phosphorus content
preparation of control solution precision weighing potassium dihydrogen phosphate control dried to constant weight at 105°C. 0, 4, 3, 9G, put it in a 50ml measuring flask, add water to dissolve and dilute to the scale, shake well, take 10ml accurately, put it in another 50ml measuring flask, dilute it to the scale with water, shake well (l per lm is equivalent to 0. 04mg of the peptide).
preparation of test solution
take about 0.1 5 g of this product, weigh it accurately, put it in a Kjeldahl Flask, add 20ml of sulfuric acid and 50ml of nitric acid, slowly heat it until the solution is light yellow, carefully add hydrogen peroxide solution dropwise, the solution was allowed to fade, and heating was continued for 30 minutes. After cooling, the solution was transferred to a 100ml measuring flask, diluted to the mark with water, and shaken.
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measurement precision
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2ml of reference solution and 2ml of test solution were placed in 50ml measuring flask respectively, and 4ml of human ammonium molybdate and sulfuric acid solution was added in turn, sodium sulfite solution 2 m l and freshly prepared hydroquinone solution (hydroquinone 0. 5G, add appropriate amount of water to dissolve, add 1 drop of sulfuric acid, dilute to 100ml)2ml with water, dilute to the scale with water, shake, and place in the dark for 40 minutes, according to UV-visible spectrophotometry (General Rule 0 4 0 1 ) , the absorbance was measured at the wavelength of 620mn, and the phosphorus content was calculated.
nitrogen content
take this product O . l g, precision weighing, according to the nitrogen determination method (General rule 07CH) determination, calculation. Phosphatidylcholine and phosphatidylethanolamine contents were determined by HPLC (General 0512).
chromatographic conditions and system adaptability test
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using silica gel as a filler (column Alltima silica, 250mm x 4. 6mm × 5 μm), column temperature 40 * C; Methanol-water-glacial acetic acid-triethylamine (85 : 15 : 0. 45 : 0 .05,V /V) as mobile phase A, n-hexane-isopropanol-mobile phase A(20 : 48 : 32, V /V) as mobile phase B; The flow rate was lm l per minute; the gradient elution was carried out according to the following table; The detector was an evaporative light scattering detector (reference conditions: drift tube temperature 72 * ~; Carrier gas flow 2.0ml per minute).
Take appropriate amounts of phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylethanolamine, phosphatidylcholine and lysophosphatidylcholine, and use three gas methane-methanol (2:1). Dissolve and make a mixed solution containing 50mg, 100, 100, 200F g, 200 and 200 of the above reference substance per lm l, inject the above solution 20 I 1 into the liquid chromatograph, and the components are eluted in the above order, and the separation degree of each component should be in accordance with the regulations. The number of theoretical plates is based on the peak of phosphatidylcholine and phosphatidylethanolamine, phosphatidylinositol should be calculated to be not less than 1500.
assay
Take appropriate amount of phosphatidylethanolamine and phosphatidylcholine respectively, weigh them precisely, and dissolve them with three gas methane-methanol (2:1), the contents of Phosphatidylcholine in each lm l prepared by dilution are 5 0 dB, 1 0 0mg, 1 5 0 WT, 2 0 0 WT, 3 0 0mg and 4 0 T, respectively, the solutions containing phosphatidylethanolamine 5 mesh, 10 t, 1 5W, 2 0W and 3 0W were used as the reference solution. Accurately measure the above reference solution and inject 20 ^ 1 into the human liquid chromatograph, record the chromatogram, and calculate the regression equation with the logarithmic value of the concentration of the reference solution and the corresponding peak area, another precision weigh the product about 15 m g, put 50ml measuring flask, add three gas methane-methanol (2:1) dissolved and diluted to the scale. 20M1 of the test solution was injected into the liquid chromatograph, the chromatogram was recorded, and the contents of phosphatidylcholine and phosphatidylethanolamine were calculated by the regression equation.
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