[Typhon]

A few days ago i extracted fluoroscein from yellow fluorescent markers with ethanol (96%) and then made the ethanol boil until i had only a little of a thick yellow/brown liquid. Since i couldn't purify the fluoroscein any more (this was done at home).

I then proceed to "experiment" a bit with my concentrated and a UV light. Everything goes as expected until i diluted a very very small portion (less than a drop) in 96% alcohol. This solution instead of being yellow as expected is nearly transparent and under a UV light it's a very clear and bright blue.

From what I've read the light that can be perceived from fluoroscein under a UV light only goes from 494 nm to 512 nm, which excludes the blue colour.

Here's a photo:


The object I'm holding below the flask is my UV flashlight.

Also, when water is added to the solution mentioned above (drop by drop) it turns to the expected bright  yellow


Does anybody have any idea of what is happening here? My guess is that it has something to do with the polarity of the solvent, probably the same mechanism as the Blue-shift in proteic solutions.

[Arkcon]

You should first start with some research on the exact markers you used.  You might even call the vendor and ask them what's in their markers.  Like you, I assumed the yellow-green fluorescent highlighter was fluorescein, its not, its usually coumarin.  A more likely explanation, than the one you gave, is that you have a very different dye.

[Typhon]

You should first start with some research on the exact markers you used.  You might even call the vendor and ask them what's in their markers.  Like you, I assumed the yellow-green fluorescent highlighter was fluorescein, its not, its usually coumarin.  A more likely explanation, than the one you gave, is that you have a very different dye.

Calling the vendor might be a tad difficult, because the markers i used are some cheap ones from  thrift store, but from what I've read coumarin is a fragant chemical, and the samples i extracted don't smell of anything but the solvent, even in the concentrated form.

But it might be a different dye, I'll try to get hold of some pure fluoroscein and test it to see if i get the same results (and do a chromatography while I'm at it).

[Arkcon]

There is a rich selection to choose from: http://en.wikipedia.org/wiki/Fluorophore#Families

[Corribus]

@Typhon,

Coumarin itself isn't used as a fluorophore but there is a whole family of coumarin derivatives very frequently used as laser dyes in the blue-green region of the spectrum (e.g., coumarin 1: http://omlc.ogi.edu/spectra/PhotochemCAD/html/045.html). I'm not aware of any that have yellow fluorescence in ethanol (these are usually more typical of xanthenes) but I'd have to check my catalog to be sure. Anyway, many of the coumarin and xanthene dyes (the latter of which fluorescein is a member) exhibit strong solvatochromism and/or polarity dependent fluorescence coloring or intensity, particularly those that have free amino groups. Though not directly related to your question, I found an old slide I presented for one of my lab's group meetings based on a photograph I took that demonstrates this kind of effect in some of the coumarin dyes, which you might find interesting (the numbers are quantum yield values I extracted from the noted citation):



No color change here for the most part (although the one on the right starts to take on a yellowish cast in water), but you can see the one with the free amino group has much higher susceptibility to fluorescence quenching in polar solvents due to what's called a twisted intramolecular charge transfer (TWICT or TICT) state.  Color shifts are usually due to solvent reorganization around the excited state.  If you're seeing yellow in water but blue in alcohol, most likely something like this is occurring.

Another possibility is concentration effects. Some highly fluorescent dyes form excimers and undergo very different luminescent color as a function of concentration. Pyrene is a classic example.

Without knowing the identity of the dye in your marker, it's impossible to say why the color change happens. Most likely it is polarity related, which either affects the intrinsic excited state relaxation properties of the molecule, or impacts the way they clump together.

[curiouscat]

Naive question.

But can you post similar photos for controls? i.e. pure water and pure ethanol?

[Babcock_Hall]

I don't have any experience working with these chromophores, but I did notice something odd in working with one carotenoid, all-trans-spirilloxanthin.  Its maximum absorption tended to shift to longer wavelength in chloroform or dichloromethane, relative either to hexane or to methanol.  The shift changed its color to from orange-pink to a more intense red.  The peaks also shift to longer wavelengths in benzene, relative to 7:2 acetone/methanol (JBC 249 6446-6453 1974).  My point is that the solvent effects in this case may have been more complex than being based upon polarity.
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