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Do the sample used to be a baseline, the sample used in the reference cell, and the sample used to zero, have to be all the same?

Question asked by josépablogf on Oct 20, 2018
Latest reply on Dec 4, 2018 by 195

Talking about a Double Beam UV-Vis spectrophotometer:


I have always placed water in a cuvette to zero the instrument. (Question 1 Please tell if there are cases in wich it has to be done different)


I have always setted up the baseline correction, and to run the baseline, I place a cuvette with all except the analyte, for example if my analyte is benzene and my sample is orange juice with benzene, my baseline would be orange juice without benzene.


(Imagine  I will add different concentrations of benzene to the orange juice and analyze them in the UV-Vis) I would run just orange juice without nitrobenzene as baseline, and i would put the same in the reference holder (the rear cell holder) and then analyze the different concentrations of benzene. Question 2. What is the purpose of placing a reference when I have already ran a baseline? are baseline and the reference always the same? On wich cases  are not they?


     From my personal experience, I have ran differente scan experiments just to see how the baseline correction works, and I basically noticed that the reference is what really matters. I ran a baseline with water and 5 ppm of an analyte, then I placed the same in the reference cell and in the sample cell, I just got a straight horizontal line at 0 abs, just as expected. Then a ran a scan of the same sample with the same baseline but in the reference cell I put water, and I got a characteristic peak even though the baseline and the sample was the same. Then I ran a scan with the same sample and reference (water with 5 ppm of analyte), and water as a baseline and a got a straight line at 0 abs . Then a ran a scan, with water as a baseline and as a reference, and a sample of water with  ppm of analyte and I got a characteristic peak as expected. After these experiments I would say that the reference sample is what really matters.


I really have been struggling with this, I hope I made myself clear