Calculating Quantum Yields for Fluorescent Dyes

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In summary, the conversation discusses a method for calculating quantum yields for samples containing fluorescent dyes. The procedure involves measuring the fluorescence and absorbance of the sample, diluting it, and repeating the process to obtain at least six data points. The accuracy of the quantum yield calculation requires cross-calibrating two different standards, anthracene and Rhodamine B, using an equation that takes into account the refractive index of the solvent. However, the speaker is having trouble determining the standard's slope (GradST) and is seeking guidance on how to properly perform the cross-calibration. The conversation also mentions that the quantum yield values obtained through this method are different from the literature values. The instructions for the procedure involve measuring the integrated emission intensity
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LtStorm
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So, I'm trying to work out a method of calculating quantum yields for some samples that contain fluorescent dyes.

I've dug around and found a procedure for how to do it, which was essentially the same as doing an extinction coefficient (take a sample, run its fluorescence and absorbance, dilute it, run it again, continue until you have at least six data points) in that you end up with a graph and its slope that you can plug into an equation.

But where I'm having trouble here is that to get an accurate quantum yield, you need to cross-calibrate two different standards. I'm using anthracene (0.27 in ethanol) and Rhodamine B (0.49 in ethanol).

The equation for doing this is;

[tex]\Phi_{X}=\Phi_{ST}\frac{Grad_{X}}{Grad_{ST}}\frac{\eta_{X}^{2}}{\eta_{ST}^{2}}[/tex]

Where 'GradX' is the sample's slope and 'GradST' is the standard's slope. The Etas are for the refractive index of the solvent, which shouldn't be an issue as I've run my standards in ethanol as that was what I could find the literature values for anthracene and Rhodamine B in.

My understanding of what I need to do at this point is thus;

For Anthracene, I use the literature value for Rhodamine B's quantum yield for [tex]\Phi_{ST}[/tex], then use my slope for Anthracene as GradX and my slope for Rhodamine B for GradST.

Then do the reverse for Rhodamine B. What I feel I'm missing here is the standard's slope, GradST. It feels odd that would also come from my data. But I don't know what else I could possibly use.

Anyone know how to do a cross-calibration for something like this properly? The literature has been little help, and my advisor hasn't been able to work out the issue either.

Also, I have not been getting garbage numbers entirely, they just are quite different from the literature values. Doing my cross-calibration, I get a QY of 0.346 for anthracene (compare to 0.27 lit value) and 0.38 for Rhodamine B (compare to 0.49 lit value).

Here is the exact passage from the instructions I have been following as well;

First, the two standard compounds are cross-calibrated using this equation. This is achieved by
calculating the quantum yield of each standard sample relative to the other. For example, if the
two standard samples are labelled A and B, initially A is treated as the standard (ST) and B as the
test sample (X), and the known ΦF for A is used. Following this, the process is reversed, such
that B is now treated as the standard (ST) and A becomes the test sample (X). In this manner,
the quantum yields of A and B are calculated relative to B and A respectively.


I'm putting this here as it isn't homework, though the moderators may feel free to move it to the homework section if they feel it is more appropriate for that forum still.
 
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The way I have always measured quantum yields is to use :
[tex]\Phi[/tex]unk = [tex]\Phi[/tex]std * (Iunk /Aunk )*(Istd /Astd )*([tex]\eta[/tex]unk /[tex]\eta[/tex]std )2
where [tex]\Phi[/tex] is the quantum yield, I is the integrated emission intensity, A is the absorbance at the excitation wavelength, and [tex]\eta[/tex] is the refractive index of the solvent
Measure an emission spectrum of the standard sample and the unknown sample, keeping all settings (like excitation wavelength, integration time, slit widths etc.) the same. Apply whatever instrument corrections are necessary. Integrate the area under both emission curves and use in the formula above. For the most accurate results choose a standard that emits in the same wavelength range as your unknown and make the absorbance of the standard and unknown samples at the excitation wavelength between .1 and .2 (optically dilute)
 

1. What is a quantum yield for a fluorescent dye?

The quantum yield for a fluorescent dye is the measure of how efficiently the dye can convert absorbed light into emitted light, or fluorescence. It is expressed as a percentage, with higher quantum yields indicating a more efficient conversion.

2. How is the quantum yield of a fluorescent dye calculated?

The quantum yield of a fluorescent dye is calculated by comparing the intensity of fluorescence emitted by the dye to the intensity of the light absorbed by the dye. This is done using the equation Φ = (number of photons emitted / number of photons absorbed) x 100%.

3. What factors can affect the quantum yield of a fluorescent dye?

The quantum yield of a fluorescent dye can be affected by factors such as the chemical structure of the dye, the solvent it is dissolved in, and the presence of other molecules that can interact with the dye. Temperature and pH can also play a role in determining the quantum yield.

4. Why is it important to calculate quantum yields for fluorescent dyes?

Calculating the quantum yield of a fluorescent dye is important because it allows scientists to compare the efficiency of different dyes and choose the most suitable one for their specific application. It also helps in understanding the physical properties of the dye and how it interacts with its environment.

5. Can the quantum yield of a fluorescent dye be improved?

Yes, the quantum yield of a fluorescent dye can be improved by modifying its chemical structure or optimizing its environment. This can be done through methods such as altering the solvent, adding co-solvents or other additives, or using a different dye altogether.

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