High-performance liquid chromatography (HPLC) is an analytical technique used to separate the components in a mixture, and to identify and quantify each component. In this article we have mentioned 39 simple tips for HPLC usage.
1. Always use high-quality solvents and reagents.
2. Use the correct column for your sample type.
3. Keep the column clean and free of contaminants.
4. Use a guard column to protect the analytical column.
5. Use the correct mobile phase composition for your separation.
6. Optimize your gradient program for best results.
7. Keep the flow rate consistent throughout your run.
8. Use a degasser to remove air bubbles from your mobile phase.
9. Check for leaks before starting your run.
10. Use a UV detector to monitor your eluent.
11. Calibrate your detector regularly.
12. Keep the detector cell clean and free of contaminants.
13. Use a reference wavelength to improve sensitivity and accuracy.
14. Avoid using buffers that can damage the column or detector.
15. Store columns properly when not in use.
16. Avoid exposing columns to extreme temperatures or humidity levels.
17. Flush the column with #solvent before starting a new run.
18. Monitor system pressure during runs to detect any issues early on.
19. Keep track of retention times for future reference and troubleshooting purposes.
20. Use a sample loop to inject precise volumes of sample onto the column.
21. Filter samples before injection to remove any particulates or impurities that could damage the column or detector.
22. Use an autosampler for consistent and accurate injections.
23. Use an isocratic method if possible, as it is simpler and more reproducible than gradient methods.
24. Check pH of mobile phase, as it can affect separation efficiency.
25. Avoid using too high of a flow rate, as it can cause band broadening.
26. Use an appropriate injection volume based on sample concentration and sensitivity requirements.
27. Check temperature settings, as temperature can affect retention times and separation efficiency.
28. Use appropriate guard cartridges based on sample matrix complexity.
29. Avoid overloading columns with too much sample, which can cause peak distortion and poor resolution.
30. Check for column degradation regularly, as it can affect separation efficiency and peak shape.
31. Use a column heater to improve separation efficiency and reproducibility.
32. Use a column oven to maintain consistent temperature throughout the run.
33. Avoid using too high of a mobile phase flow rate, as it can cause band broadening and poor resolution.
34. Use a UV detector with appropriate wavelength settings for your analytes of interest.
35. Check for baseline drift during runs, which can indicate detector or column issues.
36. Avoid using too high of a sample concentration, which can cause peak distortion and poor resolution.
37. Use an appropriate injection solvent based on sample solubility and compatibility with mobile phase.
38. Check for air bubbles in the system before starting runs, which can cause pressure fluctuations and poor reproducibility.
39. Avoid using dirty or contaminated syringes for injections, which can damage the column or detector.
Read also: 10 Important Tips for HPLC Mobile Phase
Resource person: Atefe Nasrollahi
