At 0.5 mg/ml, the concentration of the dye is so high that (1) it self-quenches, and (2) very little light will pass through it. To measure the fluorescence at such a high concentration, you should do a surface measurement using a triangular cuvette. The cuvette has a surface at a 45 degree angle to the excitation and emission monochromators.

It shouldn't be at 256 nm. Depending on solvent, it's usually at 540-560 nm.

In this article there are usual concentration dependencies for flurophores. Article Terthiophene Functionalized Conjugated Triarm Polymers Conta...

This article describes two-photon excitation of rhodamine B? that may be helpful. Article Testing Fluorescence Lifetime Standards using Two-Photon Exc...

This article is about concentration dependence of exitation in a different dye, which may help.

https://epub.uni-regensburg.de/3993/1/ubr03877_ocr.pdf

The absorption spectrum of the intra-ester of Rhodamine B has abs max @256nm(I do not think it will give very much intensity in fluorescence), the COOH has to be free (not forming intra-ester) for the abs Max @554nm.

Wish this helps!

How is the fluorescence [email protected]/ml(5000x higher in concentration, I think should be relatively low in fluorescence), compare to the fluorescence intensity@ 0.0001mg/ml?

Intra-ester structure:

https://www.sigmaaldrich.com/catalog/product/aldrich/234141?lang=zh®ion=CN&utm_medium=cpc&utm_source=bing&utm_term=rhodamine%20b:&utm_campaign=Competitor%27s%20cross-match%20(nietzsche%20Bing%20ebizpfs)&utm_content=aldrich/234141

also refer to:

https://en.wikipedia.org/wiki/Rhodamine_B

At 0.5 mg/ml, the concentration of the dye is so high that (1) it self-quenches, and (2) very little light will pass through it. To measure the fluorescence at such a high concentration, you should do a surface measurement using a triangular cuvette. The cuvette has a surface at a 45 degree angle to the excitation and emission monochromators.

that is the problem according to literature the excitation wavelength is independent of concentration of dye.

Adam, the problem with self-quenching explanation is that quenching usually does not change excitation wavelength, especially so dramatically.

There may be 2 other simple explanations for this:

1) Experimental error. Your result corresponds nicely with absorbtion curve of this dye. so it can just be wrong setting.

2) Not true solution. Your dye concentration seems very high and some dye tend to form fine suspensions of crystaline dye within concentrated solutions, so it could be an abberation caused by presence of solid in solution. Try filtering this concentrated solution (preferrably on S4 size filter) and see ifprecipitate is left behind.

Thank you very much Mikhail.