Retention time unchanged

[Rachelle]

I initially have a gradient method (i.e. 2%B / min) using an RP-HPLC (VWD).
An unidentified peak elutes @ ~7'. I have a C18 with no embedded group. I am using as MPA: Tfa/ H20 & MPB: Tfa/ACN.

I modified the method to include an isocratic step (@ the starting %B of gradient step) for 10' before proceeding with the gradient step.

I expected all the peaks to elute later by an additional 10' shift.

Almost all peaks did except for the peak at ~7'. It roughly eluted at the same RT. It eluted during the isocratic steP.

FYI: The solvent front appears at ~ 4 minutes ( column 250 x 4.6; FR 1).

Does anyone know what is causing this unusual retention response?

Thank you . Rachelle

[HPLCaddict]

What is the flow-rate and the dwell-volume of the HPLC-system? If the flow-rate is low enough and the dwell-volume high enough, the peak might have eluted even with your gradient method in an isocratic step at the beginning...

[Rachelle]

Hi:
FR=1 ml/min
Dwell vol~0.6mL

Shouldn't the dwell volume cause a later Peak elution? Im seeing the peak elute at the same RT of 7' whether I use a step gradient right away or put an isocratic hold in the beginning of the run.

One odd thing about this peak is that it has no MS detection and does not have the same absorbance as the rest of the chromatogram (Max WL 230-234nm, the rest is 220nm)

[unmgvar]

have you injected only the diluent used for the sample and ran a gradient
and have you ran the gradient without using any injection?
it might be a system peak
these 2 injections will confirm that

[HPLCaddict]

Are you sure you've got the correct flow-rate? That column should have a dead volume of roughly 2.5 mL, so at a flow-rate of 1mL/min you should see the solvent front at ~2.5 minutes. 4 minutes is a bit late...

[unmgvar]

about 4 minutes is correct for flow of 0.6ml/min,
but anyway this is the column dead volume
what is the gradient delay volume?
a 2695 waters system is about 1.3ml, agilent is about the same. so it is about 2.15, 2.5 min
4.15+2.15= is 6.3 min
if you started from 100% A to B in gradient at 2%/min, so roughly the compound was coming out in the 2.5-3% ACN composition of the column, roughly calculated.
and now you did an isocratic mode starting at 2% ACN, so you are getting the peak of the column at about roughly the same conditions. hence the little change in RT.

[Rachelle]

have you injected only the diluent used for the sample and ran a gradient
and have you ran the gradient without using any injection?
it might be a system peak
these 2 injections will confirm that

I did inject a blank/diluent and saw no peak. I did not do a non-injection run. Also, two different samples have this same peak but with different area counts.
So, it is likely from the sample(s).

[Rachelle]

Are you sure you've got the correct flow-rate? That column should have a dead volume of roughly 2.5 mL, so at a flow-rate of 1mL/min you should see the solvent front at ~2.5 minutes. 4 minutes is a bit late...

The flow rate is 1mL/min. The column is 250x4.6mm, 5um, 300Angstroms. The system is an Agilent 1200.

[Rachelle]

about 4 minutes is correct for flow of 0.6ml/min,
but anyway this is the column dead volume
what is the gradient delay volume?
a 2695 waters system is about 1.3ml, agilent is about the same. so it is about 2.15, 2.5 min
4.15+2.15= is 6.3 min
if you started from 100% A to B in gradient at 2%/min, so roughly the compound was coming out in the 2.5-3% ACN composition of the column, roughly calculated.
and now you did an isocratic mode starting at 2% ACN, so you are getting the peak of the column at about roughly the same conditions. hence the little change in RT.

FR=1.0ml/min
Gr: 15-65%B (25')--- peak at ~ 7' = ~ 21%B

Gr: 15-15% (10'); 15-65% (25)--- peak at ~7' = 15%B