Organic Chemistry Lab TLC Analysis Lab

Hi and welcome back to www.educator.com.0000

Today's topic is thin layer chromatography, also known as TLC.0002

It is typically done on something like, this is called the TLC plate.0006

What we have here is a very thin layer of silica gel which is a very polar powder.0010

That adhere to this kind of plate, in this case, it is a flexible plastic plate but it can be glass plate or even aluminum.0018

It varies a little bit, but the important part is the side that looks kind of powdery.0027

Notice how I’m handling it on the edges because my fingers have oils in them and0032

I would not want to touch it, because I could be contaminating my plate and getting some foreign organic compounds on there.0037

When I'm ready to run my TLC, what I want to do is transfer my sample onto the TLC plate.0044

We call that absorbing onto the plate or spotting the sample.0051

Let us say I have my reaction, I just finished my reaction and I have it here.0057

I have a little oil at the bottom here or maybe a solid even.0061

What I do not want to do is take my TLC spotter and spot that directly onto the plate.0066

We never take a neat boil, you know that pure sample and put it onto the TLC plate.0071

What we always need is some kind of solution.0079

What I would do is maybe before I'm putting this on to the rotovap, if let us say I finished up a reaction, this is now an ether solution or an acetate solution.0081

Before putting on the rotovap, that is a good time to take that solution and spot it onto the TLC plate.0090

But if you are done and you have an oil or a solid, and you need to put it on the TLC plate,0096

then what you are going to do if it is an oil, is just a pastor pipette and just dip it in there so you get the tiniest bit on here.0101

And then, transfer this over to a vial and rinse it in with some solvent that your sample is soluble in.0110

At last, you have ether or even acetone.0117

Transfer this into a vial.0119

Make a little solution of your sample.0120

That is the solution that we are going to spot under the TLC plate.0123

The TLC plate, we can write on with pencil because that is not going to be affected by the TLC.0126

What we are going to do is we are going to make a line down at the bottom, same where I’m going to be spotting my samples.0132

And then, you can put little cross mark where they are and you can label it saying this is product A and this is sample B.0139

Maybe I'm comparing that starting materials to my reaction mixture, or product to my reaction mixture.0145

Very often, what we do is we are going to spot, let us say we are going to compare two compounds on TLC,0151

very often what we are going to do is run three spots where we do A on the first and B on the last one.0157

The middle will be the mixture of A and B.0167

When I take my sample, I have my little vial of sample A, then I take my spotter.0170

And the spotter, just by capillary action, it is going to drop a little bit of that liquid, that solution.0175

I’m just going to touch it to the plate and you will see that plate get wet.0179

You will see that evaporate right away because we will be using a volatile solvent.0183

You are going to spot it once and then spot it on the middle channel so there is A and A.0186

And then, we let it dry and now it is ready to be run for channel A.0191

And then, we get a new spotter and our new vial for B.0195

Again, just touched to the solution in B and spot it on B, let it dry, spot it on the middle, let it dry.0198

Now it is ready to go.0204

One thing you do not know is, have I put enough sample on there?0206

We are going to be using a UV lamp later to visualize our spots.0209

What you can do is before you develop the TLC plate, you can check with the lamp to see if you can see it at the baseline.0212

If you cannot see it at the baseline, you are not going to be able to see it after the plate is developed.0220

If you need to add more sample, you simply can spot it multiple times.0225

If you have a very dilute solution, you can spot it on A and let it dry and then spot it again, and spot it again.0229

Each time, more of your compound is getting absorbed onto the sample, onto the silica gel.0236

That is a way that you can increase the amount that you have on there.0242

When we are ready to develop the TLC plate, we are going to need some kind of a TLC chamber, something like this.0246

We could use a jar, we could use a beaker with a watch glass over the top.0253

We want some kind of mostly closed system because when we put our solvent in there,0257

we do not want our solvent to all evaporate.0261

We can use a filter paper but you can just use a piece of paper towel, it is lot cheaper.0264

But we want to line the side of our developing chamber with paper.0272

Then, when we add in our solvent, that paper will be wetted with the solvent and0279

we can saturate the entire chamber with the vapors of the solvent.0283

Imagine like, if your solvent is water, you would feel like it is very humid inside that chamber0287

because it is filled with water vapor.0292

Same idea, we want it saturate with the solvent vapors.0293

As the solvent is traveling at the TLC plate, it is not going to just continuously keep evaporating off.0296

It is going to stay keeping that TLC plate wet.0302

We can make up our solvent system, typically, we are using a mixture to the solvent system,0307

a mixture of a non polar and a polar.0312

Let us say we did it at one of the 10% ethyl acetate and hexane solution.0313

We can just take 10ml graduated cylinder and pour in 1ml of ethyl acetate.0318

And then, fill up the rest of the 10ml with hexane.0324

Just pouring it, estimating it, does not have to be very precise.0327

Then, we can pour that in here, and I’m not using any solvents because my studio is not a safe place.0331

Of course, if I were using real solvents, I had my eye protection on, I would be having a lab jacket on.0339

We are just doing our dry lab here for demonstration.0343

We shake it around a little bit.0346

Make sure the paper gets all wet, wet is with the solvent.0347

Now it is ready to go.0351

Your solvent level should be just at the bottom, ¼ inch at most, of the solvent chamber, of the TLC chamber.0352

That is when we spot our TLC plate, we do not put it real close to the bottom.0360

We put it a ¼ inch up or so.0364

The goal is that our solvent is below where we spotted it.0366

If our spot is below the level of the solvent, then it is just going to wash away, as soon as we put it in there.0370

We need the spot to be above the level of the solvent.0375

Now when the solvent starts to rise by capillary action, it starts to rise at the plate,0378

it is going to carry that sample with it along the silica gel.0382

When am I ready, I can use some forceps to grab onto my plates.0387

I’m not handling it with my fingers.0391

I can place it in very gently.0393

I’m going to place it right in the middle, lean it up against the edge.0395

And then, keep its cap on it so that it does not evaporate quickly.0397

And then, we are just going to watch it.0401

We are going to watch it as the solvent slowly rises up the plate.0402

We are going to let it develop as long as possible.0406

The longer you let it go, the better the separation is going to be.0409

Now is a good time for patience.0413

But you do not want to let it go all the way to the top because0415

if it goes all the way to the top of the plate, then the solid has no one direction to go.0418

It is just going to start moving in all directions and your spots are going to start to spread out and get more diffused.0424

We want to watch it go up and we want to get somewhere close to the top.0429

Somewhere close to the top, we are going to take it out.0433

While it is still wet and while we can see it, we are going to immediately mark the level of that solvent,0436

so we know where the solvent went.0441

That is going to be important for our calculations.0443

We want to know the distance that the solvent traveled.0445

We are going to take it out, we are going to let it dry to dry pretty quickly0450

because these are volatile solvents that we are using.0452

And then, what we need to do is figure out a way to see our spots because0456

most of the organic compounds we are dealing with are colorless compounds, white solids or colorless oils, something like that.0459

The TLC plate is going to look exactly the same before developing as after developing.0468

We are going to have to see where those spots are.0472

The most common way to visualize spots on the TLC plate is by using a UV lamp.0475

UV rays are dangerous.0482

You never want to look into a lamp and these bulbs are quite expensive as well.0486

We do not want to leave them on.0490

But when we are ready, we are going to turn it on and then we are going to look at the plate.0491

What we see with the plate is, the silica gel is treated with the fluorescent compound that glows green, when you put it under UV.0497

The whole plate turns green except wherever you have organic compounds that quenches the fluorescence.0507

Any organic compounds on here are going to show up as kind of purple spots.0512

What we are going to do is we are going to look at our plate.0518

We are going to turn over to look at it and while the light is on, we are going to circle what we see.0520

We are going to circle whatever spots there are.0528

Once we turn the light off, we can still have a record of where the spots were and what they look like.0533

Those are few other ways to visualize spots.0544

Not everything is going to show up with the UV lamp.0546

This is especially good for conjugated systems, something like an aromatic ring.0549

But other compounds are not easily spotted this way.0553

You can put it in a chamber.0558

Sometimes we would take a jar like this and we fill it with iodine crystals.0560

We put iodine crystals in here and then we can put our plate in with the iodine crystals.0565

And then, just let it sit for a while and what we are going to see is the iodine adheres to organic compounds.0570

We start to see brown spots showing up.0576

That is another way that we can visualize our spots.0579

The one precaution there is that iodine is a solid but it sublimes.0582

Those vapors can be kind of, they can burn your eyes.0587

Make sure when you take the plate out, you do not pull it close to your face.0592

Give it some time for any iodine vapors to dissipate.0594

Again, circle what you see right away because eventually those spots will disappear or very likely disappear.0598

They also have a lot of stains or debts you can use to visualize spots.0606

You might take a gel like this and prepare a stain.0611

A lot of them are very caustic, very strong acids.0614

There are permanganate dips, there are either oxidizing agents, or acids, all sorts of things like that.0617

In that case, what we would do is we would dip our sample, dip our plate into the liquid, in the liquid dip.0623

And then, wipe off any excess so it is not all drippy.0631

And then, we would heat this under a heat gun.0634

When you heat it, the organic compounds will char and will leave a residue.0637

Again, that is some other way you can visualize your spots.0642

A lot of different options out there but the UV lamp is certainly the most common one.0645

What you can do too is you can visualize it with the UV lamp and circle what you see with the UV lamp.0649

And then, do a dip and sometimes new spots will show up, that were not UV active.0655

That did not show up with the UV lamp but now showed up here.0660

You can get a record.0663

What you want to do is you want to sketch this into your notebook, as close to scale as possible so that you know what your TLC plate look like.0664

Make sure you always record what your solvent system is as well, because that is important,0674

if anyone else will reproduce your work.0679

Like I said, the reason we usually do a reference sample on the same plate is0682

because the solvent system is continuously changing, things evaporating at different times.0687

It is never precise solvent system that we are using.0693

But if you spot them at the same time and run the plate together with the same solvent system,0696

then you precisely whether the RF match, the distance they travel match or not.0702

That is what is important.0707

When we go to analyze a plate, the measurement that we are going to be taking is0709

we are going to be measuring the distance.0713

Right here is where I initially spotted it.0716

There should be nothing left to that spot, unless there is some really polar component that did not traveled at all.0718

We are going to be measuring the distance from where the spot was the solvent front ended.0724

That is the distance that the solvent traveled.0731

And then for each spot, every spot has its own RF.0733

We are going measure to the center of each spot and that is going to be the distance that the spot traveled.0736

The RF is the decimal of how far the spot traveled, compared to how far the solvent traveled.0742

This one looks like it went about maybe at 20% on the way up, or 25% on the way up.0750

Its RF would be 0.25.0756

The RF range you can have gone at 0 means that the sample did not move at all.0758

If it moved all the way with the solvent front, then that we would report as an RF of 1.0765

All numbers are fractional numbers between 0 and 1, that is our RF.0771

One more thing to talk about is how do we make spotters.0778

What I have showed here to imagine transferring our sample onto the TLC plate or spotting the TLC plate,0782

I use ice for showing the capillary tube.0789

These capillary tubes, they kind of look like melting point tubes but they are open at both ends.0792

It is just a hollow tube the whole way.0798

This diameter looks very tiny but it is actually huge for the sake of a TLC.0801

If we use this to make a spot on the silica gel, it would spread out and probably like ¼ inch wide.0807

We want to make the spot as small as possible because as it travels, it will defuse out.0814

We want to minimize that.0820

At start, just small enough to see it, maybe a couple millimeters wide is the biggest you want.0825

What we can use, we have a micro pipettes for spotters.0832

We can use a capillary tube to make a micro pipette.0838

What we will do is we would do that with the Bunsen burner.0841

This is something we would all want to do as a class, while no one else has any solvents out because solvents are flammable.0845

This is an exercise that we are going to do either in a separate lab or at a time when everyone is doing the same thing.0852

We can make some micro pipettes.0857

We would light the Bunsen burner and then we would take our micro pipette.0861

Just heat it in the center and rotate a little bit.0869

When you do this, you are going to start to see that center get a little soft and smooshy because you are heating a glass.0872

And then, you remove it from the heat and pull.0877

What is going to happen is you are going to lengthen the capillary tube.0881

What that does is the middle spot, that was the hot glass that is soft, it is going to narrow.0889

It is now going to be still a tube, still a capillary tube but it is going to be really skinny in the middle.0897

All I have to do is break it with your finger nail right in the middle, against the table.0903

You just break it in the middle.0907

Now you have two micro pipettes, micro capillary tubes.0908

You can make up a bunch of those.0913

You can even, if you are good enough, which in practice, grad students get really good at this,0915

when they do not have money to buy micro spotters.0919

Is you can use a pastor pipette to make your spotters.0923

This has a lot more glass to it.0928

When you are heating this, you can stretch it a little and keep heating it, stretch a little and keep heating it.0931

Finally, when the whole center is nice and soft and hot, you can take this and you can stretch it as far as you can.0936

You just get this long, thin capillary tube.0942

And then, you can break that off in chunks and then you can use this to have multiple spotters.0948

Before doing a TLC experiment, you want to makeup a few spotters, however many you are going to need for that day.0953

If you have some extras, store it away somewhere that you have them for a later time.0958

One more thing to talk about is, what we are taking about today is thin layer chromatography TLC0964

because it has a thin layer of silica gel.0971

This is a really cheap and easy way to analyze solution, analyze a mixture and see how many components you have.0974

Maybe identify them, if you have it.0981

You can refer to something.0983

And you can actually get prepped TLC plates that are big plates, that are thick with silica gel.0986

Instead of doing a spot, you can take a pipette and0991

you can actually run all along the bottom of the TLC plate and load your sample on there.0994

And then, run the TLC so you have a band of your components moving up.1001

And then, after developing the plate, you can literally scrape off the silica gel from the glass plate wherever it was1006

and then rinse your sample off, and extract from the silica and isolate it that way.1014

TLC can be used preparative meaning to isolate and separate compounds.1020

Typically, it just uses an analytical tool to learn something about your mixture.1026

If we want to use chromatography in a preparative manner to purify things,1031

what we typically use is a flash column chromatography.1037

In a flash column, we are going to take this and we are going to load this up with silica gel.1042

Silica gel is a powder, a very fine powder.1048

When we fill those up, we are going to do that in a hood.1052

We are going to put a funnel here and shake the silica gel, and do on the hood because it is a respiratory hazard.1055

You do not want to inhale any of that.1060

This stopcock here has little pieces of cotton in here to stop anything from going down into the valve.1062

What you do is you pour a little layer of sand and then you pour in about 6 inches typically of silica gel.1072

And then, you pour in another little layer of sand and then you are going to fill this reservoir up here with solvent.1079

We are going to use an adapter here to hook up to an air line.1084

We are going to use air pressure to force the solvent through the silica gel at a rapid phase.1092

It is called flash column chromatography.1101

We do that to load up our column and get our silica all wetted with the solvent.1104

And then, very carefully with a long pipette, we are going to load our sample onto the top of that silica gel.1109

When it is all adhered onto the top of the silica gel, like spotting a TLC plate,1115

when it is on top of that silica gel, then we are going to fill out this solvent and pass it through.1120

And then, we would use test tubes to collect our samples.1126

I will put a test tube here.1133

As the solvent is coming through, I will put a test tube here and fill it up.1136

And then, as the next one comes out, I would fill that out.1141

One by one, I would continue filling up samples.1143

Now you can imagine all of our compounds are up here as a mixture.1147

But as they travel through different speeds, at different rates,1151

the one that would have the higher RF on the TLC plate that was the fastest.1154

That would be the least for our compound.1158

That moves the fastest on the TLC plate, that moves the fastest in this silica gel as well.1160

That would come off the column first.1165

What you would do is you collect 10, 20, 30 fractions one by one to keep swapping them out.1167

You are going to be hopefully, first isolating the least polar component.1174

And then, maybe some clean solvent.1179

And then, the next polar component will come off and so on.1182

You can even change the gradient of your solvent.1185

You can even start with a non polar solvent and then increase the polarity,1187

so that you really improve the separation of your compounds.1191

This is something that is usually for organic chemistry, when we finish a reaction, we want to purify our compound.1194

Doing a column on that is saner way to do it.1201

When I have these 10, 20, 30 fractions, now I could use a TLC plate to analyze each of these fractions around the TLC plate.1205

The TLC plate is going to tell me which of the 50 fractions has which components.1214

And then, let us say these first fractions, 3, 2, 7 have the component I’m interested in,1221

then I'm going to take those and I’m going to put them into a round bottom flask, a third round bottom flask.1230

When I put this in a rotovap, I’m going to have isolated in there the single component of my mixture.1236

Chromatography is a fantastic way to separate compounds.1242

We can use it both with TLC or by flash chromatography.1246

Thank you.1250