I am trying to evaluate total phenolic content using FC reagent which needs calibration of Gallic acid with FC reagent. As I am quite confused that what will be the blank in Gallic acid calibration procedure as well as for the determination of sample concentration in UV-vis Spectroscopy. In addition to it, what is the reason of taking all absorbance values at 765 nm?
In UV-Vis spectroscopy, the diluting solution required for the sample and standard preparation is used as the blank. The absorbance value is usually taken in a wavelength at which the absorption is maximum.
Sir @Kamrul Islam, the diluting solution to be used for Blank should be Gallic acid+FC ?? or can you please specify with concentration......
But in the Link provided by Sir @John Reye, Blank was taken as Air. I also had seen, someone took water as Blank. That's why I am confused
Whatever you do be consistent. Blanks are just the re fence. When I did some cell growth I used cell free growth medium (ex. LB medium) as a blank. Now what is the difference of water or air? Think about that; no much at all but be careful you are not at a wavelength where water and air give different results. Have you ever ran a wavelength scan for wafer, ethanol, or any other wavelength? Try a wavelength scan of some reacted assay solution. You will sere why 765nm was chosen. There are reasons for the chosen wavelength. They are chosen as those are where changes are being detected. Both are clear and at the wavelength of interest there is not much difference. So you are splitting hairs in some respects. Unless you are inventing a new assay I would stick with cited assays that have already been vetted. Now is there is something wrong with a method that is another ting. UV/VIS assays for all sorts of calibration are common place. I don't recall ever using air but that is not baseless. Air makes sense as well but I was always a fan of having a blank that was in a cell and had un-reacted reagent. I am not a fan of re-inventing the wheel. If others have working assays then that save you time and you can cite and use.
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