Relation between ret. time and peak area

[miro2009]

Hello,

I've been trying to develop an RPC method for separation of a certain compound using gradient elution. I've reached an acceptable gradient where my compound elutes let's say at 29 min. Finally, I just made small adjustment to the gradient slope where all components are eluting with the same separation and selectivity but all shifted 3 minutes earlier which was intended, to shorten the run time. This is all fine so far, but what I can't understand is the observed general decrease in peak area of all components by ~ 15%, shouldn't the shorter retention time be accompanied with less dilution of the eluents in the mobile phase, and accordingly higher absorbances/peak areas?
Awaiting your opinions

[Uwe Neue]

Did you increase the flow rate by 15%?

[miro2009]

Did you increase the flow rate by 15%?

No, this is what confuses me, the flow rate was kept constant 1ml/min in both methods

[adam]

If you made the gradient a little steeper, which is what it sounds like, the peaks would exit the column a little bit faster (since the organic content is higher when each peak is eluting). This could explain the drop in area count.

So, you didn't change the flow but you changed the effective velocity of the components a little bit.

[miro2009]

If you made the gradient a little steeper, which is what it sounds like, the peaks would exit the column a little bit faster (since the organic content is higher when each peak is eluting). This could explain the drop in area count.

So, you didn't change the flow but you changed the effective velocity of the components a little bit.

Dear Adam, yes the gradient was made a little steeper, so components eluted a little earlier as expected... but that should lead to a smaller elution volume as well (since retention time now is shorter) and therefore increase in peak area, not drop, or?

[adam]

I think what you're describing would effect peak height, but not peak area. If the peak elutes earlier - and therefore sharper (or in a smaller volume if you look at it that way) - then you get a greater peak height but equivalent peak area.

If you change the flow rate, then you see an effect on peak area but not peak height.

You are changing both the elution time and the effective velocity so I think you are seeing competing effects.

[Uwe Neue]

At a fixed flow rate, the peak areas should remain constant, unless something is changing in the chemistry. For example, a change in pH could create a different absobance.

[danko]

In addition to Uwe’s pH dependent extinction coefficient, the polarity of the solvent (the environment) and in this context mobile phase composition i.e. more organic solvent at the moment of elution, is a factour that might affect the UV absorbance too.
Never the less, if I were in the situation, I’d run a blank injection following my sample injection and see whether or not some of the sample stays on the column. Very steep gradients might result in partial elution of the analyte/s – especially in protein separations.
Even better test would be to run a sample with the lates gradient followed by a blank with the previous gradient (the shallower one).

Best Regrads

[miro2009]

Dear Adam and Uwe, both the flow rate and mobile phase A and B composition were kept constant, maybe you're right I should be looking on the height on not the area, in reply to Danko the slope is 0.32%/minute which I believe isn't really steep, but I'll make the blank run idea to exclude incomplete elution..

Going back to varying the flow rate, of course it affects peeak area as Uwe said, and I'm actually using this concept for increasing sensitivity of another method I'm developing for a finished product of a very low concentration of protein and need to ask your opinions, it's also a gradient method, and keeping buffer composition (A:0.1%TFA, B:0.1%TFA in ACN) and gradient slope constant, I halfed flow rate to 0.5 ml/min and observe ~ doubled peak area (a little less than exact double) at same %B causing analyte elution, is it common that low flow rates could cause incomplete elution? is the only way to exlude it by making a blank run with another method of a higher flow rate for eg.?
Also, I'm already using 100ul inj volume on a 4.6x250mm C18 column and using WL 214 nm. Narrow bore columns cause us a lot of problems as some samples have a lot of impurities and backpressure overshoots with these columns.
Unfortunately with proteins WL range used is usually 210-220 nm, rarely up to 230nm, so you could already imagine baseline noise in this region, does anyone have other ideas to go around this? ie. increased sensitivity vs. lower noise
Sorry for all the questions, but I find having these discussions and sharing these "practical" opinions more useful than "textbook" facts

Awaiting your opinions

[danko]

Hi Miro,

Halfing the flow rate results in doubling the peak area – not because you elute more but because you elute the same amount over longer time. If you compare the peak heights at flow rate 1 mL/min and 0.5 mL/min you’ll observe approximately the same heights but the peak width with the 0.5 mL/min flow rate will be the double of that when the flow rate is 1 mL/min.
That leads me to the sensitivity thing: Acquiring larger peak area does not mean improving the sensitivity. The sensitivity is peak height dependent (actually dependent on the signal to noise ratio, but in your case you need to think peak height).
Finally, if you inject particles or whatever, that causes pressure increase, it’s not only a problem for narrow bore coluns but for wider diameter columns as well – it just take longer time for the bill to arrive to you.

Best Regards

[miro2009]

Hi Miro,

Halfing the flow rate results in doubling the peak area – not because you elute more but because you elute the same amount over longer time. If you compare the peak heights at flow rate 1 mL/min and 0.5 mL/min you’ll observe approximately the same heights but the peak width with the 0.5 mL/min flow rate will be the double of that when the flow rate is 1 mL/min.
That leads me to the sensitivity thing: Acquiring larger peak area does not mean improving the sensitivity. The sensitivity is peak height dependent (actually dependent on the signal to noise ratio, but in your case you need to think peak height).
Finally, if you inject particles or whatever, that causes pressure increase, it’s not only a problem for narrow bore coluns but for wider diameter columns as well – it just take longer time for the bill to arrive to you.

Best Regards

Thanks Danko for your reply, for the sensitivity issue I might have another opinion, first I see the doubling in both peak area and peak height, again the retention time is almost constant because the eluting factor here is %B (remember the gradient is constant),, have you tried making this direct comparison before and not notice this? I think your explanation may apply mainly to isocratic methods?
as for our sample impurities, of course we don't inject particles , it's just samples heavily loaded with dissolved electrolytes,vitamins, peptides and peptones mixtures in addition to our analyte of interest, so I just assume a bigger column would have a higher capacity..

has anyone else tried this flow rate lessening and observed increased peak area and height, using the same gradient?