Study On CE Method With Dual C~4D/UV Detector For Simultaneously Determining Major Metal Cations And Whey Proteins In Milk

The most important nutrients in milk are minerals and proteins.Minerals in milk are indispensable nutrients for regulating physiological function and promoting growth and development of human beings.The amount of whey proteins in milk often directly determines its nutritional value and quality and can also provide important information about adulteration,production techniques and cow diseases.Therefore,the study of metal cations and whey proteins content in milk has become a hot topic in the field of food.At present,the traditional analysis technologies for the determination of metal ions and whey proteins in milk have two main defects:complicated operation procedures and high analysis cost.However,capillary electrophoresis technology can provide a rapid,simple and low-cost method to determine the major metal ions and whey proteins in milk.A reliable and simple CE method for simultaneous determination of major metal cations and whey proteins in milk samples is developed and it is discussed in detail in this thesis.Firstly,the thesis introduces the basic theoretical knowledge of capillary electrophoresis（CE）and capacitively coupled contactless conductivity detection（C⁴D）.Secondly,the role of metal cations and whey proteins in milk on human body,the significance of the study of metal cations and whey proteins in milk and the commonly used detection methods for determining metal cations and whey proteins in milk are introduced.Finally,the purpose and significance of this research are introduced.A reliable and simple CE method with dual C⁴D and UV detection modes for simultaneous determination of major metal cations in milk samples was developed.Sample pretreatment comprised dilution,acidification to p H 4.55 with 10 m M HAc and centrifugation.The complete separation of metal cations K⁺,Ca²⁺,Na⁺,Mg²⁺could be achieved within 10 min in a simple BGE composed of 1.0 M HAc and 12 m M L-His with p H 2.74 under+15 k V.The samples were injected for 5 s at 50 mbar pressure,the excitation voltage and excitation frequency of the C⁴D detector were 80 V and 1000 k Hz,respectively and the detection wavelength of UV detection was set at 200 nm.In cation analysis,the range of detection limit was 0.05～0.10 mg/L for C⁴D detection and 0.10～0.50 mg/L for UV detection,respectively.It is found that the working ranges of four cations have satisfactory linearity and low quantitative limits and the correlation coefficients are better than 0.995 under both detectors,which is suitable for milk sample analysis.And the relative standard deviations（RSD%,n=5）of intraday and interday analysis were 0.35～0.52%and 0.47～0.69%for relative migration time,2.42～4.16%and 4.07～5.60%for peak area,respectively.Finally,the contents of four major metal cations in milk samples of various brands and prices were obtained.Whey proteinsα-Lac,β-Lg in milk are successfully determined within 20 min by using the same BGE solution,the same electrophoretic conditions and the same sample pretreatment procedures.In whey protein analysis,satisfactory linearity were obtained for both whey proteinsα-Lac,β-Lg analysis,and correlation coefficients（R²）were both better than 0.995 and the detection limits ofβ-Lg andα-La analysis were 5 mg/L and 3 mg/L,respectively and the relative standard deviations（RSD%,n=5）of intraday and interday analysis were 0.25～0.33%and 0.42～0.45%for migration time and 2.88～3.22%and 3.64～4.16%for peak area,respectively.Finally,the content of whey proteinβ-Lg andα-Lac in milk samples of various heat treatments were obtained.Furthermore,this CE method can realize the simultaneous determination of metal cations and whey proteins by single injection analysis in the analysis of pasteurized milk and INF milk.An economical,simple and environmentally friendly,and rapid CE analysis method was developed,which can effectively determine the three major whey proteins（α-lac,β-lg,BSA）in milk.The influence of the concentration of BGE,the kinds and the concentration of buffer additive on the separation effect of whey proteins was studied,and the optimal BGE was determined to be 1.6 M HAC,20 m M L-His,0.1%HPMC,p H 2.70.The sample was injected for5 s under a pressure of 50 mbar,the operating voltage was+15 k V,the temperature was 25°C,and the detection wavelength of the UV detector was 200 nm.The three kinds of whey proteins have a good linear relationship in the range of 30～1000 mg/L,and the linear correlation coefficient（R²）is better than 0.994,and the detection limit of the method ranges from 5～15 mg/L.The relative standard deviations（RSD%,n=5）of the intra-day analysis of migration time and peak area ranged from 0.34～0.47%and 2.67～4.28%,respectively,and the relative standard deviations（RSD%,n=5）of the inter-day analysis of migration time and peak area ranged from 0.44～0.61%and3.44～5.47%,respectively.The CE method has been applied to the determination of whey protein contents in different types of heat-treated milk.