Determination of the Content of Recombinant Collagen using Xtimate Bio SEC-300

1.1 Solution Preparation

1.1.1 Preparation of mobile phase:

Weigh 6.9 g of sodium dihydrogen phosphate and 11.36 g of disodium hydrogen phosphate, dissolve in 1300 mL of ultrapure water, adjust the pH to 6.7 with phosphoric acid, mix well and filter under vacuum. Take 475 mL of the above buffer solution, add 25 mL of HPLC-grade isopropanol, mix well, degas by ultrasound, and the mobile phase is ready.

1.1.2 Preparation of recombinant collagen protein standard solution:

Weigh an appropriate amount of recombinant collagen protein solid provided by client, dissolve it in water to make a solution of approximately 2mg/ml, and shake well to obtain the solution.

1.1.3 Preparation of sample solution:

Weigh 0.68 g of potassium dihydrogen phosphate provided by client, add 39.5 ml of 0.1 mol/L sodium hydroxide solution, dilute with water to 100 ml, and add 0.2 g of recombinant collagen protein. Dissolve and stir the mixture evenly.

Then, weigh 0.9 g of sodium hyaluronate provided by client and evenly spread it on the surface of the prepared solution. Let it stand for more than 8 hours until the sodium hyaluronate is fully swollen and turns transparent.

Stir the solution evenly, take an appropriate amount of the solution, dilute it 10 times with water, shake well, and filter. The filtrate is collected for use.

Using the Welch Xtimate Sugar-Ca (7.8×150mm, 8μm) columns, the analysis was performed under the chromatographic conditions. The main component can be completely separated from other substances, meeting the detection requirements.