Determination of rapeseed glucosinolates - Photometric method

1. range

This standard specifies a method for the determination of sulfur and germanium in rapeseed by photometry.

This standard applies to conventional rape breeding, hybrid three-line, two-line breeding, radiation breeding and biotechnology breeding in the detection of erucic acid glucosinolates and rapeseed and commercial rapeseed acquisition during the rapid determination of sulfur content.

2. principle

Using the specificity of the enzyme, the glucosinolate reacts with the specific enzyme (Gln MEN) in the rapeseed, and the resulting product reacts with a dedicated color developing agent (Chrom G) to form a colored product having a characteristic absorption peak, and a sulfur standard is determined based on a standard curve. content.

3. Reagent

All reagents were of analytical grade (AR), except for special labeling, and water was secondary water specified in GB/T 6682.

3.1 Sulfur assay A fluid.

3.2 Sulfur assay B fluid.

3.3 Sulfur test plate.

4. equipment

Laboratory routine instruments and the following items.

4.1 Mini crusher.

4.2 Electronic balance (range 0-200 g, minimum reading 100 mg).

4.3 40-200 μL micropipette.

4.4 1000 μL micropipette.

4.5 Absorbent cotton.

4.6 quartz cuvette (18 mm x 18 mm).

4.7 erucic acid sulferometer.

5. sampling

According to GB5491, rapeseed samples should be taken and the foreign impurities should be removed when sampling.

6. Steps

6.1 Preparation of Samples

Divide the rapeseed samples taken according to GB5491. Take about 10 g of one sample and pulverize it in a micro-pulverizer for 20 seconds. Mix the crushed material and pulverize for 5 seconds. (You can also pulverize the sample with a mortar. 40 mesh sieve).

6.2 Samples

Accurately weigh 0.50 g (accurate to 100 mg) in a 5 mL stoppered tube.

6.3 Extraction

Pipette 3 mL of glucosinolate to measure A solution, close the lid tightly, shake the tube vigorously, and mix and react for 8 minutes (mix twice more in the middle).

6.4 Filtering

Use a glass rod to slowly pour fluffy, uniform texture, and appropriate amount of absorbent cotton into the test tube. When the absorbent cotton contacts the liquid surface, place the test tube on the table top and allow the absorbent cotton to automatically absorb fluid. After the suction is complete, lift the centrifuge tube and use glass. The rods are lightly pressed down with absorbent cotton until the supernatant is filtered out, and the supernatant can also be obtained by centrifugation or filter paper.

6.5 Enzyme-Color Reaction

Remove the sealed and stored glucosinolate test plate (strips can be used, other sealed, protected from light), use a micropipette to take 50 μL of the supernatant in the test plate wells, and select a well to add 50 μL of sulfhydryl B liquid as a blank; enzymatic reaction 8min, halfway blowing with a suction head twice, so that the enzymolysis reaction is uniform.

6.6 Dilution of developing solution

With a micropipette, 150 μL of sulfonium was used to measure the B solution, which was added to blank wells and sample wells respectively. The reaction was continued for 2 minutes, followed by one-stage insufflation to make the system even and stable.

6.7 Determination

In the erucic acid thioxane speed tester, use the reagent blank as a zero point, select “calibration”, memorize the zero point value, and then select “sample measurement” to perform sample test analysis. The reading value is gram per gram of rapeseed meal. The total number of moles of glucosinolates contained (μmol/g).

7. Precision and tolerance

Repeated determination of the relative standard deviation of less than 6%; two parallel determination results erucic acid difference of less than 0.8%; the results of the two parallel determination of sulfur and hydrazine difference of less than 4.0μmol/g. The measurement results are expressed as an arithmetic average.

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