SN/T 5270-2019Rubber and its products. Determination of ethylene thiourea. High performance liquid chromatography (English PDF)

6.1 Sample preparation

Cut the sample into small pieces below 3 mm×3 mm, and then freeze and crush them to particles with a particle size of not more than 1 mm, ready for use.

6.2 Sample processing

Accurately weigh 1 g of the sample (accurate to 1 mg) into a 50 mL plastic centrifuge tube (5.5) with a stopper, add 30 mL of methanol (4.2), Add 0.5 mL of BHA solution (4.6), ultrasonically extract at 40 °C for 60 min, centrifuge at 3 000 r/min for 3 min, take all the supernatant, Re-extract once, combine the extracts in a concentrating bottle (5.6), and concentrate at 40 °C to about 2 mL.

Activate the solid phase extraction column (4.8) with 5 mL of acetonitrile (4.1), discard the eluent; place the activated solid phase extraction column (4.8) in another on a concentrating bottle (5.6), take the above concentrated solution and apply it to the column, wash the concentrating bottle (5.6) with 3 mL and 2 mL of acetonitrile (4.1) and wash the purified acetonitrile was applied to the column, and then the solid phase extraction column (4.8) was eluted with 5 mL of acetonitrile (4.1), and all the eluate was collected.

The eluent was concentrated to dryness at 40 °C, and 2.00 mL of mobile phase (4.7) was accurately added to dissolve the ethylene thiourea in the residue, over 0.45 µm Filter membrane (5.10), to be analyzed.

6.3 Matrix calibration working curve

Take an appropriate amount of 4.5 medium ethylene thiourea standard stock solution, dilute with acetonitrile to obtain the concentration of 2.0 mg/L, 4.0 mg/L, 10.0 mg/L, 20.0 mg/L, 50.0 mg/L and 100.0 mg/L serial dilution standard solutions, respectively take 1.00 mL each of the above diluted standard solutions and add to 1 g (precise to 1 mg) blank sample matrix, follow the steps specified in 6.2 to obtain the matrix calibration curve of ethylene thiourea, and the final instrument the analyzed concentrations of ethylene thiourea were 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/kg, 10.0 mg/kg, 25.0 mg/kg and 50.0 mg/kg.

6.4 Blank experiment

Except that the sample is not weighed, the rest are carried out according to the steps in 6.2.

6.5 Determination

6.5.1 Chromatographic detection conditions

Since the test results depend on the instrument used, it is impossible to give general parameters for HPLC analysis. Set parameters it should be ensured that the measured component and other components can be effectively separated during chromatographic determination. The following parameters have proved feasible.

a) Chromatographic column. C18 column, 250 mm×4.6 mm, 5 µm, or equivalent;

b) Chromatographic column temperature. 35 °C;

c) Mobile phase. Acetonitrile. water = 30.70, volume ratio;

d) Flow rate. 1.0 mL/min;

e) Detection wavelength. 234 nm;

f) Injection volume. 10 µL;

g) Running time. 15 min.

6.5.2 High performance liquid chromatography determination

According to the chromatographic detection conditions of the high performance liquid chromatograph set in 6.5.1, after the instrument is stable, the matrix calibration work curve Make solution (6.3), blank solution (6.4) and sample constant volume solution (6.2) equal volume interspersed sample injection determination. Take the chromatographic peak area as the Y axis, Taking the concentration as the X axis, draw the standard curve, the matrix calibration working solution and the response value of the ethylene thiourea in the sample to be tested should be Within the linear range detected by the instrument, the chromatographic peak area is quantified by external standard method.

The typical liquid chromatogram and ultraviolet spectrum of the standard solution are shown in Figure A.1 and Figure A.2.in Appendix A.