I recently observed the shift of the whole profile little by little due to the passage of time / long term use of the column. In 2 weeks, the shift became 0.4min much later and the back pressure had increased from 1200psi to 1700 psi. My best guess of the shift was due to the clogging. Flushing may be needed.
Anyone has a better explanation?
Typical clogging effects.
Flushing out can help if what clogged the colum is soluble in the mobile phase.
If not you can perform carefully these operations:
1 invert the column (inlet become outlet)
2 flush out the column with the mobile phase (isocratic) for some hours at reduced flow rate (let's say not more than 30% of normal flow rate), take note of the initial pressure and the pressure each hour so you can see when the pressure does not change anymore once the pressure remain constant for about 2 hours you can consider step 2 ended.
3 at this point change again the inlet outlet of the colum to restore the original configuration
4) flush out with mobile phase the column gradually starting from 20% of the normal flux up to the 100% of the work. Flow increases of 10% each hour should match
5) let flow at working flow overnight.
6) check the column performances
Best
Most HPLC back pressure problems are cause by blockages in the system or guard col. In order to be sure that, check pressure with/without column. If the column pressure is high, back flush the column. For the shift in retention, this can be cause by Column aging, Column contamination, A poor column/mobile phase combination for your analytes, Insufficient column re-equilibration, Changes in flow rate or column temperature. Change in mobile phase may change your retention. Sometimes mobile phases can differ because of the way they are mixed in the lab, so Be sure you use a consistent method for mixing your mobile phase.
0.4 min shift may be of no concern. Can not answer the question w/o knowing what the expected retention time of the peak is/was. Examples: Small shift over a 30 minute run time would be normal. Small shift over a short 8 minute run would indicate a problem. The back pressure increase you have observed is far more significant and indicates a more serious problem with the method or sample introduction.
Perhaps this attached and linked article will help you? "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it."
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Hi,
shift of 0.4min is very small if your run time is more so need not to worry about the same and follow some good flushing or washing practise for column after use. You can go through following method if you find your column id clogged,
1.Introduce Water: ACN (80:20) at 1.0 mL/min for at least 30minutes.
2.Introduce 0.1% OPA in H20: ACN (80:20) at 1.0 mL/min for at least 40minutes.
3.Introduce 0.1% OPA in 100% ACN at 1.0 mL/min for at least 40minutes.
4.Introduce Water: ACN (80:20) at 1.0 mL/min for at least 30minutes.
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