Chromatography Forum

My peaks are shifted to right i.e their retention time is increased. What can be possible reasons. I was using all the same conditions as previous one. it can be problem in pH meter which I used to adjust the pH of my buffer ? (i was using pH of 2.3 adjusted with 85% orthophosphoric acid). What can be other possible reasons ?

Ur valuable suggestions will really help me to solve my problems.

Perhaps, you have a leak somewhere in your system. Check at the flow at the outled of your system, by timing how long it takes for a certain volume to come out. As an example, use a 10 ml volumetric cilinder and record the volume after 10 minutes. Then compare this to the flow rate of your pump. If the volume is less than 10 ml, you probably have a leak. Using this procedure, you can check evry component of your system.

Good luck,
Gilbert Staepels

In addition to Gilbert's suggestion, look at your dead time (t0). In particular, if all the peaks, including t0, have increased by the same percentage, a low flow is almost certainly the problem. If peaks have shiffted by different percentages, then flow is *not* the problem.

You have already considered pH. Other possibilities include temperature, evaporation of organic solvent from your mobile phase (or incorrect proportioning if using on-line mixing) or a change in your column (this more commonly results in decreased retention, but an increase *is* possible)

There was no leakage and no pressure change, so no fluctuation in flow rate also. Yes all the peaks are proportionately shifted to right (higher retention time).

I want to know, whether lower pH causes higher retention timer or higher pH causes.

Impact of pH on retention times is analyte dependent, so no generalizations can be made, except that changes in pH should not impact retention time of an un-retained solute.

If you running a gradient, how long do you (re)equilibrate with starting mobile phase between injections?

If tR for all peaks increased, including that if an un-retained solute, then it sounds like the extra-column volume in your system has increased. Have the connections to and from the injector port to the column, and from column to detector been re-plumbed, perhaps with longer or larger ID tubing? Are you using a different sample loop than before? Do you make manual injections or use an autosampler?

I suggest running a gradient to 1% acetone (no column) to measure dwell volume, and ensure your pump can make a linear gradient.

Yes all the peaks are proportionately shifted to right (higher retention time)

To repeat what DJ said (in different words): if an unretained peak (t0) shifted, there are only two possible explanations:
1. your column got larger (not highly likely), or
2. your flow rate changed.