Normalizing peaks to migration time?
New user here. I'm also somewhat new to CE so please bear with me. I have a question about how I should be reporting my data.
We use a 3130xl to run sugars cleaved from glycosylated proteins. We isolate the sugars, label with a fluorescent dye, and run the samples in water. The output is an FSA file that we convert to XML for use of our in-house analysis software which was written in MatLab. The software shows us a spectrograph of the sugar peaks which corresponds to certain complex sugar structures (fluorescence units on y-axis, migration time on x-axis, obviously). Our software then allows us to select peaks, then integrates the area. We report the relative areas of the peaks as representing relative abundance of each sugar structure in the sample.
My question is, should we be normalizing the data to the migration time? If you look at a representative sample that we run on the machine, you'll see that the structures appearing on the left of the spectrograph are the ones hitting the detector first - these peaks are generally very tight at the base. The structures appearing on the right of the graph, however (the ones with longer migration times) generally have broader bases. So is integrating these areas under the curves really representative of relative abundance of these structures in our samples, or should we only be looking at rfu (peak height)? Or should we be normalizing the data to the migration time and then integrating? Note: we don't care about really quantifying the amount of each sugar structure in the sample, just relative abundances.
Additional question: I know there's a quick and easy experiment to test this...we have labeled standards we've purchased from a company, so I have access to 'peaks' that would run at either end of the spectrum we're looking at...I can run the same amount of each 'peak' and use the software to integrate and see if the output is the same. If I want to try this experiment, is it best to run the same molar quantities of sugar, or go by weight (since the structures have different dalton weights)?
Thanks for reading - I appreciate any and all input. Please let me know if anyone has any questions.