Well - the most definitive article on quantum yields I have ever seen is the chapter by Knut Rurack (attached).  In that he lists quantum yields for fluorescein in NaOH around 90% (depending on the lab) and for rhodamine B in water above 95% (again depending on the lab).  These values pretty much correspond with my recollections of the literature.  But on another note, I don't understand how you are comparing fluorescence yields carried out at two different excitation wavelengths since the energy reaching the sample will be different for these two cases.  Usually one excites the sample and standard at the same wavelength to ensure that the photon density reaching the cuvettes are the same.  Also, absorbance readings are not very precise at such low values - it is usually better to make standard solutions at higher absorbances (OD ~ 1 is best) and then to dilute.

Well - the most definitive article on quantum yields I have ever seen is the chapter by Knut Rurack (attached).  In that he lists quantum yields for fluorescein in NaOH around 90% (depending on the lab) and for rhodamine B in water above 95% (again depending on the lab).  These values pretty much correspond with my recollections of the literature.  But on another note, I don't understand how you are comparing fluorescence yields carried out at two different excitation wavelengths since the energy reaching the sample will be different for these two cases.  Usually one excites the sample and standard at the same wavelength to ensure that the photon density reaching the cuvettes are the same.  Also, absorbance readings are not very precise at such low values - it is usually better to make standard solutions at higher absorbances (OD ~ 1 is best) and then to dilute.

Several things wrong in my experience:

-The absorbance values are a bit low. You have a good correlation coefficient for your fluorescein data, not so much with first three vs last three of your rhodamine. You want to keep your absorbance below 0.1 to avoid skin effect on QY measurements but not to low to minimize noise contribution on your absorbance measure.

- You excited at the maximum absorbance of your molecule and recorded your fluorescence spectrum too close to the excitation. I don't know what apparatus You're using, but most spectrometer uses slits to filter the excitation and emission wavelength. To avoid any overlap between excitation scattering and fluorescence reading, you want to excite at "Lower Emission Wavelength - Excitation slit - Emission slit" (minus an additional 5 for security sometimes). Slits being 5 by default, that would mean exciting at 486 or lower and 504 or lower.

- You need comparable emission wavelength for your reference and sample. Detectors have their own quantum yield of detection which generally gets lower as the waveleght increases, so your ref and sample should ideally fluoresce in the same range of wavelength to avoid this effect. I would suggest you compare rhodamine B to another rhodamine or FITC to GFP.

- Another reason for using a ref with a similar spectral range is that it is IMPERATIVE you excite the ref AND sample at the same wavelength: the excitation power is not homogeneous on all wavelengths and your excitation source might have huge variations depending on what part of the spectrum your using. As a general rule for any type of experiments, you want to avoid changing too many variables at once when it is not absolutely necessary.

- You NEED to excite where you measured absorbance. Since you can't really use different excitation for a same measurement, here you would need to excite your rhodamine at 496 (well, 486 as seen before) where its absorbance is much lower thant the one recorded, this would mean underestimating your QY.

I hope that will help, don't hesitate to ask questions if I wasn't clear.

To David: are you sure about rhodamine B QY ? 0.95 would seem right rhodamine 6G or sulforhodamine 101 but I thought rhodamine B had around 0,6-0,7 in ethanol (its best solvent).

I am certainly not positive about the quantum yield or rhadamine B - in ethanol or water. Unfortunately, like so many quantum yields, the published values vary considerably.  The literature values for rhodamine B in ethanol, for example, range from 0.5 up to 0.99.  The variation seems to depend in part on the method utilized (as discussed in detail be Rurack).  Perhaps it would be better to use rhodamine 6G for which  the literature range seems tighter,  Rhodamine 6G was carefully studied by Magde et al (Photochem Photobiol. 2002 Apr;75(4):327-34.) and found to have a QY of 0.95 as you said.  These authors also reported fluorecein in 0.1N NaOH to be 0.925.

about the quantum yield of Rhodamine B: Rhodamin B can take two prototrope forms. Depending on pH, it can exist as cation or as neutral form. The quantum yields differ:

 - cation: ~ 49% at room temperature in ethanol

 - neutral form: ~ 68% at room temperature in ethanol

This is often overseen by physicists... A big deal of the literature discrepancies com from this issue. Additionally, the Rhodamine B quantum yield is strongly temperature-dependent at room temperature...

more on the topic of quantum yield reference dyes in my PhD thesis, unfortunately in German only (chapters 6.3.7 and 6.4.2)

Article Mehrdimensionale Fluoreszenzspektroskopie : eine instrumente...

about selecting parameters:

 - excitation wavelength: my be different for reference and sample, but you have to care for the fluorescence spectrometer's difference in excitation intensities at those wavelength. This correction is built into some spectrometers, and can be measured for others (look for "quamtum counter")

 - absorbance value: might be choosen much higher as 0.05, which brings a big plus in accuracy. You just have to account for the inner filter effects.

EXPLANATION: fluorescence intensity is NOT proportional to absorbance intensity! This is just an approximation made by Parker&Rees in the 1960's, to prevent exorbitant calculations (they were too lazy to calculate by hand and computers were rare this time). Even for absorbance values as low as 0.05, the systematic error made by this approximation is already several percent.

What really happens: fluorescence intensity is proportional to the amount of light absorbed within the cuvette region seen by the fluorescene spectrometer's detection optics. You need the absorbance spectrum together with two parameters describing the fluorescence detection geometry in order to correctly calculate the factors. With this approach, I could routinely determine quantum yields from samples with absorbance up to ~1.0, resulting in a much lower influence of absorbance measurement uncertainties.

To Prof. David M Jameson , really thanks for your kind help. Your information is helpful for me. Yes, the same excitation wavelengths could be used for both standard and sample in this QY measurement and I didn't realize this before the measurement. It has been advised that the solutions could be determined with a low absorbance (typically A

To Dr. Julien Gravier, 

Thank you so much for your detailed answer, it would be very helpful for me.

Thermo Scientific Lumina fluorescence spectrometer was used to record my fluorescence spectrum. Attached files are instrument setting and fluorescence spectrums of fluorescein and Rhodamine B. Just as what you said, I should excite both the two samples at the same and a shorter wavelength to avoid some negative effect. Since I haven’t another rhodamine dye, maybe I will try FITC for my later experiment.

To Dr. Peter Kapusta,

Thanks a lot for your helpful answer. It's a pity that spectra correction haven't been done for my experiments since I didn't realize the importance of the correction before your answer. I will try to do the correction later to reduce some unwanted effect.  

To Dr. Julien Gravier, 

Sorry for forgetting the attachment. 

To Friedrich Menges,

It has been widely advised that the solutions could be determined with a low absorbance (typically A

You misunderstand me.  Of course you cannot do the fluorescence measurements at ODs near 1.  What I meant was that you can determine the OD of a stock solution with great accuracy near 1 and then quantitatively dilute it - for example 20x - to get your OD of 0.05.  This is a more accurate way to determine the OD of your final samples.

To expand on a point made by Friedrich, many people make the mistake of using OD values for sample and standards when they should be using the fraction of light absorbed.  Here is the relevant paragraph from my book on fluorescence:

The classic approach for determination of a quantum yield using the "relative" method is to select a fluorophore, a standard, which has absorption and emission properties that roughly match those of your sample.  For example, tryptophan or N-acetyltryptophanamide (NATA) are common standards for proteins, while quinine sulfate and fluorescein are often used when the excitation is in the mid-UV or visible regions, respectively.  Then, one makes up solutions of the standard and sample such that the optical densities are similar.  Now, one simply measures the emission spectra of both standard and sample, applies correction factors to get true molecular spectra, and integrates the area under the spectra to get the total intensity of the emitted light.  One then uses the following equation to calculate the quantum yield of the sample:

QYsample = QYstandard (IT sample/IT standard)(1-10-ODstandard/1-10-ODsample)(nsample/nstandard)2                             7.3

The term (1-10-OD) represents the fraction of light absorbed at the excitation wavelength.  I should note that, in the literature, one often sees these terms replaced with absorbances or optical densities, which is only a good approximation if the optical densities are very low.  The IT terms are, of course, the total intensities (more about these terms soon), and n refers to the refractive index.  When publishing quantum yields, it is important to describe the methodology in detail and especially to indicate the quantum yield assumed for the standard.  That way when a skeptical reader (like me) happens along, he or she can substitute his or her own preferred value for the quantum yield of the standard.

Many thanks for Prof. David M Jameson. I think I will find a suitable way to measure QY of my sample after all your help. 

Hi all,

a PDF file of the Demas & Crosby Review from 1971 can be downloaded from here: http://files.instrument.com.cn/bbs/upfile/200882684459.pdf

It is really worth reading and contains nearly everything relevant. Todays' reviews on fluorescence quantum yield determination don't contain substantially more or better information. On pages 999-1000 (chapter 2. Optical Dilute Measurements) it is clearly explained, that quantum yield is proportional to fraction of absorbed light and it is also explained how it came that instead the optical densities are commonly used. This goes back to the Parker & Rees, who first described in 1960 their method of  determination of quantum yields in optical dilute solutions by using a quantum yield reference dye.

http://files.instrument.com.cn/bbs/upfile/200882684459.pdf

I am very new to this field....can anybody tell me in detail procedure for quantum yield determination for turn on flurescent probes....what excitation wavelenght we mist use ...should I use absorbance maxima of probe or absorbance maxima of fluorphore after turn on release

for proper quantum yield calculation, always use the absorbance characteristics of the fluorescing species, unless you want to investigate any kind of energy transfer between absorbing species and another fluorescing species...

Thank you ..Friedrich menges...but my fluorscent probes show very low value at the excitation wavelenght of the fluorophore which release uppon turn on reaction

Thank you all yayy

Dear friends

I have read all the conversations. I have a query related to the same question asked by Zhan Jinsheng.

Any correction is required for measuring the QY, if I maintain the following:

1. Considering the reference standard as Rhodamine 6g; QY = 0.95 in ethanol and excitation wavelength 488 nm.

2. The fluorophore (sample) has abs range 435 to 595 nm with maximum wavelength 552 nm. Rhodamine 6g is suitable for this dye?

3. Abs (OD) at excitation wavelength ~0.01 to 0.1 (Is it suitable?)

4. Fluorescence excitation for sample and ref (both) 488 nm.

5. Quartz cuvette with path length 1cm (3 ml volume).

6. Absorption instrument: JASCO V-670 Spectrophotometer

7. Fluorescence instrument: Varian Cary Eclipse Fluorescence Spectrometer

8. gradient method for QY calculation.

Here I have attached the spectrophotometer setting

The lowest ODs possible would be best - as long as you have determined them very accurately by a dilution method. Also remember to correct the emissions for the instrument response - i.e., use true molecular spectra for your determination. Then you should be OK.

Good luck!