1ng/mL,22.4 ng/mL and 15.3ng/mL,.These methods could be used for the determination of trace amounts of Resveratrol.In this work, the effects of the reaction of resveratrol with HAuCl₄ on RRS,SOS and FDS spectral characteristics,intensities and the optimum conditions were investigated.Meanwhile, the influences of coexisting substances were tested and the results showed that the method had a good selectivity.Based on the above researches,a high sensitive,simple and rapid method for the determination of trace amounts of resveratrol by resonance light scattering technique has been developed.It could be applied to the determination of resveratrol in polygonum cuspidatum samples with satisfactory results. 6.Rsveratrol - potassium permanganate systemIn pH 1.8-2.5 BR buffer medium,there was a oxidation-reduction reaction between potassium permanganate(KMnO₄) and resveratrol(Res),which resulted in the fading of KMnO₄.The maximum fading wavelength was at 525 nm,and the apparent molar absorptivity(es25) was 3.27×10⁴ L·mol^-1·cm^-1.A high sensitive,simple and fast method was established and could be applied to the determination of resveratrol in some natural medicines.