Thursday, 26 April 2012

Help on: Retention Time Change


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“I am working with TFA in my mobile phase and my analyte elutes at 13.5 minutes. In the same day the retention time has changed by 0.2 minutes. It is normal? I'm using an isocratic method.”

"If you experience a change in retention time during a run then it indicates that something in the method conditions is changing, most likely a temperature or mobile phase pH shift. In my opinion, 0.2 minutes is a very small change for a peak at 13.5 minutes (~1.5%) and may not necessarily be a big problem. However if the shift continues, it may become an issue. I suggest that you check the following:
  • Is the column temperature controlled? If not, then that is probably the source of the RT shift and if you have no way to introduce temperature control then you will experience RT shift. If you do have a column oven, then ensure that it is working correctly and that there is a suitable length of tubing to and from the column also being temperature controlled.
  • Is your analyte ionisable? TFA is a very useful acid modifier for HPLC mobile phases and is successful for a range of molecules. However it is an acid modifier rather than a buffer and so some ionisable analytes may not be suitably pH controlled using only TFA.
If the problem does not escalate then it is probably fine to continue. I suggest that you monitor the retention time change. For example, does it slowly change over the course of the run or suddenly shift, or does it always change in one direction (increasing or decreasing retention time)?"

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