One of my colleagues raised the question why does she see a difference in peak area for the same sample on the same instrument with the same method but different columns. She had noticed about 1.5 difference in the area between two columns. Could a difference in back pressure generated by the columns affect the speed the analyte moves through the flow cell after it has come off the column? By the way this is a UV method. Thanks for any input.
Daren

Are you using the same flow rate on the different columns?

yes everything is identical but the columns.

Are the dimension of the columns the same? I had a similar case that I switched from a 4.6x150 to a 4.6x75mm column, everything else the same but the areas are different. It puzzled me.

I understand the area is a function of flow but... somehow the theory of mass conservation keeps ringing in my mind: If I inject the same mass into the column, should I get the same area regardless of the condition? Unless there is a leak but whatever goes in should come out right?

Daren,

I'm not sure what you mean by a difference of 1.5 in the area. Is this a response ratio or a difference of 1.5 peak area units.

I don't think the difference is due to back pressure or flow rate. All HPLC pumps are positive displacement pumps and the solvent compressibility due to a difference in column back pressure is probably small.

What your colleague is seeing is probably an effect of the different columns producing different peakshapes and thus different areas. The different columns probably differ in plate count, tailing factor, etc.

It is a single peak. The flow rate is the same. You are using a UV detector. How wide are the peaks? Is there a drastic difference in retention? What is the sampling rate of the detector, i.e. is it high enough so we get a good integration of the peaks? Tom suggested problems with tailing. You should see this if you look at the integration marks on the peaks. Can you give us some idea, what the analyte is? Also, just some other silly possibilities... what is the mobile phase? Is this a gradient? Is your sample ionizable and - if yes - are you using a buffer?

Daren,

Tom M. is right when he said it could be related to plate height.
If everything is the same and the areas under the curves are different. The plate counts/efficiency/HETP (different terms for the same measurement) of that column are different from the first column. I bet the peak width at 10% peak height is wider and the max. Height is reduced.
With a less efficient column (this refers to the packing of the column) there will be more axial and eddy diffusion and increased mass transfer (the 3 terms of the van Deemter equation) causing greater dilution and peak broadening resulting in the same amount of product coming out in a greater area.

Dannie - so what? Daren said that he is measuring peak area. This should remain constant, even if the plate count is changing. A peak that has a lower plate count is - at the same retention - wider, but not as high. At different retention times - as it is most likely - the peak area should still come out as a wash, unless there are issues around the peak integration. Or - there is something completely different going on.

Uwe Neue,

Oops, my bad... or maybe not....
Could it be that the laws of mass conservation are being broken by this disagreeable column?

Maybe Daren will give us some more information (if he hasn't got bored and left us)

Daren, you mention a high back pressure on one of the columns, what type of media are you using, what type of columns, and what pressures are you generating on each column. Are you seeing any difference in total product eluted from the column?

Thanks for all of the responses,
I just asked to look at the chromatograms and found that one of the peaks of interest is not homogeneous. The detection was at 215 nm and as the wavelength approaches 270 it begins to shoulder and split. Sorry about the ghost chase I assumed peak purity was already checked.
Thanks again
Daren