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Detailed procedure for using high performance liquid chromatography

Time:2018/08/10   Pageviews:8    Share:
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1. Start setting parameters (detection wavelength, flow rate), replace the required mobile phase, and check if the column is correct.
2. Discharge the mobile phase in the drain line, rinse the injection valve, and wash the syringe.chromatography vials
3. Flush the column for equilibration, during which the sample can be processed in advance and the sample information is registered.
4. After the balance is good, enter the blank test to eliminate system pollution.
5. After the column is stable, the sample is injected, waiting for the end of the operation (during cleaning the syringe), calculating the points, and issuing a report.

The detailed steps are as follows:

1, boot operation:
(1), turn on the power, when connected with Harb, pay attention to the Harb power supply, turn on the computer, open the Bootp Server (usually turned on when starting);
(2), open the power of the component from top to bottom, when there is a signal in the Bootp Server (with six lines of characters), open the workstation (open On line first);
(3) Open the switch of the 10% isopropyl alcohol solution of the flushing pump head (need to use the needle pump) to control the flow rate, which is based on the minimum flow rate that can be discharged;
(4) Pay attention to the volume setting of the remaining solution of each mobile phase. If the set volume is lower than the minimum limit, the pump will be automatically stopped. Pay attention to the volume of the pump solution and add liquid in time.chromatography vials
(5) During the use process, the working state of the instrument should be observed frequently, and various emergencies should be handled correctly in time.
2. First flush the system with the mobile phase used for a certain period of time (if the mobile phase used is a salt-containing mobile phase, it must be rinsed with water for more than 20 minutes and then replaced with a salt-containing mobile phase). The D-lamp or W is turned on about 30 minutes before the formal injection analysis. Lights to extend the life of the lamp;
3, establish a chromatographic operation method, pay attention to save the Method named for yourself, do not cover or delete other people's methods and experimental results;
4. When using the manual injector to inject, the syringe should be washed with the needle washing solution before and after the injection. The needle solution is generally selected to be the same solvent as the sample solution. The sample must be used before the injection. Clean the syringe barrel more than 3 times and remove the air bubbles in the syringe;
5. The sand core filter head in the solvent bottle is easy to be broken. Pay attention to the protection when replacing the mobile phase. When the filter head is found to be dirty or long bacteria, it can not be washed by ultrasonic. It can be washed with 5% dilute nitric acid solution and then washed.
6. After the end of the experiment, generally rinse the entire pipeline with water or a low concentration of methanol aqueous solution for more than 30 minutes, then rinse with methanol. Turn off the D lamp and W lamp during the flushing process;
7. When shutting down, first turn off the pump, detector, etc., then turn off the workstation, then shut down, and finally turn off the components of the chromatograph from bottom to top, and turn off the switch to wash the pump solution;
8. Users must conscientiously perform the instrument registration system and do not disassemble the instrument.

Mobile phase:
1. The flow should be selected from chromatographic pure reagent, high-purity water or double-distilled water. The acid-base solution and buffer solution should be filtered. Pay attention to distinguish the use range of water-based membrane and oil-based membrane when filtering;chromatography vials
2. The mobile phase of the water phase needs to be replaced frequently (generally no more than 2 days) to prevent deterioration of the long bacteria;
3. When using a double pump, in the four phases A, B, C, and D, if there is a salt-containing mobile phase in the mobile phase used, then A and D (the inlet port is located below the mixer) place the salt-containing mobile phase, B. C (the inlet is located above the mixer) to place the salt-free mobile phase;
One of the four reservoirs A, B, C, and D is a brown bottle for storing the aqueous phase mobile phase.
1. Treat the sample by filtration or centrifugation to ensure that the sample does not contain solid particles;
2. Prepare the sample solution with a mobile phase or a weaker mobile phase (if the reverse phase column is larger than the mobile phase; if it is a normal phase column, the polarity is smaller than the mobile phase), prepare the mobile phase as much as possible. Sample solution
When manually injecting, the injection volume should be as small as possible. When using quantitative tube, the injection volume should be 3 to 5 times of the quantitative tube;
1. Read the instructions attached to the column carefully before use, pay attention to the applicable range, such as pH range, mobile phase type, etc.
2. Use a mobile phase that meets the requirements;
3. Use a guard column;chromatography vials
4. If the mobile phase used is a salt-containing mobile phase, use the reversed-phase column with water or low-concentration methanol water (such as 5% aqueous methanol) and then with methanol.
5. When the column is not in use, it should be washed with methanol, and then removed and tightly closed at both ends for storage;
6, do not wash the column with high pressure;
7. Do not use silica gel bonded phase chromatography column for a long time at high temperature;

 This is the end of the introduction of Detailed procedure for using high performance liquid chromatography. I hope it can help you.


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