Calculating Quantum Yields for Fluorescent Dyes

[LtStorm]

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;

\Phi_{X}=\Phi_{ST}\frac{Grad_{X}}{Grad_{ST}}\frac{\eta_{X}^{2}}{\eta_{ST}^{2}}

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 \Phi_{ST}, 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.

 

[fliflek]

The way I have always measured quantum yields is to use :
\Phi_unk = \Phi_std * (I_unk /A_unk )*(I_std /A_std )*(\eta_unk /\eta_std )²
where \Phi is the quantum yield, I is the integrated emission intensity, A is the absorbance at the excitation wavelength, and \eta 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)