Fluorescien's a pretty interesting molecule with quite a wide variety of
uses
because it's quite fluorescent but has a very low toxicity. In particular this low
toxicity allows it to be used for everyday things as well as in the
medical field for diagnostics.
I actually find it pretty interesting that the toxicity is low enough that it
can be injected into people to help map the vascular system and diagnose
diseases.
I should note though that just because of toxicity is low it doesn't mean that
people don't have bad reactions to it.
Another common use for the fluorescein is actually in highlighter markers.
I won't be doing an extraction from highlighter markers because it's already
another YouTube channel that has done this and I'll provide the link below.
All of these uses of fluorescein are pretty interesting but I'm going to be
totally honest and say that I want to make it just so I can play with it.
So with that being said let's get started.
I start out by adding two grams of phthalic anhydride to a small round bottom flask
and then on top of this I add three grams of resorcinol
and then to this I add five drops of concentrated sulfuric acid.
Ideally the chemicals would've mixed up before adding the acid, but oh well.
It's then lowered into an oil bath and heated to 190 to 200
degrees Celsius.
As the oil heats up and the chemical start to react you'll notice that a red
color starts to form.
This is actually the first bit of fluorescein being made.
Slowly as the oil gets hotter and hotter more and more will dissolve and the
solution will get redder and redder until it eventually becomes nearly black.
I had a stir bar at the bottom and eventually became liquefied enough that
I was actually able to stir it.
So we want to heat it at this temperature of around 200 degrees
Celsius for at least 30 minutes.
It's important not to heat it up too much because the product can decompose.
Above I've included the overall reaction that's occurring between the phthalic
anhydride and the resorcinol.
So as you can see one molecule phthalic anhydride will react with two molecules
of resorcinol. The mechanism behind this reaction is pretty complicated and
there's three distinct steps that occur. Without going to too much detail the
phthalic anhydride undergoes a Friedel-Crafts reaction twice with each
molecule of resorcinol. These two Friedel-Crafts reactions result in the
production of one water molecule but they also create an intermediate
molecule that can undergo a Fischer esterification with itself, so it does
just that
and in doing so it forms the final fluorescein molecule and also expels one
water molecule and the scheme I've color coded everything so you can see where
things end up. All of the hydrogens and the waters come from resorcinol
but in each water one oxygen will come from the resorcinol and the other
will come from the phthalic anhydride. The sulfuric acid that is used is a
catalyst and it will catalyze both the Friedel-Crafts reactions and Fischer esterification.
If you'd like to know more about the mechanism I've included a good link
below in the description.
After 30 minutes is up it's removed from the oil bath and allowed to cool to room temperature.
At this point you can do two things if you don't want to isolate the fluorescein
you can simply dissolve this crude product in some sodium hydroxide solution.
This would give you a nice stock solution that you can start playing with immediately.
This is what is done in most of the videos I've seen online but I actually
wanted to isolate the fluorescein itself.
And to do this we're going to need to do a little bit of a workup.
It's taken inside and stir bar is added and you can see the crude product is now solidified.
And then added about 15 milliliters of acetone to dissolve the fluorescein.
Fluorescein is very soluble in acetone, but we do have a hard block on the
bottom so it still takes a little while to fully dissolve.
Slowly as more fluorescein dissolves into the acetone it should get darker and darker red.
Eventually what it doesn't seem like much more is dissolving the acetone is
decanted into a beaker.
The process is then repeated by adding more acetone until everything in the
flask is fully dissolved.
In the end I use a total of about 50 milliliters of acetone
but the exact quantity really doesn't matter.
Even when there's very little fluorescein left in the flask it might be dyed
yellow and it's kind of a waste of acetone to try to fully remove it.
Here's the beaker which looks like it has a bit over 50 milliliters of acetone.
The heat is then turned up and the acetone is removed.
The use of the acetone is really nothing more than to just transfer it to a larger container.
By doing this the surface area is increased and it makes for the next step
where we dissolve in ether go much faster.
In theory though you can skip this step it just might take a lot longer to dissolve in the ether.
When the acetone's all gone were left with a syrupy mix on the bottom that slowly
solidifies as it cools down.
Once it is fully cooled down we're left with a nice red glass on the bottom of the beaker.
To the cool glassy mixture i added about 20 milliliters of diethyl ether and a
few milliliters of distilled water.
The fluorescein is actually not soluble in water but the addition of it greatly
increases the rate that it's dissolved into the ether.
Another stir bar is placed into the beaker because the old one is kind of stuck in the mixture.
A watch glass is then also placed on top because ether evaporates pretty quickly.
At some points when it didn't seem like a lot of the fluorescein was dissolving
adding more water seemed to be more beneficial than adding more ether.
And to speed things up a little more i added a further 20 milliliters of
diethyl ether followed by a little bit of water again.
After about 10 minutes a lot of it was dissolved except for some goo on the bottom.
The diethyl ether and the water was then transferred to a separatory funnel.
And the dissolving step was repeated by adding another 40 milliliters of diethyl
ether to the beaker followed by again several mils of water.
You'll notice that this time the ether seems to be a lot less concentrated with fluorescein then before.
And if you look at the bottom there's still a lot of that black stuff.
Anyway the diethyl ether and the water was then transferred to a separatory funnel.
More diethyl ether is added but you'll notice that there's still quite a bit of
that black stuff at the bottom.
I found this stuff was stubborn and it really wouldn't go into the ether layer
unless i added a bunch of water.
Keep adding water until it fully dissolves into the ether layer.
In this step I added a lot more water than i did in the previous one and you
can see the ether is taken up a lot more fluorescein.
And after just a few minutes of stirring it seemed like everything was taken up into the ether
So again just like before it was added to the other extractions in the separatory funnel.
And then with a small amount of ether and water final washing was done of the beaker.
And then for final time this was added to the separatory funnel as well.
Once all the extracts were together I mix them around thoroughly.
Before proceeding on i drain away the lower water layer that was left over from the
extraction steps.
And now to our fully loaded separatory funnel with our product i added 50 milliliters of water.
This washing step serves mostly to wash out sulfuric acid and leftover resorcinol.
So as we always do with the separatory funnel we cap, shake, and vent and then
we put it back on the stand for the layers to separate.
Once the layers have separated we can discard our lower water layer.
The washing step is repeated but this time we use 50 milliliters of saturated salt solution.
You'll notice that before adding it the top ether layer is a little cloudy.
However after the capping, shaking and venting, you can see it's cleared up a lot.
So now the bottom layer which is mostly water is drained away into a beaker.
The upper ether layer is poured into an Erlenmeyer flask and it's dried using
molecular sieves. This is just to pull out any of the water that might remain.
I also could have used another compatible drying agent like sodium sulfate,
but I just like molecular sieves.
After letting it dry for several minutes it's directly gravity filtered into a round bottom flask.
I and then set up a little simple distillation to remove and recover my ether.
Near the end of the distillation it looks kind of like red paint.
It was actually really hard to dry and at this point it didn't really seem like any
more ether is coming off. To solidify it i actually had to pull a small vacuum on
it while heating.
Eventually we were left with this very dark red crust.
However it was still a little bit damp despite the vacuum and the heating so I
took it out and put it in an oven.
Be careful if you do this though because there will still be some phthalic anhydride
left over which might sublimate.
After the oven as left with about three grams of nice dry fluorescein.
To test it out I then prepared a sodium hydroxide solution.
Once it had fully dissolved I added a little bit of fluorescein to it.
You might notice the sodium hydroxide solution is a little bit green before I add it.
This is because the spatula used to put in the sodium hydroxide was contaminated
with a little bit of fluorescein.
Anyway then I turn on the stirring to fully dissolve the fluorescein and you can see the solution quickly
turns to a dark red.
However you might notice at the top it's actually a little bit green.
From the stock solution of fluorescein it can then dropwise be added to a beaker
of water and I do this under UV light for added effect.
At this point you might find it interesting how the solution of the
fluorescein is red but when it's dropped into the water it's green
you also might remember me pointing out like 20 seconds ago that the top of the
fluorescein solution was green.
this is a good example that highlights the major difference between transmitted
light and reflected light a small amount of light is reflected at the surface and
we see it as green but the light that keeps going and makes it through the
solution comes out as red.
this is because if the concentration is high enough the fluorescein will quench its
own fluorescence.
So now we'll mix it up a little so we have a nice solution of glowing water.
Actually kind of fun to play with and it's
kinda cool to pour glowing liquid. However you'll probably notice that the glow
seems much less on the smaller beaker.
This is pretty much just because it's further away from the backlight and you
can see that when we slide it closer it gets much more intense.
Interestingly enough it glows quite well during the day as well and you don't
really need a black light to see it.
It might just be the lights that i use but during the day it's actually still
pretty cool to see.
The black light really does provide better contrast but if you don't have
access to one i think this is still cool enough.
In this shot is the effect is highlighted even more where you can see
it's red liquid in the pipette
but the moment it goes into the water it turns green.
In the pipette the concentration is high enough for it to quench its own
fluorescence but when it's dropped into the water
the concentration is decreased and we can see the nice green fluorescence.
So in the end we left with
three grams of fluorescein contaminated with a little bit of phthalic acid or
phthalic anhydride.
Remember that if you're just looking for a quick and easy way to get the
fluorescein solution
you don't have to go through all of the purification steps.
However I just did it because I found it more interesting and I wanted the
fluorescein powder.
Anyway that's it for fluorescein but in the future I do plan to make a video
where I go into more depth on fluorescence in general.