5mL of a solution A (unknown concentration) was transferred into sic 25mL volumetric flask. The following volumes of a standard solution of A with with a concentration 75ppm were added to the flask: 0mL, 0.5mL, 1mL, 1.5mL, 2mL, and 2.5L. The excitation spectrum and and emission spectra is provided. Determine the unknown concentration of A in that solution?

Question

I was given excitation spectrum of A and fluorescence spectra of solution #1 to #6

DrBob202. Here is my answer so far

-From the excitation spectrum, the wavelength at which maximum intensity is obtained is 375nm
-From the emission spectrum, the wavelength at which there is maximum fluorescence is 472nm

My spreadsheet
Standard volume Concentration Intensity
1 0 0 0
2 0.5 1.5 19.979
3 1 3 29.986
4 1.5 4.5 39.958
5 2 6 49.947
6 2.5 7.5 60.000

Then I plot a graph between intensity and concentration

Equation is y=7.617x+4.7499

Then I stuck from here.
Furthermore, this is how I find a concentration. Can you possibly check that?
Stard 2: 0.5*75ppm/25=1.5
Stard 3: 1*75/25 = 3

Thank you in advance for your help

Answer 1

I commented in my first response that your concentrations were calculated correctly. However, I don't know what the 19.979; 29.986, etc are. These may be the intensity. Also, I note you have a b of 4.7499 which I didn't see in the first post. At least I don't remember it in the post. I suppose that's immaterial.
What are you plotting? Is that intensity of the excitation or intensity of the fluorescence? And I don't understand the first entry. I assume sample 1 is the unknown, it is "zero" concentration, but how can the intensity be zero? Also, although you don't have an absorbance, I think intensity, in one form or another, takes the place of absorbance.

Answer 2

Thank you so much DrBob222. I see what I did wrong. Yes you are right. These number 19.979, 29.986 is intensity from fluorescence (emission) spectra data. The intensity of standard 1 is not 0. It is 9.990. So after correction, the equation I got is y= 6.6656x + 9.9777 and R^2 is perfectly 1.

So now how can I find the concentration of the unknown? I think I need to know y but I am not sure

Answer 3

ok. good. Now you understand that "standard 1" really isn't a standard. That's the unknown. If you were doing this on graph paper (I don't know how you do it on Excel), you would extrapolate that straight line starting at the 9.990 point and moving to the left and down until it crosses the x axis. That will be the concn then of the unknown. Of course it will read a negative number and you change that to a + number since the negative sign is the result of the extrapolation. I believe you do that mathematically by setting y = 0 and solve for x in your straight line equation. Change sign to read + and correct for the dilution. (The concn in the original sample will be (25/5) x the concn you find here.

Answer 4

DrBob222, thank you for your answer
Thanks to your suggestion, my final answer is 7.48447 ppm (unknown concentration)
However, I am just wondering why Prof. gave the graph of excitation spectrum of A. One reason I can think of is that I use the highest intensity which is 100 and then plug it in the equation to find x. If I did that, my final concentration would be 70. Is it possible to do so

Furthermore, the second question is "If a synchronous experiment is to be performed, what would be the offset that you would use?
Thank you in advance for your help