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在pH 3.00的柠檬酸-Na2HPO4缓冲溶液中,偶氮胂(III)与La3+发生结合反应生成蓝色螯合物,此时溶液的pH恰恰低于蛋白质的等电点。因此,血清蛋白带有正电荷,并且由于静电作用力的存在,2 min之内即可结合若干La3+-偶氮胂(III)螯合物生成高分子化合物。该反应能引起La3+-偶氮胂(III)螯合物在650 nm处吸收的减少,减少的吸收直接正比于血清蛋白的浓度。同时,高分子化合物的生成使得蛋白分子尺寸增大,更利于血清蛋白从水相中分离出来。据此,建立了基于上述结合反应修饰血清蛋白的浊点萃取-激光热透镜光谱法测定人血清白蛋白和牛血清白蛋白的新方法。在室温条件下,Triton X-114和十二烷基苯磺酸钠共同萃取并富集了痕量的非电中性的血清蛋白。最后,剩余的表面活性剂相被稀释,并且以热透镜光谱法测定。各种影响萃取效率及热透镜信号变化的因素也被考察及优化。方法已用于人血清样品中蛋白质总量的测定。
In citric acid-NaH2PO4 buffer solution of pH 3.00, the arsenazo (III) complexed with La3 + to form a blue chelate, and the pH of the solution was just below the isoelectric point of the protein. As a result, the serum protein is positively charged, and due to the electrostatic forces, several La3 + -Arsenazo (III) chelates can be bound within 2 minutes to form the polymer compound. This reaction can cause a decrease in the absorption of La3 + -arsaner (III) chelates at 650 nm, with a reduced absorption directly proportional to the serum protein concentration. At the same time, the formation of polymer compounds makes the protein molecular size increases, more conducive to the separation of serum proteins from the water phase. Based on this, a new method for the determination of human serum albumin and bovine serum albumin by cloud point extraction - laser thermal lens spectroscopy was established based on the above binding reaction. At room temperature, Triton X-114 and sodium dodecyl benzene sulfonate were co-extracted and enriched in trace amounts of non-neutral, neutralized serum proteins. Finally, the remaining surfactant phase is diluted and determined by thermal lens spectroscopy. A variety of factors that affect extraction efficiency and thermal lens signal changes have also been investigated and optimized. The method has been used for the determination of the total protein in human serum samples.