Chromatography Forum

Hi all

I'm in the process of optimizing a RP-HPLC method. So when running the sample on a 4.6x250mm column (C18, 5um, 100Å, 1ml/min) I get a fairly narrow peak.


However when running on a semi-prep column (10x250mm, 5 ml/min), with only the diameter being different, I get a peak displaying a much lower area.




The samples are injected from the same vial. The 2 columns are located on the same HPLC system (agilent 1260).

Is there a rational explanation to this?

In advance thanks
Erik

Flow-rate increased five-fold -> Peak-area decreased five-fold.

Increase sample size proportonal to column volume in order to maintain surface

Thanks guys, basic stuff I guess

However I only observe a 3.2 fold decrease in peak-area!

\Erik

Thanks guys, basic stuff I guess

However I only observe a 3.2 fold decrease in peak-area!

\Erik

Oh, I read the first number of the analytical run's peak area as "8", so ~18,000 x 5 ~ 87,000 looked quite good Now I see it's indeed a "5", correct?

First of all, those two runs were performed on the same HPLC? If you used different machines, don't try to compare absolute peak areas.
If it indeed was the same HPLC, first thing that comes to my mind is that you might be out of the linear range.

Hi
Yes it's a "5", a bit blurry sorry for that.

Yes the 2 runs where performed on the same system, utilizing a column switch valve though. The 4.6mm column goes trough one column heater (3um) and the 10mm column goes trough the other column heater (6um).

Would the different volumes (3um vs 6um) of the individual column compartments have an effect?

Any input is appreciated.

\Erik

Different extra-column-volumes might have an impact on peak-shape, but not on absolute peak areas. On a given HPLC peak areas should (!) only depend on sample concentration, injection volume, flow-rate and detection wavelength (did I forget something?).
How does the peak area look like when you increase the injection volume five-fold for the prep run?

Finally got to test an five fold increase of the injection volume on the semi-prep column, and the peak areas are now comparable.

thanks
Erik

Thank you for the feedback! It's nice to hear that a problem could be solved .