Determination Of Sodium Thiocyanate In Milk By Spectrophotometry And Reversed-phase Iron-pair Chromatography

Sodium thiocyanate is often used as a preservative in milk. It can extend the shelf life in milk collection and transportation effectively. While, sodium thiocyanate is a kind of toxic chemical reagent. Cyanide ion it produces is easily combined with trivalent iron in cytochrome oxidase, causing tissue oxygen using dysfunction. When intakes, the body will show acute and chronic toxicosis and a series of symptoms. It has been added as banned substances into the list of non-food substances illegallly added in food. But it has no national standard exactly. According to the structural properties of sodium thiocyanate, This research optimizes the existing spectrophotometry. Reversed-phase ion-pair Chromatography is established for determination of sodium thiocyanate in milk.The main research results are as follows:1.The optimum single factor conditions of sodium thiocyanate determination by Spectrophotometry in milk are optimized in this article. The best chromogenic reagent is determined by contrasting sensitivity of three chromogenic reagent including ferric chloride, ammonium ferric sulfate and ferric nitrate. Meanwhile, the pretreatment conditions of trchloroacetic acid concentration, refrigeration extraction time, ultrasonic-assisted extraction time, centrifugal rotational speed and chromogenic conditions of nitric acid and chromogenic reagent dosage, selection of stabilizing reagent and color developing time have been optimized. The experiment have been methodologically investigated including standard curve linear relation, the lowest detection limit, precision, repeatability, stability, recovery and sample verification, improving accuracy and sensitivity of the method. The results indicate that ferric nitrate reagent of 80 g/L, trichloroacetic acid concentration of 200 g/L, refrigeration extraction time for 10min, ultrasonic-assisted extraction time for 10min, the centrifugal rotational speed of 3000 r/min, nitric acid dosage of 2.0mL, chromogenic reagent of 2.0mL, stabilizing agent of 1% gum Arabic 1mL, chromogenic time for 15min. The methodological investigations are as follows:the good linear relation in 0.0000g/L-0.0500g/L, the linear regression equation for Y=29.897×X+0.0222(R2=0.9993). The lowest detection limit of 2.2476mg/L, recovery between 90.1% and 103%.RSD 0.556%-2.009%.This method has advantage of simpleness, good precision. It is suitable for small business application.2.The Reverse-phase Ion-pair Chromatography is researched and established. After protein precipitation by acetonitrile extraction, supernatant fluid is filtered by filter membrance for detection. Influence factors including sample load, mobile phase composition proportion, column temperature, maximum absorption wavelength, velocity, mobile phase pH, ion-pair reagent concentration are screened in this research. Chromatographic conditions are as follows:chromatographic column Eclipse XDB C18(150×4.6mm,5μm), sample load 5uL, mobile phase of methanol:0.20% hydroxide tetrabutyl ammonium solution(v:v)=30:70, column temperature of 35℃, maximum absorption wavelength of 210nm, velocity of 1.2 mL/min,8.5% phosphoric acid regulates pH, mobile phase pH 5.0,0.20% hydroxide tetrabutyl ammonium solution. The good linear relation in 0.0000g/L-0.0200g/L, the linear regression equation for Y=1.0×107×X-492.6(R2=0.9995). The lowest detection limit of 0.0144mg/L, recovery between 95% and 101%,RSD 1.730%-1.780%.3.The methods contrast of spectrophotometry and Reversed-phase Iron-pair Chromatography are researched. Sensitivity, the lowest detection limit, precision, repeatability, stability, average recovery, sample processing time,sample testing time and analysis cost are compared in two methods. The results are as follows:the latter has the higher sensitivity, the former and the latter the lowest detection limit of 2.2476mg/L, 0.0144mg/L; precision RSD 1.741%,1.499% respectively; repeatability RSD 1.939%, 1.687%; stability RSD 1.298%,1.547%; average recovery of 96.4%,98.7%. Sample processing time of 40 min,60 min. Sample detection time of 10s,6.382min. As for analysis cost, the latter is higher. Verification tests are conducted by sampling dairy products in market. The results relative standard deviation(RSD) are within 5%.