About the Reason and Solution of the HPLC FAQs
1. Why does the retention time sometimes drift and sometimes change rapidly when analyzed by HPLC( such as SCX HPLC Column, NH2 HPLC Column)? How to solve it?
① The temperature control is not good. The solution is to use a constant temperature device to keep the HPLC column temperature constant.
② The mobile phase changes and the solution is to prevent the mobile phase from evaporating, reacting, etc.
③ The column is not well balanced, and the column needs to be equilibrated for a longer time.
About rapid change
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. The air bubbles can be expelled by exhaust operation.
③ The mobile phase is inappropriate. The solution is to change the mobile phase or make the mobile phase properly mixed in the control room.
2. Main reasons and solutions for insufficient HPLC sensitivity.
① Insufficient sample size, the solution is to increase the sample size.
② The sample does not flow out of the column. The mobile phase or column can be changed depending on the chemistry of the sample.
③ The sample does not match the detector. Adjust wavelength or change detector based on sample chemistry.
④ Too much attenuation. Just adjust the attenuation.
⑤ The time constant of the detector is too large. The solution is to reduce the time parameter.
⑥ Detector’s pool window is polluted. The solution is to clean the pool windows.
⑦ There are air bubbles in the test cell. The solution is the exhaust.
⑧ Improper pressure measurement range of the recorder, just adjust the voltage range.
⑨ The mobile phase flow rate 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 redo the calibration curve.
3. Why is the column pressure unstable when doing HPLC analysis? How to solve it?
① There is air in the pump. The solution is to clear the air in the pump and degas the solvent.
② The proportional valve is invalid, just replace the proportional valve.
③ The pump gasket is damaged, just replace the gasket.
④ The solution of air bubbles in the solvent is to degas the solvent and change the degassing method if necessary.
⑤ Check the system for leaks, find the leaks, and seal them.
⑥ Gradient elution, pressure fluctuation is normal at this time.
4. The column pressure is too high during the HPLC column acceptance test. Why?
Excessive 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 column itself. You can check the cause of the problem by following these steps:
① Remove the protection pre-column to see if the column pressure is still high, otherwise, it is a problem with 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 volume of the column. (Do not connect a 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. Only for used columns.
④ Replace the column inlet sieve plate. If the column pressure drops, it means that your solvent or sample contains particulate impurities. It is these impurities that will block the sieve plate and cause the pressure to rise. If the column pressure is still high, contact the manufacturer. In general, connecting an in-line filter between the injector and the guard column can avoid excessive column pressure.
5. What are the reasons for bubbles in the mobile phase?
① Bubbles are often formed in mobile phase solutions due to dissolved oxygen or air.
② The liquid path resistance is relatively large, and vacuum bubbles appear when the liquid is absorbed.
③ When the system starts to work, the air in the flow path cannot be exhausted.
④ Air was mixed during sample injection.
6. The amino column hurts the column when entering the acid sample. If the column efficiency is reduced and the peak shape is changed after using for a period of time, how to recover?
Flush the column with 5-10 times the column volume of 0.5-1.0% NH3 in acetonitrile-water (50:50) solution (after washing, wash the excess ammonia with an alkali-free mobile phase), and then proceed. When analyzing such acidic analytes, it is recommended to add a little ammonia such as 0.1% to the mobile phase.