Chromatography Forum

Hi I was wondering if anybody can enlighten me as to the exact reason why you see a decrease in signal with an increase in flow rate. I understand there is a lower residence time within the flow cell of the sample, but how does this exactly translate into a lower signal? Thanks for your help.

You are talking about UV detection? I always thought that this was due to a change in refractive index, sure would like to hear about any "educated" thoughts on this.

Higher flow rate = lower analyte time in detector = less time for analyte absorbance to be monitored = smaller peak areas

That would make sense to me, but that doesn't mean it's right.

Does the refractive index of a solution really change at higher flow rate? You learn something new every morning!

Mags,

H/u curve does it ring a bell ?

higher flowrates, less efficiency, broader peaks, so lower peak heights ( = lower signal, if this is what you mean ??)

pHilippe

Yes it is UV detection.

HW: Refractive index change--hadn't thought about that. I know changing the flow rate in the middle of a run will cause blips and what not in the signal due to refractive index changes, but I was not aware it could affect the abosrbance of an anlayte, can you elaborate on this for me>?

PJ8: The underlying factor of the absorbance change is the fact that the analyte spends less time in front of the detector, this is indeed a direct result of the increased flow rate. However, what is the fundamental explanation for this? Does it relate to the speed at which the detector is capturing the data? Or is it at the molecular level, i.e. there are fewer molecules are actually absorbing the light because they are travelling through the detector faster? My first inclination is that the absorption process happens on a much faster time scale than the time it takes for the analyte to cross the 1cm flow cell length. With that in mind I changed the collection rate of the detector and the signal was exactly the same. The leads me to believe that the fundamental cause is on the molecular level. I'm sure I heard the explanation in chemistry class, but have just forgotten. I've tried digging up the information, but it's suprisingly hard to find, so I thought I'd ask here.

Philippem: I should have clarified more, not only does the height decrease, but the area also decreases, so I would think that it is not an efficiency issue. The funny thing is, the decrease seems to be linear. I.e. 2x increase in flow rate = half the signal. I just can't put it all together to come up with an explanation.

Thanks again guys

Yes it is UV detection.

HW: Refractive index change--hadn't thought about that. I know changing the flow rate in the middle of a run will cause blips and what not in the signal due to refractive index changes, but I was not aware it could affect the abosrbance of an anlayte, can you elaborate on this for me>?

PJ8: The underlying factor of the absorbance change is the fact that the analyte spends less time in front of the detector, this is indeed a direct result of the increased flow rate. However, what is the fundamental explanation for this? Does it relate to the speed at which the detector is capturing the data? Or is it at the molecular level, i.e. there are fewer molecules are actually absorbing the light because they are travelling through the detector faster? My first inclination is that the absorption process happens on a much faster time scale than the time it takes for the analyte to cross the 1cm flow cell length. With that in mind I changed the collection rate of the detector and the signal was exactly the same. The leads me to believe that the fundamental cause is on the molecular level. I'm sure I heard the explanation in chemistry class, but have just forgotten. I've tried digging up the information, but it's suprisingly hard to find, so I thought I'd ask here.

Philippem: I should have clarified more, not only does the height decrease, but the area also decreases, so I would think that it is not an efficiency issue. The funny thing is, the decrease seems to be linear. I.e. 2x increase in flow rate = half the signal. I just can't put it all together to come up with an explanation.

Thanks again guys

mags, sorry I misunderstood you, thought you where talking about a dip in the baseline one can sometimes (when mobile phase strongly absorbs) see when speeding up the flow.
A peak area obviously changes proportional to the flow rate because the area is proportional to Absorbance x time. The hight schouldn´t change, though, unless your response time is set way too low, or the flow rate is far from optimal.

Thanks HW:

"A peak area obviously changes proportional to the flow rate because the area is proportional to Absorbance x time"

TIME.

The UV detector sends out a certain number of photons, then records how many are absorbed. The signal you see is the number of photons absorbed over a certain amount of time as the analyte passes through the detection window. So even if the analyte is the same concentration, it spends less time in front of the detector, a fewer absolute number of photons are absorbed. (The lamp did not send out the same number of photons in the shorter time)

This makes sense because even if I did increase the collection rate, the same number of photons are still being absorbed by the analyte. That number is lower in the case of the faster flow rate.

I think I made sense of it, if I'm still wrong or somebody can explain it to me, please do, it's driving me insane.

Hi mags,

The lower flow rait, the smaller volume the anayte elutes in.

Best regards

mags, a mental prop might be: If one stops the flow during a peak the peak area will approach infinity as the stop time approaches infinity.....its that simple.

danko, if you stay near optimal flow than the elution volume stays about the same (flow x time ~ constant?)?

Hi HW Mueller,

That’s right regarding the column efficiency part; witch is of grate importance to the peak height as an inverse function of its width. But I was referring to the conditions in the flow-cell. If the flow rate is too high and the flow-cell volume is too small, then the first fraction of a peak (especially a broad one) will be out of the detector before the last fraction of the peak arrives in the flow-cell. Thus it will not reach “fullâ€

Absorption is not the absolute number of photons absorbed. It is defined as (the log of) the ratio between light intensity before and after the detection cell. The absorption is directly proportional to the concentration, the cell length (here constant), and the absorbtivity of the analyte (here constant). Thus, as HW Mueller wrote, if you stop the flow rate, the concentration will remain constant and the absorption signal will be constant, as long as the light is on.

For a concentration sensitive detector (eg. UV) the peak height is independent of the flow rate whereas the peak area is inversely proportional to the flow rate due to the lower residence time. Conversely, for a mass sensitive detector, the peak height is proportional to the flow rate, whereas the area is constant.

On top of this comes the decrease in column efficiency (broader, lower peaks) that typically is observed at higher flow rates according to the van Deemter H/u curve.

I responded already to the other post, but looking at the replies here, it seems that it is not clear what is going on. So I will try to explain again.

If I have a hypothetical ideal column whose plate count does not change with flow, then the hieght of the signal will not change with flow for all concetration-sensitive detectors, which means UV and RI detectors. If I increase the flow 2-fold, the peak area decreases 2-fold, but the height of the signal remains the same.

In real life, the column performance, i.e. the plate count, changes with flow rate. Under normal circumstances, the peak width (inside the column) gets larger with increasing flow. Or, the peak width measured by the detector or on the computer screen does not decrease 2-fold, when I increase the flow 2-fold. It decreases less than 2-fold, due to the fact that peak inside the column got somewhat wider, since the plate count at higher flow is lower. Since the peak inside the column is wider, the concentration is lower, and you get a lower signal in your UV detector. However, the peak area multiplied by the flow rate will remain constant.

HW: Thanks for the simple explanantion, My problem was I kept trying to paint a mental picture in my head of the problem, and your mental prop was a very good one.

Tobais, thank for getting that incorrect picture out of my head. Although, I'm still curious as to why both the area and height decreased. It doesn't appear that there is any significant change in efficiency.

I had forgotten the fundamental difference between mass and concentration sensitive detectors, thanks all for reminding me.

I strongly doubt that the rise time of your UV detector is set too low, but maybe your integration parameters (number of points per time) are wrong?
On the other hand, "feeling/believing" and "knowing" are often two different things (regarding "efficiency" here).