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Hi! I’m Deboki Chakravarti and welcome to Crash Course Organic Chemistry!
When you get down to it, all life is kinda similar.
From plants to fungi to humans, we share many of the same biological organic reactions
that break down molecules for energy and build the molecules that make you... you.
But we are different from a mushroom or a tree.
And those differences allow some organisms to produce molecules that may have wildly different effects in other organisms—
sometimes harmful, but sometimes beneficial.
For example, willow leaves contain salicylic acid that acts as a plant hormone,
and as far back as 4000 BCE the Assyrians used this extract to treat joint pain in humans.
Natural products like salicylic acid can be medicines themselves,
or they can be inspiration for more effective treatments.
Like in 1853, French chemist Charles Frédéric Gerhardt synthesized a similar compound,
acetylsalicylic acid, which we know today as aspirin.
Gerhardt's aspirin wasn’t pure acetylsalicylic acid, though,
which brings us to the topic of this episode.
When we’re running reactions in a lab,
we need to purify them to isolate the chemical we're trying to make from any other reactants or side products hanging around in the mixture.
And one method to separate chemicals is chromatography!
[Theme Music]
Many reactions produce mixtures,
sometimes due to competing side reactions and sometimes because the starting material didn’t completely react.
For example, let’s look at a synthesis of the antidepressant medication paroxetine,
also called Paxil.
We’re starting with an alcohol and our goal is to turn it into an ether.
Remember that hydroxide is a poor leaving group, so in this synthesis,
we react the alcohol to form a mesylate.
Now we have a great leaving group,
and can use the Williamson etherification reaction to make the ether.
Unfortunately, those reactions aren’t super efficient,
and the ether is only 56% of our products.
So before we move onto the last step to make Paxil,
we’ll need to separate the ether we want from side products that we don't.
This is where chromatography comes in.
Chromatography is what we call a group of different techniques that let us separate the components of a mixture,
usually to quantify or identify them.
Chromatography always involves two key pieces to help separate different molecules in a sample:
there's a stationary phase or adsorbent, and a mobile phase or eluant.
The inventor of chromatography, Russian-Italian botanist Mikhail Tsvet,
first used solid calcium carbonate as a stationary phase to separate a sample of colored plant pigments.
That’s how chromatography got its name!
We can do a simple kind of paper chromatography using a piece of paper as our stationary phase,
to separate the component pigments in washable marker ink.
Paper is made up of cellulose, which is a bunch of sugar molecules strung together.
All of those hydroxyl groups hanging off the sugars have dipoles,
which means this paper is polar and interacts a lot with molecules that are also polar.
We're not just tearing off a piece of our notebook here.
Notebook paper is often coated to prevent ink from running, and we need the ink to spread out.
Now, I’ll draw a little line on the bottom of our chromatography paper in pencil to mark the origin,
which is where we put our washable marker ink sample.
This ink is a mixture made up of several different colored compounds that have different polarity.
Then, we can stand the paper up in a solvent chamber.
Here, I just have a jar with our solvent,
or mobile phase: a mixture of isopropyl alcohol and water.
We have to make sure the marker ink spot is above the solvent,
or our sample will just diffuse into the solution at the bottom of the chamber,
giving us a murky solution and no separation on the paper.
Oops.
What we want to happen here is for a process called capillary action to move the solvent up our chromatography paper.
We actually saw capillary action way back in Episode 6 when we looked at candle wax, too!
Polar compounds in the marker ink sample spend a lot of time interacting with the polar stationary phase,
and stick to the cellulose molecules at the bottom of the paper.
Less polar compounds don’t interact as much with the paper molecules and move up with the mobile phase more easily.
Different colored marker inks have different compounds,
so let’s check out the beautiful color separations from some paper chromatography!
Like I mentioned, our solvent chamber was filled with isopropyl alcohol
and water because that's safe and easy to do in a Crash Course studio.
But in organic chemistry labs, diethyl ether and hexane is a common chromatography solvent system.
Also, because paper is porous, the colors in the paper chromatography experiment were pretty spread out,
which isn't that useful for working with organic chemicals.
So our stationary phase in the lab is usually silica gel or alumina,
which has the consistency of very fine sand and gives a better separation.
A thin layer of the stationary phase is adhered to a glass, aluminum,
or plastic-backed plate and we call the technique thin layer chromatography, or TLC for short.
Now that we've got a new experimental setup,
let’s look at what happens to a sample of an organic compound when we alter the ratios of solvents.
We’ll start with an equal mixture of diethyl ether and hexane as our mobile phase,
a 1:1 solvent system.
We can quantify the movement of a spot of organic compound on the plate by calculating its retention factor or Rf –
a way to describe the eluting power of a solvent system,
or how much the solvent can move a compound up a stationary phase during chromatography.
To calculate Rf, we measure the distance traveled by the spot from the origin,
and divide it by the distance from the solvent front to the origin,
which is how far we let the solvent move up on the plate.
We have to remove the plate from the chamber before the solvent reaches the top,
otherwise the spot tends to spread out too much!
Doing the math in this 1:1 solvent system, our spot has a retention factor of 0.41.
Now let's try a different ratio by increasing the hexane, the nonpolar solvent in our mobile phase,
for a 1:5 ratio of ether to hexane.
After doing the chromatography experiment, measuring, and calculating Rf, we only get 0.16!
So we can say that the 1:5 solvent system has less eluting power than the 1:1 solvent system.
The nonpolar solvent hexane is less effective at pulling polar organic compounds away from the polar stationary phase and moving them up the TLC plate.
However, if we switch the ratio and increase the ether, the polar solvent in our mobile phase,
we'll get different results.
With a 5:1 ratio of ether to hexane, we’ll expect polar organic compounds to interact to a greater extent with the more polar mobile phase,
increasing the eluting power.
Let's do the math and, what do you know, in this solvent system our Rf is 0.70.
In the lab we're not just calculating Rf values of spots of pure organic compounds in different solvents.
We use chromatography to separate reaction products,
like the ether from the side products when making Paxil.
And that requires us to examine, and actually physically separate, mixtures from a sample.
We use TLC to monitor our reaction, and,
using a thin capillary tube, we put a spot of our starting material in lane A on the left.
We spot our reaction mixture, which usually contains multiple compounds that we want to separate from each other, in lane C on the right.
In the middle lane B, we spot both on top of each other – a cospot.
If all the lanes look like lane A after our chromatography is done, we could have made
a completely pure product from the reaction with an identical Rf to the starting material.
However, it’s more likely that something's gone wrong with the reaction.
So basically, TLC lets us preview if our reaction actually worked,
or if we're stuck with the same molecules that we started with!
Lane B, the cospot, helps us make sure there really is a difference between our starting material and our reaction mixture,
and that we know our starting material was used up in the reaction.
For example, if lane B looks like it’s missing the spot from lane A, but lanes B and C look the same,
a starting material could've hitched a ride with the reaction solvent to move further up the plate and fool us into thinking it's a new compound when it's not.
But if lane B includes the same separations as both lane A and lane C, we're good.
It's really important to triple check all this with these three lanes!
Now, we’re not always working with colored compounds in plants like Mikhail Tsvet,
or separating bright marker ink with paper chromatography.
Often with TLC, our reaction mixtures are light yellow or clear which can make them hard to see.
Luckily, many organic compounds have aromatic rings, double bonds,
or triple bonds, which absorb UV light.
So we keep a UV light in the lab to shine on TLC plates and reveal our separated components, so we can circle our spots with a pencil.
If we're working with compounds that don't show up under UV light,
we use chromatography stains that can undergo chemical reactions with the compounds on our plate.
Conveniently, the effects of these stains can be seen with the naked eye.
For example, a potassium permanganate stain reacts with compounds by oxidation,
and makes the oxidized compound spots appear yellow, on a pretty purplish background… kind of like our set!
TLC gives us a great preview of how our compounds will separate,
but a tiny plate isn't practical to purify grams of our reaction mixture.
For that, we’ll need a bigger, badder technique called flash chromatography.
To set up our flash chromatography,
we actually need to play around with TLC to find a solvent system that gives the component we want to separate from the reaction mixture an Rf of about 0.2.
We also need to know the mass of the unpurified sample we want to separate,
and from there we can pick an appropriately sized flash column with a simple table –
thanks to the work of chemists before us!
So let's say that we've figured that out, and now we’ll head into the Thought Bubble Lab!
To start, we'll push a plug of cotton firmly into the bottom of the chromatography column,
and cover the cotton with a little sand for reinforcement.
We're gonna use a method called slurry packing, where we mix the mobile phase solvent with silica gel as our stationary phase,
and pour that into the column through a funnel.
Next, we’ll apply pressure to the top of the column using an air hose,
until the solvent is level with the silica gel.
Let’s add a little more sand to keep the silica gel even,
and then we're ready to load our sample into the column!
With a long Pasteur pipet, we add our reaction mixture to the column.
Then, we can fill the reservoir with our mobile phase and run the column.
We’ll apply pressure with an air hose again,
forcing the liquid through all the silica gel, sand, and the cotton plug.
And we'll collect fractions of the mobile phase in test tubes.
We have to be careful not to let the solvent move below the level of the silica gel in the column –
that would mess up our carefully balanced mixture of mobile and stationary phases.
So we can add solvent to the top to prevent the column from going dry!
The less-polar compounds move quickly through the silica gel in the flash column into the first few test tubes.
After we've collected all the fractions, we can check if our compound has eluted by doing TLC.
The less-polar compounds will also move fastest via capillary action upward on the TLC plate!
Lastly, we’ll combine the fractions that have the compound we want in a round bottom flask and concentrate using a rotary evaporator.
A vacuum reduces the pressure inside the rotovap,
which lowers the boiling point of the solvents to remove them easily from the sample.
We spin the flask to create more surface area and encourage the solvent molecules to move into the vapor phase.
Hopefully we've isolated a good-sized, pure sample of the compound we want!
Thanks, Thought Bubble!
With a purified sample, we can calculate a percent yield to see how efficient our reaction was,
analyze our compound using spectroscopy, and then move on to the next reaction in our synthesis.
Maybe now we have our purified ether and can move onto the final step to make Paxil,
or we're doing any number of other reactions.
Flash chromatography is pretty common in organic chemistry!
In this episode we learned that:
Chromatography separates organic compounds based on polarity
Thin layer chromatography tells us how well our reaction went,
and Flash chromatography lets us separate larger quantities of compounds
In the next episode, we’ll talk about how we can use proton NMR,
one of the most important spectroscopy techniques to an organic chemist, to see if we really made our desired product.
Until then, thanks for watching this episode of Crash Course Organic Chemistry.
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