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?
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
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.
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
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.
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
To determine the unknown concentration of solution A in the 25mL volumetric flask, you can use the intensity values obtained from the fluorescence spectra and the equation you derived.
From your spreadsheet, you have recorded the intensity values corresponding to different volumes of the standard solution added to the flask. Let's use this information to calculate the unknown concentration.
First, let's calculate the intensity ratio for each standard volume. To do this, divide the intensity value by the concentration of the standard solution.
Standard 1 (0 mL): The intensity ratio is 0 because the concentration is 0 ppm.
Standard 2 (0.5 mL): The concentration of the standard solution is 75 ppm. So, the intensity ratio is 19.979 / 75.
Standard 3 (1 mL): The concentration of the standard solution is 75 ppm. So, the intensity ratio is 29.986 / 75.
Standard 4 (1.5 mL): The concentration of the standard solution is 75 ppm. So, the intensity ratio is 39.958 / 75.
Standard 5 (2 mL): The concentration of the standard solution is 75 ppm. So, the intensity ratio is 49.947 / 75.
Standard 6 (2.5 mL): The concentration of the standard solution is 75 ppm. So, the intensity ratio is 60.000 / 75.
Now, plot a graph with the concentration values of the standard volumes on the x-axis and the intensity ratios on the y-axis. Fit a line through these data points to obtain an equation of the form y = mx + c.
It seems like you have already done this step and obtained the equation y = 7.617x + 4.7499. This equation represents the relationship between the intensity ratio (y) and the concentration (x) of the standard solution.
To find the unknown concentration, you can substitute the maximum intensity value obtained from your fluorescence spectra into the equation and solve for x. In this case, the maximum intensity value can be obtained by finding the fluorescence intensity at the wavelength of maximum fluorescence (472nm).
Once you have the maximum intensity value, substitute it into the equation y = 7.617x + 4.7499 and solve for x, which represents the unknown concentration.
To calculate the concentration according to your original method, you can use the equation:
Concentration = (Standard volume * Concentration of standard solution) / Volumetric flask volume
For example, for Standard 2 (0.5 mL), the concentration would be (0.5 mL * 75 ppm) / 25 mL = 1.5 ppm. Similarly, you can calculate the concentrations for the other standard volumes.
I hope this helps you determine the unknown concentration of solution A. Let me know if you have any further questions!