Chromatography Forum

I have run the same concentration standards in HPLC with two different columns, one is the Shim-pack VP-ODS (150 x 4.6 mm, particle size 4.6 microm) and another one is the Cronusil-M C18 column (250 x 4 mm, 5 microm), and the peak area was very different.

The Shim-pack's peak area was almost twice compared to Cronus'. Is there any possible reasons for such results?

Thank you!

If the other factors are all the same, the retention time should be shorter on the 150mm length column, that will affect areas.

Erm, no, it should not affect peak areas. It will affect peak HEIGHTS.
All other factors being the same, peak areas should be the same on both columns.
Did you use the same flow-rates on both columns? And double-check that you used the same injection volume and detection wavelength.

Hi Miniteh,

I agree with HPLCAddict on this one. With the same ID, the shorter column should afford a taller peak given same eluent conditions, certainly for isocratic conditions...but I'll ask also if these separations are both isocratic or gradient? And, if they are gradient, were they scaled to the different column lengths? Agree also with HPLCAddict's and Consumer Product Guy's further cautions (all conditions for injections on both columns would have to be the same as possible for an apt comparison).

I've a question, have you tried an injection of the same standard (at the same concentration) without a column in the HPLC, and compared the area(s) of the injection without a column in place to the injections with each different column in place? Perhaps the peak area(s) without a column in place and the Shim-pak may be the same?

One possibility may be adsorption of the analyte on the Cronus column. Don't know much about that manufacturer, myself.

Please, see what you think, and thank you.

I have run the same concentration standards in HPLC with two different columns, one is the Shim-pack VP-ODS (150 x 4.6 mm, particle size 4.6 microm) and another one is the Cronusil-M C18 column (250 x 4 mm, 5 microm), and the peak area was very different.

The Shim-pack's peak area was almost twice compared to Cronus'. Is there any possible reasons for such results?

Thank you!

Is this reproducible?

The linear flow through the 4.6 mm column is slower than that when 4.0 mm is utilized.
Slower linear flow = longer time for detection of the compound = broader peak (whilst roughly the same height ) = greater area.

Best Regards

Danko, we don't know if the flow has been scaled. From the original post, I'd suppose no.
And even if it has been scaled, going from 4.6 to 4.0 won't give you double the peak areas, but only ~32% more.

I took for given (a precondition) that the volumetric flow (the pump parameter) remaned constant.
And that would've resulted in a slower linear flow through the column with a larger diameter.
So it's the other way around the area in the thicker column would be larger. That's also what has been observed by Miniteh.

If you like to play, you could confirm the above by using the same column and only set the flow rate down by 15%.

Best Regrds

Erm, I think you're on a wrong lead .
You're right, with the same flow-rate (in mL/min), linear flow in a 4.6 mm column will be slower than in a 4.0 mm column. But we're not measuring peak area in the column . In the detector flow-cell, it's 1 mL/min in both cases, so no difference in peak areas. Peak area will change if you change the flow-rate, no matter what the column diameter is.

As I mentioned before, if you like to play, then do the experiment I suggested to you earlier.
If not then do the following mental experiment: If you stop the flow while the compound you're measuring is in the flow-cell you'll get a never ending peak/area. You see?

Yes, Danko, you're absolutely right. I didn't doubt your suggestions.

I took for given (a precondition) that the volumetric flow (the pump parameter) remaned constant.
And that would've resulted in a slower linear flow through the column with a larger diameter.

This is the part is was commenting on. Changing the linear velocity in the column (!) will NOT change peak areas. Peak areas will change only if you change the flow-rate (the pump parameter).
So, concerning the peak areas it doesn't matter if you use a 4.0 or 4.6 mm column if the flow-rate is kept constant.
I understood the original poster did NOT change the flow-rate but just swapped the columns. This definitely does not explain a change in peak-areas.

I'd suggest to you to carry out the experiment I mentioned earlier and then we will talk about the reasons for what you'll observe.

Best regards

Danko, I'd suggest you lean back, take a deep breath and re-read all posts . And don't try to see something that's not there.

All your statements about changed peak areas when changing flowe rate are right, the mental experiment thing is a classic and you´re right in mentioning it.

All I was commenting on is this one:

The linear flow through the 4.6 mm column is slower than that when 4.0 mm is utilized.
Slower linear flow = longer time for detection of the compound = broader peak (whilst roughly the same height ) = greater area.

and this one:

I took for given (a precondition) that the volumetric flow (the pump parameter) remaned constant.
And that would've resulted in a slower linear flow through the column with a larger diameter.

And that's simply wrong! What you're saying is that changing column diameter while keeping flow-rate (in mL/min) constant will change the linear velocity in the column and thus peak areas. The first part is true (linear velocity in the column will change with diameter) but the second not! The linear velocity in the column will change, but not in the detector's flow-cell, so peak areas are constant.
Peak areas will change only if you change the flow-rate and thus the linear velocity in the flow-cell, too.
Think of it this way: If you have a flow-rate of 1mL/min, the mobile phase will run at 1 mL/min through the flow-cell, no matter what size the column in between is (or even if there's no column at all).

HPLCaddict, You're right. Maybe more detailes are needed in order to find out what's happened.

Best Regrds

Hey, Miniteh, are you still out there? Did the peak area differences resolve themselves?