Aqueous two-phase system–ion chromatography for determination of thiocyanate in raw milk

Acetonitrile (ACN) was HPLC grade, KBr was SP grade, and all other reagents were AR grade. Sodium thiocyanate was obtained from Aladdin Industrial Corporation (Shanghai, China). ACN was provided by Dikma Technology Co. Ltd. (Beijing, China). KBr was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Other reagents were from Fuchen Chemical Reagents Factory (Tianjin, China).

IC analysis was performed on a 930 (Metrohm AG, Herisau, Switzerland) ion chromatography system. Chromatographic separation of sodium thiocyanate was carried out using a Metrosep A Supp5 (250 mm × 4.0 mm, 5 μm) anion chromatographic column, which utilized 5 mM Na₂CO₃ containing 5% (v/v) acetone as eluent under isocratic conditions at a flowrate of 1.0 mL/min. The temperature of column and detector was maintained at 35 °C using a chromatography oven. The suppressor regeneration solution was H₂SO₄ solution (10 M). A SC-3610 low speed centrifuge (Zhongke Zhongjia Scientific Instrument Co., Ltd., Hefei, China) and 0.22 μm water membrane filters (Jinteng Laboratory Equipment Co., Ltd., Tianjin, China) were used for sample treatment. IR spectra were examined with an ALPHA-T Fourier transform infrared spectrometer (Bruker Daltonics, Karlsruhe, Germany)

Raw milk (40 g) was weighed into a colorimetric tube, where 9 g of trichloroacetic acid aqueous solution (50%, w/w) and 1 g of hydrogen peroxide aqueous solution (1%, w/w) were added and mixed. Then, the sample was centrifuged (5000 rmp, 10 min), the supernatant was filtered by a 0.22 μm water membrane filter [25], and the filtrate was reserved.

The sodium thiocyanate standard was dried in an oven at 80 °C for 3 h. The dried sodium thiocyanate was accurately weighed to 1.397 g in a 1000 mL volumetric flask, fixed to the mark with ultrapure water. Then, the solution was mixed and stored at 4 °C. It was valid for 3 months.

Phase systems were prepared by adding predetermined quantities of organic solvent (acetonitrile and acetone), (NH₄)₂SO₄, and the filtrate. The pH of the systems was adjusted with hydrochloric acid or sodium hydroxide. The compositions of ATPS are shown in Table 1. The phase-forming components were mixed using a vortex mixer for 2 min, then performed centrifugal separation at 2000 rpm for 3 min. The volume of the top phase of ATPS was recorded. The top phase was collected and concentrated by nitrogen blowing, then the volume of the concentrated solution was recorded and the concentrated solution filtered with the organic filtration (0.22 μm). Finally, IC was used to detect the SCN⁻ content in the concentrated top phase solution. The average of three replicates is reported.