How I normalized arbitrary unit in EEM of Fluorescence measurement for each of this condition:
a. If I measure an unknown mixture of compounds (example from plant extract) and there are no standard sample since the mixture of the compound is varied.
b. If I measure an unknown mixture of compounds for a different sample that varies through the time (I measure for example every one week to see the change of fluorescence compound of the sample).
c. If I measure solid sample using Front Face measurement, are there any differences in measuring solid sample compared to liquid sample?
a) you can normalize the data by using the area of the Raman scattering peak: https://www.researchgate.net/publication/26740308_Fluorescence_Intensity_Calibration_Using_the_Raman_Scatter_Peak_of_Water
b) here you shouldn't normalize, as the changes in sample will result in intensity changes
c) Depending on the sample concentration, the shape of emission spectra can differ from front-face vs. rectangular detection. Additionally, spectral shape of solution might be quite different from solid state spectra. It is unlikely that you can compare those spectra straightforward...
Article Fluorescence Intensity Calibration Using the Raman Scatter P...
Thanks a lot for the valuable answer Dr. Menges. I really appreciate it. Previously I little bit confuse whether it is necessary to normalize my data or not. I will learn more.
Regards.
you're welcome:-) The only stupid question is the one that you don't ask, and your questions were far awy from stupid.
a) further explanation: depending on the emission intensity, your data might have been acquired with different instrument settings (like gain etc.). The Raman peak of water is some kind of "constant", because water is always the main content with the same concentration (55.41 mol/l). So, by npormalizing to a constant area under peak for the Raman band, you can take out all influences from instrument and instrument settings.
addendum for b) if you can't make sure to do all measurements under same conditions & settings, a Raman band normalizaiton might also be helpful for better comparison and accurate time-dependent data series.
Thanks again! That motivate me more to learn.
I have read the mentioned paper above, It solve some of my problems. I think I will use the method to calibrate my data and make the intensity unit in my research more "global". It leads me also to read further literature regarding spectral data correction.
(a and b) To make it sure, so I need to get water Raman data for each instrument setting (if I change the setting, for example, I change the sensitivity of the measurement from high to low) and then I calibrate my data to water Raman of each correspondent instrument setting?
c) Besides measuring a liquid sample, I also measure solid sample (peel of fruit and vegetable) using front face measurement. As you mentioned before that I can not compare those spectra straightforward. Maybe I can not compare it quantitatively but maybe I can compare a kind/type of compounds in the liquid and solid sample qualitatively. One of my goals is to develop an approach to relate the fluorescence intensity of solid sample with the sample quality parameter (it can be a concentration of a compound per unit area; area of defects per total illuminated area or other kinds of a quality parameter - I will develop a machine vision system in this case). But before I continue to the next stage, I wondering are there any standard for Front-Face Fluorescence for solid sample measurement that I should follow?
Dear Dr Ivanova,
What do you mean by method validation? Could you explain more about it? Thanks a lot.
Thanks Mas Dheni for your answer,
c. Do you mean for the machine vision system that I will construct later? I agree with that.
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Pharmaceutical Botany
- Plant Extracts