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Adoption of standard: GB/T 22492-2008

1 Test Principle:

It was determined by high performance gel filtration chromatography. That is to say, using porous filler as stationary phase, based on the difference in the relative molecular mass size of the sample components for separation, detected at the peptide bond of the ultraviolet absorption wavelength of 220nm, using the dedicated data processing software for the determination of relative molecular mass distribution by gel filtration chromatography (i.e., the GPC software), the chromatograms and their data were processed, calculated to get the size of the relative molecular mass of the soybean peptide and the distribution range.

2. Reagents

The experimental water should meet the specification of secondary water in GB/T6682, the use of reagents, except for special provisions, are analytically pure.

2.1 Reagents include acetonitrile (chromatographically pure), trifluoroacetic acid (chromatographically pure),

2.2 Standard substances used in the calibration curve of relative molecular mass distribution: insulin, mycopeptides, glycine-glycine-tyrosine-arginine, glycine-glycine-glycine

3 Instrument and equipment

3.1 High Performance Liquid Chromatograph (HPLC): a chromatographic workstation or integrator with a UV detector and GPC data processing software.

3.2 Mobile phase vacuum filtration and degassing unit.

3.3 Electronic balance: graduated value 0.000 1g.

4 Operating steps

4.1 Chromatographic conditions and system adaptation experiments (reference conditions)

4.1.1 Chromatographic column: TSKgelG2000swxl300 mm×7.8 mm (inner diameter) or other gel columns of the same type with similar performance suitable for the determination of proteins and peptides.

4.1.2 Mobile phase: Acetonitrile + water + trifluoroacetic acid = 20 + 80 + 0.1.

4.1.3 Detection wavelength: 220 nm.

4.1.4 Flow rate: 0.5 mL/min.

4.1.5 Detection time: 30 min.

4.1.6 Sample injection volume: 20μL.

4.1.7 Column temperature: room temperature.

4.1.8 In order to make the chromatographic system meet the detection requirements, it was stipulated that under the above chromatographic conditions, the gel chromatographic column efficiency, i.e., the theoretical number of plates (N), was not less than 10000 calculated on the basis of the peaks of the tripeptide standard (Glycine-Glycine-Glycine).

4.2 Production of relative molecular mass standard curves

The above different relative molecular mass peptide standard solutions with a mass concentration of 1 mg / mL were prepared by mobile phase matching, mixed in a certain proportion, and then filtered through an organic phase membrane with the pore size of 0.2 μm~0.5 μm and injected into the sample, and then the chromatograms of the standards were obtained. Relative molecular mass calibration curves and their equations were obtained by plotting the logarithm of relative molecular mass against retention time or by linear regression.

4.3 Sample treatment

Accurately weigh 10mg of sample in a 10mL volumetric flask, add a little mobile phase, ultrasonic shaking for 10min, so that the sample is fully dissolved and mixed, diluted with mobile phase to the scale, and then filtered through an organic phase membrane with a pore size of 0.2μm~0.5μm, and the filtrate was analyzed according to the chromatographic conditions in A.4.1.

5. Calculation of relative molecular mass distribution

After analyzing the sample solution prepared in 4.3 under the chromatographic conditions of 4.1, the relative molecular mass of the sample and its distribution range can be obtained by substituting the chromatographic data of the sample into the calibration curve 4.2 with GPC data processing software. The distribution of the relative molecular masses of the different peptides can be calculated by the peak area normalization method, according to the formula: X=A/A total×100

In the formula: X - The mass fraction of a relative molecular mass peptide in the total peptide in the sample, %;

A - Peak area of a relative molecular mass peptide;

Total A - the sum of the peak areas of each relative molecular mass peptide, calculated to one decimal place.

6 Repeatability

The absolute difference between two independent determinations obtained under conditions of repeatability shall not exceed 15% of the arithmetic mean of the two determinations.