Measuring fluorescence quantum yield using a reference compound with a known fluorescence quantum yield.

Decide on a single wavelength of excitation that allows for excitation of both reference and sample.

Make sure the reference is dissolved in the solvent for which its quantum yield has been determined. Look up the refractive index for this solvent.

The sample can be dissolved in any solvent of which the refractive index is know. The fluorescence quantum yield is a property of the compound in a given solvent. Hence, the determined value is only valid for the solvent used.

Dilute sample and reference solution to obtain an optical density

That's basically the process. However, take care, you have to prepare your reference sample so it has the same absorbance than your compound at the selected wavelength, NOT the same concentration.

There are other methods, but not as simple and require special instrumentation.

https://gair.media.gunma-u.ac.jp/dspace/bitstream/10087/5145/1/Ph.D_E1-396.kobayashi.pdf

The comparative method is the best! Except if you have an integration sphere, in this case you don't need to use a reference.

Also, you don't want to have the same concentration of reference and your compound. You need the same absorption (typicaly OD = 0.1) at the wavelength of excitation.

No, at the same absorbance so that you not saturate the absorption in a small slab at the front of your cuvette.

You need to record the absorption spectra for each compound and solvent, then, perform fluorimetry using same slits and as M Vazquez said, setting concentrations to the same absorption at the selected excitation wavelength. Note that you may perform a baseline for each solvent, to discard scattering.

I agree with the previous answers. You have to strictly comply to a few basic constraints.

To avoid autoabsorption effect you should use an OD = 0.1 or lower. This means that you can compare quantum yield only if they were obtaiend at those concentrations or lower. Since it is hard to obtain exactly the same absorbance for the two samples to be comapred it is better to prepare 5 soltuions for every sample whose OD is around 0.02-0.1 (and also the solvent alone) and to measure them for both absorbance and fluorescence. By reporting in a plot the Integration of the fluorescence spectrum vs the absorbance you should obtain a linear behaviour, whose slope is the Gradient relating absorbance and fluorescence. Having the gradients for the two samples (unknown and reference) you can use those gradients in the formula that gives you the Quantum Yield.

Please note that it is compulsory to apply corrections in fluorescence spectra for both the excitation and the emission. This gives you the possibility to compare results using different excitation wavelenghts for the reference and the sample. Also note that you cna compare results only if the excitation and emission slits were setted in the same conditions for both the reference and the sample.

Here I only stressed some crucial steps to be taken into account. A free excellent guide to perform fluorescence quantum yield measurements is available on the HORIBA (formerly Jobin Yvon) site:

http://www.horiba.com/scientific/products/fluorescence-spectroscopy/application-notes/quantum-yields/

Please download the guide PDF file and follow carefully the indicated steps. If you can acquire the S1c/R1c signal (S1c is the fluorescence corrected signal and R1c the signal from the excitation lamp output, measuring the flux of photons at every wavelenght and continuously) you can use this signal to obtain the integration of fluorescence. This is straightforward for Horiba instruments, but most of the high level fluorometers performs in the same way.

I forgot... To have good sensitivity on low asborbance signal, it is better to sue 5 cm pathleng cuvettes, for which the classical OD = 0.1 givies a signal having OD = 0.5. This should minimize absorbance errors. Please also note to check (every day in which you will perfom measurements !!!!) two reference compounds for each other congurent behaviour, otherwise you will waste a whole day of measurements, de to instrumental problems or wrong applciation of the method...

Good luck!

Yeah, previous answers are absolutely ok and it is normal practices of molecular spectroscopiests. It is also called seconday standard method calculating fluorescence quantum yield. Absoulte fluorescence yield can be measured with special accessor attached with fluorescence spectrometer but it is not so commenly used.

Harry is right, the same absorbance at the excitation wavelength was my meaning

Yo might fin useful this guide. I agree with all the previous answers

THe procedure above is the correct one

Actually, as long as you keep the absorbance at the excitation wavelength low (

Piere-Henri

I was not reacting on your reply. They came at the same time

Measuring fluorescence quantum yield using a reference compound with a known fluorescence quantum yield.

Decide on a single wavelength of excitation that allows for excitation of both reference and sample.

Make sure the reference is dissolved in the solvent for which its quantum yield has been determined. Look up the refractive index for this solvent.

The sample can be dissolved in any solvent of which the refractive index is know. The fluorescence quantum yield is a property of the compound in a given solvent. Hence, the determined value is only valid for the solvent used.

Dilute sample and reference solution to obtain an optical density

Santu

Before you post an entry you should please have a look at earlier replies, especially that of Preus who presents even free software:

In the comparative way absorbance should be a factor of 10 lower and it does not have to be the same as the reference

Hello, thank you very much for all.

The best way to prepare the reference example Quinine sulfate and it does not matter that your organic compound should have the same concentration. Measure the absorption at different point say 6 or 7 by changing the concentration of your compound and at the same measure PL each time you change your absorption. Then get area under the PL curve and also the absorbance value.The absorbance should be maximum under 0.10 to avoid re-absorption effect. Then apply in quantum yield equation.

Correction for differences in refractive index is problematic and should better be avoided. Reason: only a small part of the optical pathway is filled with solvent.

Essential is a correct calibration of the fluorescence spectrometer, employing a calibrated lamp.

Further: try to measure flu. quantum yields of compounds reported to have a quantum yield equal or close to unity, and see what you get.

There is also the integrating sphere equiupment from Hamamatsu.

Interesting test: compare the fluorescence spectra of e.g. quinine sulphate published in the literature. You will be in for a surprise.

I concure with this answer

how to theoretically find excitation and emission of fluorescence in organic compound.are you know tell me the reference books.