Monday 19 October 2009

Help on: Retention time shift in HPLC analysis


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"I am using 0.1 % TFA in water + 100 % Acetonitrile as mobile phase in gradient RP-HPLC. But since last month I have problem of RT shift. I am not able to rectify it. Please suggest solution for the same. "

"Some of the most common problems which can cause a shift in retention time are temperature, mobile phase composition and equilibration of the column. I shall discuss each of these first and then move on to other potential causes of your problem.
Is your column in a column oven and maintained at a constant temperature throughout your analysis? Temperature is a variable in HPLC analytical methods (we often use it to adjust retention during the method development process) and changes in temperature can result in shifts in retention time. A method that runs at ‘ambient’ temperature can be subject to large variations in the room temperature of a laboratory. So even if the method says ambient it is best to control the temperature.
Mobile phase composition:
How do you create the gradient for your method? If you premix your mobile phases of 0.1% TFA in water and 100% acetonitrile to result in one reservoir which corresponds to the method starting conditions and another which corresponds to the end conditions then it is possible that changes in the volatile components of these mixtures due to evaporation could lead to shifting retention times. However, if you use online mixing and simply have 0.1% TFA in one bottle and acetonitrile in the other then this cannot be the source of the problem.
Column equilibration:
Generally, when using RP-HPLC methods the column can be equilibrated relatively quickly. Check that you have allowed enough time at the end of each injection before the next injection. Typically 10 column volumes will be sufficient (refer to the previous post on column equilibration). An exception to this is when using ion-pairing reagents, these can take a significantly longer time to equilibrate due to the adsorption of the ion-pairing reagent on the surface of the stationary phase. Although TFA can act as an ion pairing reagent I have never had an issue with equilibration at the concentration you are using.

If you can rule out these common causes then we need to look more closely at both the HPLC method that you are using and the problem that you are experiencing. Some questions to consider:
You say your problem started last month, was the method working well before this, and for how long?
If the method has been trouble free for a significant length of time then it indicates that the problem is more likely to be due to a problem with the HPLC system or the column. Consider carefully, are you certain that you haven’t changed any of the method parameters? Review the method thoroughly. Run the method on a different column (preferably new) and see if the drifting retention time is observed.
If the method hasn’t been proven to work then the problem could be due to the method parameters, e.g. sample preparation could be inadequate leading to progressive contamination of the column, or the column and analytes may not be completely compatible.
Are all the peaks in the chromatogram moving (including the solvent front) or is it restricted to certain peaks only?
If all the peaks, including the solvent front, are shifting then the problem may be due to system leaks, or air bubbles in the pump. If the solvent front does not move then the change may be linked to the mobile phase and method conditions. If the shift in retention time is only observed for certain peaks then it may be due to a change in the column which only effect certain types of functional groups , e.g. subtle pH change.
What is the nature of the retention time shift, changing within a run, changing between runs, increasing with time, decreasing with time, no pattern in the drift?
The way in which the retention time is shifting will provide extra information on potential causes and may support a diagnosis based on the discussion above.
A note regarding your mobile phase. TFA does not actually have much buffering capacity, it is very convenient for acidifying the mobile phase when you need a volatile buffer. Depending on the parameters of your gradient it may be that there is very little acid at the end of your gradient. You can add TFA to the organic portion of your mobile phase, i.e. 0.1% TFA in acetonitrile and thus the amount of TFA present throughout the gradient remains constant. This may result in some improvement in your chromatography, however I don’t think that this would explain the shift in retention time that you are experiencing.
Please use the comments if you would like to continue this dialogue and provide some more information which may help to diagnose your problem.”

Do you have anything to add? Feel free to leave a comment.


  1. I'm having this problem, since about a month ago, and all peaks are very consistently delayed ~60%, with no apparent change to the method, solvents, columns, compounds, etc.

    I've changed columns to no effect. I assume that this has something to do with the pumps, but the pressure is constant and at the same level as before the shift, as well as the external flow rate. I'm not quite sure where to go from here, but I'll try to flush any air from the lines which may be causing the delay.

    1. A delay of 60% is quite large, I think that the most likely cause is related to an increase in the organic content of the mobile phase composition. Although you believe that there has been no change to the conditions, I would check this thoroughly.

      It is unlikely that the change would be due to the pump if the flow rate and pressure are satisfactory but it could be due to a problem with the mobile phase mixer.