Shift of Peaks (higher retention time)

[mobunvar]

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.

[gstaepels]

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

[tom jupille]

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)

[mobunvar]

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.

[DJ]

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.

[tom jupille]

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.