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signal vs flow rate
Posted: Wed Nov 08, 2006 8:39 pm
by mags
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.
Posted: Fri Nov 10, 2006 1:17 pm
by DR
Your peak area is a function of absorbance and time. The length of time a given slug of sample is absorbing light is proportional to the total area. Think of it like a large person getting between you and a TV. Your level of irritation will increase in inverse proportion to the speed of the person.
Posted: Sun Nov 12, 2006 8:07 pm
by Uwe Neue
DR explained the reason why the peak area gets smaller when you increase the flow rate. However, the signal does not become lower with this effect. The reason that the signal becomes lower with increasing flow rate is a decrease in plate count. The decrrease in plate count makes the peaks proportionally wider, and therefore also with a lower peak height.
Posted: Tue Nov 14, 2006 10:55 pm
by unmgvar
it is divided into 2 reasons.
1 column plate count decreasing like Uwe described. it is possible to see it by looking at the Van Deemter plot. but if you use a good monolith column or a 3 or less micron particle size column the effect becomes less to almost negligible.
2. the speed at wich your detector is set. the baseline in your chromatogram is not really a line. it is actually a very large number of connected dots. this is beause your detectors does not collect data continusly. he does it at a given rate. generally called the sampling rate and the units are Hz.
the UV detector is able to correctly see a peak if it get 20 to 40 data points (depends on who you ask) when the compounds get to it. if the detector is not fast enough then your compounds elute too fast for it to be picked up by the detector.
BTW you don't only get a smaller and false peak area many times if you have close eluting peaks you will get a bad resolution as well, and it is not because the column is not efficient enough but because the detector is working too slow. a lot of people working with monolith columns at high flow rates make that mistake everyday.