| A method for quantitative analysis with substitute standard to the unstable compound by high performance liquid chromatography (HPLC) was mainly established in this article. We investigated the compounds’ stability in different solvent backgrounds by HPLC and ultraviolet spectrophotometer (UV spectrophotometer), which the compounds had the maximum absorption wavelength (λmax) in the ultraviolet and visible region respectively, and thereby investigated the feasibility that alternate standard used in the HPLC quantitative analysis. A test method which detects lutein in foods without lutein standard was provided in this article, and the results obtained from this method were as much accurate as the traditional external reference method. Besides, this method would save more money.The studies in this thesis are states as follows:1.Eight target compounds’response stability which had the λmax in the ultraviolet region (four pKa<7and four pKa>7) in different mobile phase and solvent background were detected by UV spectrophotometer and HPLC. The result showed that the stability of compounds which had the λmax in the ultraviolet region was influenced by it’s own pKa and the kind of additive; benzocaine, procaine, paracetamol and chlorogenic acid had stable response under different solvent backgrounds, which were fit to be the substitute standard under different detect background. The slopes of the standard curve of benzocaine and paracetamol on HPLC almost had no change. The results showed that the chromophore groups of the compounds which had the in the ultraviolet region were suit to be the substitute standard on HPLC when the same additive was added and different mobile phase were changed.2.The target compounds’response stability which had the λmax in the visible region in different solvent background (nonaqueous system) was detected by UV spectrophotometer, and the stability of the correction factor of each compounds to lutein were detected by HPLC. The experiment result showed that, the compounds which had the λmax in the visible region were much stable under different mobile phase and solvent backgrounds compared to the target compounds in the ultraviolet region, the correction factor on HPLC were totally stable when showed as the substitute standard. It was feasible to be the substitute to the unstable compounds which had the similar maximum absorption wavelength. During the process of the detection of lutein contents, we should try to avoid the solvent to be acidic environment, because lutein could degradation easily on this condition, which would lead the inaccuracy result. Among the rest, the correction factor of para red and sudan Ⅰ were the most stable, which were the most suitable to be the substitute standard.3.Sudan Ⅰ and para red were used as the substitute standard to lutein to quantitative analysis lutein in foods with the aforementioned experimental conditions. Besides, the lutein extraction process was improved in this article. The result showed that, the relative error between the calculated lutein results and the detected actual concentration of the chosen sixteen kinds of foods were less than3.5%, which explained that sudan Ⅰ and para red were feasible to be the substitute to the unstable compounds. It could overcome the shortage of lutein instability, reduce analysis costs. Compared with the traditional ethyl acetate abstract method, the method which uses acetone as the extraction solvent had more advantages because of its simple and convenient, and the extraction efficient would be much higher, the analysis results were stable. |
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