Research Of Quantitative Method On In-situ Thin Layer Reflectance Spectroscopy

Quantitative analysis of TLC mostly use reflection method,behaviour of diffused reflection light is complex,and optical conduction of equipmet is more complex.Reflective absorbance is used to quantitative analysis only for solution of lower concentration,the scope of linearity is narrow.Date by means of single-wavelength or dual-wavelength were collected, small data size,low precision,and the high-resolution multi-wavelength TLC scanner prices are generally more expensive.Therefore, TLC scanner urgent need to provide higher optical resolution,decrease the impact of stray light, reduce hardware costs and research quantitative analysis method.The measurement system of in-situ thin layer reflectance spectroscopy based on lighting source, optical fiber conduct technology,CCD micro-spectrometer and stepping motor has been designed.The substitution of the measurement system for Thin-layer chromatogram scanner can be used in TLC scaning.The error sources of TLC determination and TLC quantitative analysis is analyzed.The study aim to build a quantitative analysis based on in-situ thin layer reflectance spectroscopy.The content of study include the following four parts:(1) To establish a measurement system based on in in-situ thin layer reflectance spectroscopy.Spots were scanned by mean of two-dimensional movement,the scanning track was designed as"S".Spectral response of spots correspondence position in the multi-wavelength were collected on the different position of recording duration.Caffeine and phenacetin were acted as study sample.The results showed that linear correlation coefficient of between absorption of reflectance spectroscopy or Kubelka-Munk(K-M) and content of phenacetin spots in a range of 2.00-24.00μg were 0.9805 and 0.9990.When the scope of phenacetin spot content were narrowed to 2.00-10.00μg and 8.00-24.00μg,linear correlation coefficient were more than 0.9933.Linear relationship of K-M and content of phenacetin spots was much better relativity.The influence of scanning rate on quantitative analysis were studied. K-M of caffeine spots decreases with increasing sweep speed, and the effect of linear relationship between K-M and content of spots is not remarkable in scanning rate range of 0.962-3.282mm/s.(2) Chlorogenic acid as the research object aims to investigate analytical accuracy of in-situ thin layer reflectance spectroscopy.Relationship of between spectral response and spots correspondence position were analyzed to correctly recognize error sources for quantitative analysis.Linear correlation coefficient between contents of chlorogenic acid and K-M of spot planimetry was 0.9995 in the wavelength coverage of 230.49 -374.00nm.The accuracy of the method was validated by adding standard recovery experiment,the recoveries was 100.67% and relative standard deviation(RSD) was 1.61% , the result suggested that the method was sensitive and precise, which could satisfy the quantitative detection request of sample.(3) To establish a multi-wavelength thin layer chromatograph method based on in-situ thin layer reflectance spectroscopy for determination of chlorogenic acid in flos lonicerae.Chlorogenic acid in flos lonicerae extracts were separated by thin layer chromatography on polyamide film with 75% water mircroemulsion and methanoic (6:1)as the mobile phasea. Spectral response of chlorogenic acid spots were collected by the measurement system. Experimental results show that linear relations of contents of chlorogenic acid and K-M of spot planimetry were good.The conetent of chlorogenic acid were calculate by external standard two-point working curve method.The conetent of chlorogenic acid in flos lonicerae was 4.983mg/g and RSD is 2.35%(n=6),the average recovery is 98.87% and RSD is 2.90%, detection limits(S/N=3) is19ng. The conetent of chlorogenic acid in flos lonicerae was 5.158mg/g by HPLC. The measurement shows that the measured results by using the system basically agree with HPLC analysed ones.(4) To analyze and discuss the separation process of flos lonicerae extracts separated by TLC.There were 10 spots separated from flos lonicerae extracts by thin layer chromatography on polyamide film,and spectrogram were extracted from the curves of chromatographic peak of samples.Developing process were analyzed to provide the basis of the optimized separation conditions of TLC.