HPLC procedure

1) Filter the mobile phase and select different filters as needed.

2) Ultrasonic degassing of the filtered mobile phase for 10-20 minutes.

3) Open the HPLC workstation (including computer software and chromatograph), connect the mobile phase pipe, and connect the detection system.

4) Go to the main menu of the HPLC control interface and click manual to enter the manual menu.

5) If it has not been used for a while, or if a new mobile phase is replaced, the pump and injection valve need to be flushed first. Flush the pump directly at the pump's water outlet and draw with a needle. To flush the injection valve, you need to click on the purge menu and then click start. The speed should not exceed 10 ml/min.

6) Adjust the flow rate. When using the new mobile phase for the first time, you can try the pressure first. The larger the flow rate, the higher the pressure, generally not more than 2000. Click injure, select the appropriate flow rate, click on, go to the baseline, and observe the baseline.

7) Design the method of aliasing. Click on file and select select users and methods to select various existing aliasing methods. To create a new method, click on new method. Select the required accessories, including injection valves, pumps, detectors, etc., as needed. After the selection, click on the protocol. A complete method of aliasing needs to include: a. steady flow before injection, generally 2-5 minutes; b. baseline return to zero; c. loading-inject conversion of injection valve; d. sample time, with different samples different.

8) Operation after injection and injection. Select the aliasing method and click start. For injection, all samples need to be filtered. After the method is finished, click postrun to record data and mark it. After all the samples have been finished, use the above method to take a baseline and wash off the residue.

9) When shutting down, turn off the computer first, then turn off the liquid chromatograph.

10) Fill in the registration form and sign it by the person in charge.

Precautions:

1) The mobile phase requires chromatographic purity, and water uses 20M deionized water. The mobile phase after degassing should be carefully shaken to avoid air bubbles.

2) The column is very fragile. For the first time, do not let the liquid pass through the column.

3) All liquids passing through the column must be strictly filtered.

 The pressure should not be too large, preferably no more than 2000 psi.