High Performance Liquid Chromatography(HPLC) as an important branch of chromatography, is an analytical technique to separate, identify, and quantify components in a mixture. using a high-pressure infusion system, the single solvents with different polarity and different proportion of mixed solvents, such as mobile phase, buffer pump into a stationary phase chromatographic column, each component is separated in the column, enter the detector for testing, so as to complete the analysis of the sample. This technique has become an important application of separation analysis technology in chemistry, medicine, industry, agriculture, commodity inspection.
Frequently asked questions
1 Why does retention time drift or change rapidly?
- Temperature control is not good, the solution is to use a constant temperature device, keep the column temperature constant;
- The mobile phase changes, the solution is to prevent evaporation and reaction of the mobile phase;
- the column is not well balanced, needs to balance the column for a longer time.
On the question of rapid change
- The flow rate changes, the solution is to reset the flow rate, so that it remains stable;
- there are bubbles in the pump, can be driven out of the bubble by exhaust operation;
- The mobile phase is not appropriate, the solution is to change the mobile phase or make the mobile phase properly mixed in the control room.
2 The cause of towing or double peaking?
- the sieve plate is blocked or the column fails, the solution is to reverse wash the column, replace the sieve plate or replace the column;
- There is a disturbance peak, the solution is to use a longer column; Change flowing phase or replace the selective column;
- The column may be overloaded, reduce the amount of sample.
3 Main reasons and solutions for insufficient HPLC sensitivity
- Insufficient sample size, the solution is to increase the sample size;
- The sample did not flow out of the column. The flowing phase or column can be changed according to the chemical properties of the sample.
- The sample does not match the detector. Adjust the wavelength or change the detector according to the chemical properties of the sample;
- Detector decay too much. Adjust the attenuation;
- The detector time constant is too large. The solution is to reduce the time parameters;
- Detector pool window pollution. The solution is to clean the pool window;
- There are bubbles in the test tank. The solution is exhaust;
- The recorder pressure measuring range is inappropriate. Adjust the voltage range;
- The improper flow rate of the flowing phase. Adjust the flow rate;
- The attending detector and recorder exceed the correction curve. The solution is to check the recorder and detector and recalibrate the correction curve.
4 When doing HPLC analysis, the column pressure is unstable. Why? How to solve it?
- there is air in the pump, the solution is to clear the air in the pump, the solvent degassing treatment;
- If the proportional valve fails, replace the proportional valve;
- pump gasket damage, replace the gasket can;
- the bubble in the solvent, the solution is to the solvent degassing, when necessary to change the degassing method;
- System leak detection, find out the leak point, the seal can;
- Gradient elution, when pressure fluctuations are normal.