HPLC retention times

[Savepuck60]

Hello,

Our lab has been running a Thermo Vanquish HPLC for vitamin A complex using a C30 column, mobile phase A 5% UPC in methanol and B MTBE. The peak grouping nicely elutes from 6-12 minutes.

We are now transferring the method to an Agilent 1260 system for backup. Analyzing the same sample, the peak grouping elutes from 12 to 28 minutes, much later, using the same eluents, column etc.

Where should we start to look at first?

Thanks

[TylerSmith123]

You may have to update your methods for the new system. It may be impossible to exactly match the RT of your first trials on a completely new LC due to a variety of factors. Simply think of the tubing between the two instruments, the plumbing itself will be very different between them and even this volume can have a large impact on your retention times. In my opinion, it would be simply best to validate a method on this new system and ensure it's of the quality you desire.

[Hollow]

Maybe some more details?
is the method isocrstic or gradient?
Is the column the same (same-same or same-new)?
What about the column thermostates / temperature?
What is "UPC"?
Are the peaks separated the same just with higher retention times or is the separation changed?
Do you also see a change of t0 (the first baseline distortion)?

> only change in retention times >> check/compare flow rates of both systems

> if t0 is +/- the same, then check the mixing accuracy of the mobile phase.
> If isocratic and online mixing, try a run with a premixed mobile phase on both systems to compare
> are both systems of the same mixing type; high pressure vs low pressure mixing?
> if gradient, take into account the gradient delay volumes of the two systems

[DR]

My bet is on differing dwell volumes.

Once you confirm the flow accuracy of each system, calculating Vd is usually a matter of measuring the discrepancy between the programmed start time of a 10-100% B gradient and the time in your data when it can be seen as a deflection of your baseline (using water, and 0.3% acetone in water for your A, B MPs, no column). That time x your glow rate is your Vd a.k.a. gradient delay.

I think a Thermo system using viper connectors may have a lower Vd than a 1260, especially if the 1260 is a binary, high pressure mixing setup.

[per_oxid]

If you use the same column, mobile phase and flow I cant see how such a large shift is possible. Verify that you have correct flow rate and no leaks. Maybe measure volume of eluent to confirm the flow is accurate.

[Multidimensional]

A "Thermo Vanquish" vs some version of an Agilent 1260 system (you do not state which pump and there are many types) are huge variables in method development. It is always unreasonable to expect identical results from one system to another. The method may not even be running correctly on the first system, so besides obvious differences in plumbing, tubing ID and length, flow cells, dwell volume etc, far too many reasons for different Rts to provide an answer on a forum like this one. Please consult a professional chromatographer who can come on-site and go over the TWO HPLC system and the method used. An experienced chromatographer should be able to easily identify areas that need to be addressed.

And what exactly is "UPC" ?