high performance liquid chromatography, broad peaks

HPLC column care and maintenance

The column is a key factor in HPLC chromatography separation hence preventive care and maintenance of HPLC column required for better performance and increasing span life of a column. In HPLC analysis every chromatographer know the importance of column, because of the theoretical plate number, resolution, tailing factor, peak symmetry, and system suitability depend on the column performance. 90% of analysts work in HPLC chromatography carried out by the reverse phase chromatography, therefore, some important points consider that to protect and helpful in growing the life and performance of the HPLC column are mentioned here.

1. Install the column appropriately when you replace or change it.

2. Confirm the performance of new column supplied by the vendor.

3. Always use guard column to protect from impurities and other foreign particulates.

4. Protect the column from bumps, knocks and mechanical shock.

5. Always use HPLC grade solvents and water for analysis.

6. Always use fresh water and buffer for analysis.

7. To prevent impurities and foreign particles, filter the mobile phase by using 0 .45 μ filter

8. Filter the sample using 0 .2 μ filter before injection.

9. Degas all mobile phase and samples.

10. Periodically check the column performance.

11. Keep the back pressure below 4000 psi.

12. Avoid making changes in any sudden pressure.

13. Always saturate the column with the mobile phase before sampling.

14. Maintain the temperature of the column oven if available.

15. The pH of the mobile phase or buffer should be in between 3.0 to 7.5 pH.

16. Avoid use of phosphate buffer more than 20 mM.

17. Avoid using a viscous buffer.

18. Do not overload the column.

19. Avoid injecting biological samples directly into HPLC injectors.

20. Avoid injecting of high acidic or basic sample directly in the sample loop.

21. Avoid store the column with buffer.

22. Never let the column dry, it should be wet with the appropriate solvent

23. Avoid working with high flow rates, it creates pressure

24. Wash the columns properly after every use with HPLC grade water and methanol/acetonitrile.

25. Store the column in a solvent like methanol/acetonitrile.

To improve column performance, follow the above points in your regular practice.


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