Common questions and answers about HPLC
1. Q: When using HPLC for analysis, the retention time sometimes drifts and sometimes changes rapidly. What is the reason? How to solve?
Answer: Regarding the drift problem:
1. Poor temperature control, the solution is to use a constant temperature device to keep the column temperature constant
2. The mobile phase changes, the solution is to prevent the mobile phase from evaporation, reaction, etc.
3. The column is not well balanced, the column needs to be equilibrated for a longer time
About rapid changes
1. The flow rate changes, the solution is to reset the flow rate to keep it stable
2. There are air bubbles in the pump, which can be expelled through operations such as exhaust.
3. The mobile phase is not suitable, the solution is to change the mobile phase or make the mobile phase properly mixed in the control room
2. Q: What are the causes of peak tailing or double peaks in liquid chromatography?
Answer: 1. If the sieve plate is clogged or the column fails, the solution is to flush the column back, replace the sieve plate or replace the column.
2. There are interference peaks, the solution is to use a longer column, change the mobile phase or replace the column with good selectivity
3. Q: The main reason and solution of insufficient sensitivity of HPLC
Answer: 1. Insufficient sample size, the solution is to increase the sample size
2. The sample did not flow out of the column. The mobile phase or column can be changed according to the chemical nature of the sample
3. The sample does not match the detector. Adjust the wavelength or change the detector according to the chemical nature of the sample
4. The detector has attenuated too much. Just adjust the attenuation.
5. The detector time constant is too large. The solution is to reduce the time parameter
6. The detector pool window is contaminated. The solution is to clean the pool window.
7. There are bubbles in the detection cell. The solution is exhaust.
8. The pressure measurement range of the recorder is inappropriate. Just adjust the voltage range.
9. Mobile phase flow is not suitable. Just adjust the flow rate.
10. The detector and recorder exceed the calibration curve. The solution is to check the recorder and detector, and make the calibration curve again.
4. Q: When doing HPLC analysis, the column pressure is unstable, what is the reason? How to solve?
Answer: The reasons may be:
1. There is air in the pump, the solution is to clear the air in the pump and degas the solvent;
2. If the proportional valve fails, replace the proportional valve;
3. Pump seal is damaged, just replace the seal;
4. The solution to the bubbles in the solvent is to degas the solvent, and change the degassing method if necessary;
5. Check the system leaks, find the leaks and seal them;
6. Gradient elution, pressure fluctuation is normal at this time.
5. Q: The column pressure is too high during the acceptance test of the HPLC column I purchased. Why?
Answer: High column pressure is the most common problem encountered by HPLC column users. There are many reasons for this, and it is often not a problem with the pillar itself. You can check the cause of the problem by following the steps below.
1. Remove the protection pre-column to see if the column pressure is still high, otherwise it is the problem of the protection column. If the column pressure is still high, check again;
2. Remove the chromatographic column from the instrument to see if the pressure drops, otherwise the pipeline is blocked and needs to be cleaned. If the pressure drops, check again;
3. Connect the inlet and outlet of the column to the instrument in reverse, and flush the column with 10 times the column volume of mobile phase (do not connect the detector at this time to prevent solid particles from entering the flow cell). At this time, if the column pressure still does not drop, check again;
4. Replace the sieve plate at the inlet of the column. If the column pressure drops, your solvent or sample contains particulate impurities. It is these impurities that block the sieve plate and cause the pressure to rise. If the column pressure is still high, please contact the manufacturer. In general, an online filter is connected between the injector and the guard column to avoid the problem of excessive column pressure. The Rheodyne 7315 filter provided by SGE is the best choice to solve this problem.
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