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Lecture Comments (4)

1 answer

Last reply by: Professor Starkey
Fri Sep 22, 2017 10:53 PM

Post by Maryam Fayyazi on September 20, 2017

Professor I have couple of questions about my lab;
1)given the structure of benzophone and biphenyl:
which structure would you be expected to have higer Rf value on a polar TLC plate(silica,alumina)why?

2)why is it recommended to spot the analyte on the TLC plate in the position that it will not be immersed eluent solvent?

3)why it is recommended to use pencil to mark your TLC plate instead of pen?
thanks alot

1 answer

Last reply by: Professor Starkey
Thu Mar 31, 2016 2:08 PM

Post by Tammy T on March 31, 2016

Why Silica gel SiO2 is polar? I drew out Lewis structure and VSEPR of SiO2, and the 2 dipole moments toward the 2 O on SiO2 molecule cancelled each other out. I thought SiO2 molecule has no net dipole moment overall. Please explain. Thank you great lecture!!

Introduction to TLC (Thin-Layer Chromatography)

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Chromatography 0:06
    • Purification & Analysis
    • Types of Chromatography: Thin-layer, Column, Gas, & High Performance Liquid
  • Theory of Chromatography 0:44
    • Theory of Chromatography
  • Performing a Thin-layer Chromatography (TLC) Analysis 2:30
    • Overview: Thin-layer Chromatography (TLC) Analysis
  • Step 1: 'Spot' the TLC Plate 4:11
  • Step 2: Prepare the Developing Chamber 5:54
  • Step 3: Develop the TLC Plate 7:30
  • Step 4: Visualize the Spots 9:02
  • Step 5: Calculate the Rf for Each Spot 12:00
  • Compound Polarity: Effect on Rf 16:50
    • Compound Polarity: Effect on Rf
  • Solvent Polarity: Effect on Rf 18:47
    • Solvent Polarity: Effect on Rf
    • Example: EtOAc & Hexane
  • Other Types of Chromatography 22:27
    • Thin-layer Chromatography (TLC)
    • Column Chromatography
    • High Performance Liquid (HPLC)
    • Gas Chromatography (GC)
    • Preparative 'prep' Scale Possible

Transcription: Introduction to TLC (Thin-Layer Chromatography)

Hi, welcome back to

Today, we are going to be talking about thin layer chromatography also known as TLC.0002

Chromatography is used to separate mixtures of compounds.0007

It can be used for two different purposes.0010

Either for purification, in other words to separate the mixtures of compounds,0012

or simply bought for analysis where you can find out how many components you have in a mixture,0016

maybe the identity of those components, so on.0022

There is a wide variety of chromatography methods that are available.0026

The C in TLC stands for chromatography.0031

TLC is thin layer chromatography.0034

There is also column chromatography, gas chromatography or GC,0036

and high performance liquid chromatography also known as HPLC.0040

They all have the same general theory of chromatography.0045

What is going on is, we have taken extra components and we add it to some kind of stationary substrate.0049

We call that the stationary phase.0055

There is going to be some kind of mobile phase that moves past this stationary phase.0058

Maybe a gas, maybe a solvent, something like that is a mobile phase.0063

And then, the components of our mixture are going to partition between those two phases.0067

You have done a liquid-liquid extraction, like for the sep funnel.0072

We know that the components of the mixture partition between those two different layers.0077

The same thing is going on here.0082

We have a stationary phase and a mobile phase.0083

Everything that is added to the stationary phase is going to move.0085

It is going to partition between the stationary and the mobile.0091

It is going to do so by different affinity such as polarity,0095

sometimes chromatography, separates components based on polarity.0100

Something that is more polar might stick to the stationary phase more.0103

Something that is less polar will spend more time in the mobile phase.0107

Because of this difference in affinity for the two phases, we are going to end up with traveling at different rates.0110

Some things are going to move through the column faster.0116

Or whatever stationary phase you have, it is going to move through faster.0121

Other compounds are going to move through more slowly.0125

Therefore, we are going to get a separation.0127

Once our components are separated, once again, we can either analyze them,0130

detect their presence, detect their amount, maybe run them through our mass spec to find out what their mass is.0136

Or maybe we can even collect them, as a way to separate and end up collecting the different components.0144

There are several steps in performing a TLC analysis.0153

I will go through them overall and then we will look at each step independently.0156

The first thing we are going to do is we are going to spot our TLC plate.0161

This is what a TLC plate typically looks like.0163

We have our stationary phase, a powder like silica gel.0167

It is mounted to some kind of plate, either plastic or aluminum, or glass plate, something to hold onto that silica gel.0171

The first thing we need to do is transfer our sample onto the plate.0180

We call that spotting the TLC plate.0185

Then, we are going to prepare our developing chamber and this is maybe a beaker with a watch glass over the top or some other jar.0188

We are going to put a little solvent at the bottom of this.0197

We are going to develop the TLC plate by standing it in the jar, so that the bottom is resting in the solvent.0200

And then, just by capillary action, the solvent is going to rise upwards on the plate and wet it.0209

Once the TLC is done, we then take it out and we mark where the solvent front was.0217

And then, we do some kind method to visualize the spot and see where they are on the plate.0223

And then finally, we are going to measure the RF.0228

The RF is a measurement of how far the spot has traveled, in comparison for the solvent has traveled.0230

We are going to measure the distance for spot A here.0239

We are going to measure the distance that the solvent travel, by dividing those two we are going to get our number for RF.0243

We will look in each of these steps one by one.0249

To spot the TLC plate, we can use a pencil to very lightly write on a TLC plate.0253

Now this is a powder, like I said, notice I’m holding on its edge,0259

we do not want to touch the TLC plate because then I can get organic oils on there and contaminate my sample.0263

If you write very hard with your pencil, it can cause the powder to flake off.0268

We do not want to disrupt the powder.0275

If you write very lightly, it is possible to mark it.0277

You would not want to use a pen or marker because those have organic compounds that will contaminate our sample.0279

But a pencil mark would not move, you can use a graphite to mark our plate.0284

We are going to take a spotter, a micro capillary tube, micro pipette.0288

Just touching it to a liquid solution is going to drop a little bit.0294

And then, touching our plate is going to transfer a small drop of that on to our plate.0298

We are going to spot it and we will see a little wet spot.0303

And then, as the solvent evaporates, our sample will be transferred onto that.0305

We will always going to be using a solution of our sample, whether our sample is a liquid or a solid or an oil.0312

In any case, what we are going to do is develop our sample in some kind of solvent.0321

It is that solution that we are going to spot on there.0327

We just want to add the tiniest amount for analysis.0329

We do not want, if we were to take a liquid sample and spot it on the TLC plate,0332

it would just be overwhelmed via the small thin layer of silica gel that is there.0336

You would overload the plate and then you just get a big blob going up.0341

You would not be able to identify anything.0344

Make sure you always dilute your sample in a solution and that solution is what you spot there.0346

Your developing chamber, like I said, might be some kind of jar with a lid on it or you can just use a beaker with a watch glass.0355

We need something to cover it because we do not want the solvent to be evaporating at a large rate.0361

We can also use a filter paper to line the sides of the jar.0367

And then, when we add in a small amount of solvent, we are going to shake it,0372

we are going to wet the filter paper with that solvent.0375

What we are doing is we are saturating the TLC chamber with the vapors of that solvent.0378

If there is some water, you can measure being really humid in the chamber.0385

We are doing that with our solvent vapors.0390

The reason we are doing that is as the solvent is rising on a TLC plate,0391

we do not want it to just continuously keep evaporating.0394

We want it to stay wet on the plate.0397

You do that by having plenty of solvent vapors already in the gas phase,0399

so that it is already saturated and there is no place for the liquid to evaporate to.0404

Or it is doing so but it is doing so in equilibrium.0409

We are going to add a small amount of solvent there, how much do we want?0412

We want to cover the bottom completely but we do not want it too high0415

because wherever we spotted our TLC plate, this solvent layer has to be below that.0420

If we spotted our plate, maybe 1/4 inch up the plate, the solvent level needs to be less than 1/4 inch.0428

That is because if we put this in and our solvent level is up here,0433

then our spots would simply dissolve into the solvent, if they were below the solvent level.0437

We have to make sure our spot is higher than the solvent.0441

As the solvent starts to rise, then it hits the spot and it is going to be carrying the components of that spot with it.0443

We are going to a place the TLC plate into the chamber.0453

Forceps are very handy to do that.0457

We are going to cover it and we are going to slowly watch as the solvent front rises.0461

With it, we are going to be carrying our spots.0466

We are going to watch it.0469

The longer you let it develop, the further you let this solvent go, the better the separation you are going to get.0470

Imagine we have 1000 runners about to compete in a race or maybe marathon.0478

They are all going to be traveling at different speeds.0485

But if you stop the race a 100 yards out, they are all still going to be clustered together.0487

You are not going to get that much separation.0493

But if you let them go a mile or 2, or 10 miles, eventually, you are going to get a much better separation of your components.0494

The longer you let it go, the better.0501

But you do not want to let it hit the top.0504

Because if it hits the top, now there is no place for the solvent to go anymore.0506

Then, the solvent is going to be moving in all directions and you are going to get diffusion of your spot.0510

Watch it come close to the top.0514

Before it hits the top, we want to remove it.0516

And then immediately when we remove it, we are going to use a pencil to mark that solvent front.0519

Because once the solvent evaporates, we will not remember how far it went.0523

Remember, we are going to have to measure that distance that the solvent travelled.0527

Immediately, take it out and either mark with a pencil or scrape off a little of the silica gel at the proper height,0531

so that you will know later how far your solvent travelled.0538

You need to visualize your spots because your TLC plate, most organic compounds are colorless.0544

Your TLC plate, before and after you developed it, looks exactly the same.0549

Just looking in this plate, I would not know if I have done it or not.0555

Actually, I already sketched a line here where my solvent went, so I would actually guess that this is a developed plate.0557

You do not know where the spots are because they are colorless.0564

Typically, the way that we visualize our spots, one good method is to use a UV lamp.0569

Now this is a lamp that has ultraviolet light.0574

This is a hazard because ultraviolet light is hazardous.0577

We want to make sure we do not look at it or expose ourselves to it.0581

Also, make sure you turn the lamp off when you are done because they are quite expensive and they do not have a very long life.0586

When we expose it to a UV lamp, when we shine it under a UV lamp, the silica gel, the whole plate is going to glow green.0596

That is because a fluorescent component has been added to the silica gel that will fluoresce under UV light.0602

You will see the whole plate turn green.0611

But wherever there is an organic component, it will quench that first and shows up as kind of a bluish or purple spot, a dark spot.0614

What we are going to do is, while we are shining the UV lamp,0624

we are going to take our pencil and we are going to circle everything we see.0628

Once we turn the lamp off, we are back to a colorless plate and we would not know where the spots were.0630

Make sure you circle what you see and then when you turn off the light,0636

what is left is you have outlined the spots that were there.0639

We can analyze it now.0645

There are some other methods we can have because not everything works with the UV.0647

It is really good for conjugated systems like if you have an organic ring,0652

a benzene ring in your structure that should show up with the UV lamp, but not everything does.0655

There are some other things you can do.0660

You can take some iodine crystals and put them in a chamber, and put through your plate in there and wait.0662

And then, the iodine will deposit itself whether they are organic compounds.0668

You will see some brown spots appearing.0672

Again, you can circle those when they come out.0673

Iodine also sublimes, kind of an irritant to your eyes.0675

After you take the plate out, make sure you circle it in and let it ventilate for a while to get rid of those iodine vapors.0681

There are also dips and stains and sprays that you can do, where you can make a mixture and dip your plate in there0687

and heat it on a hot plate or heat it with a heat gun and that will char any organic components.0693

That is another way to visualize your spots.0700

A lot of different methods, it depends on what function you have, depends on what kind of compound you have.0702

But start with the UV lamp, a lot of times we start with the UV lamp, circle whatever you see.0708

Then, maybe we will also move onto a dip or stain to see if anything else shows up.0713

Maybe combine those methods.0719

How about calculating the RF of the spots?0722

This is where we measure the distance that each spot traveled.0724

We are going to measure the distance that the solvent traveled.0729

And then, that ratio is going to be the RF.0732

The RF, first spot A looks like it has traveled about 1/3 up the plate.0735

Spot B looks like it has traveled about ¾ up the plate.0741

We will report those numbers, if I did the math here, 0.37, 0.76.0743

All numbers for RF have to be between 0 and 1.0748

If it does not travel at all, that is an RF equals 0, stays at baseline.0754

That is 0 because this number, the spot distance is 0.0762

If it travels all the way as far as the solvent goes, that is an RF of 1 with solvent front.0767

Why is that comes out to 1, because the distance that the spot traveled0778

is the exact same number as the distance that the solvent traveled.0781

When you divide those two numbers, you get a 1.0784

All our numbers are between 0 and 1.0787

We report it as a decimal, in decimal form.0789

What I have not mentioned here is this particular plate that I setup, it looks like I spotted three samples here.0794

You can see that I labeled them on the first spot.0803

In the first slot I spotted sample A.0805

And then, in the last slot, I spotted sample B.0808

It is very common to run a TLC with more than just one solution that you are analyzing.0812

It is very common to compare two solutions because maybe you have a starting material vs. a reaction.0819

Make sure you want to see if you have any un-reactants material left.0825

Or maybe if you have a sample of your pure thing and you try to isolate,0828

you can do that standard compared with your reaction mixture, or whatever you are trying to purify to see how they compare.0832

But running two samples side by side is not always helpful.0840

Because let us say you are on A here or we could call it x and y so I’m not confusing it with the A and B review.0846

This is x and this is y.0857

Let us say you have this spot and this spot.0859

That is not a useful TLC plate because you cannot really tell if those are identical compounds.0864

If they have the same RF then we would say that they are the same compound.0870

Or it is good evidence that they could be the same compound.0873

But sometimes there may be a solvent that does not rise perfectly, evenly up the plate.0875

Is that enough of a difference to confirm that all those cannot be the same compound or those must be the compound?0880

It is very difficult.0886

What we do instead is, when we are comparing two compounds by TLC,0887

we are going to not only spot them, just spot them side by side.0893

But in the middle here, we are going to spot both x and y.0898

That means, I’m going to take my solution of x and I’m going to spot it here and in the middle,0901

and then, let those dry.0907

And then, I'm going to take my solution y, and I’m going to spot it here and here, and let those dry.0908

What I have done in this chambers, I have simulated what a mixture of x and y would look like.0913

Now if I ran that TLC and I got this and this and this,0919

now I have pretty good evidence that x and y are in fact the same compound because that middle mixture only gave one spot.0927

That is pretty good evidence that they are both containing the same compound.0936

Where if I tried another one and I mixed some in the middle, and I had this and this, that is a very different story.0943

Even though x and y have very similar RF, when I mix the two, I did not get one clean spot.0955

I got two spots kind of on top of each or kind of lumpy, something different.0960

That is an indication that you have a mixture of compounds, you do not have one compound.0965

Here x does not equal y and here x does equal y.0970

Note here on y, I have this setup here.0975

A has just a single component.0978

B has just a single component.0981

What do we expect in the middle chamber, in the middle column,0984

we expect to have both A, see how they match, and B in the middle one because we spotted both solutions in that middle.0987

This is just kind of a handy way.0995

When you go to measure the RF for spot A, you can pick any of the places0997

where you had A spotted and pick anywhere of those spots.1002

Pick the middle of that spot and then calculate that distance.1006

Let us talk about how the RF, the distance that it travels, how that varies by the sample that you are using.1013

It depends on the polarity of that sample.1020

Let us compare a mixture of two compounds A and B.1024

If A is more polar, it is going to have a stronger affinity for the silica gel.1028

Silica gel is the stationary phase we are using.1033

It is SiO₂, it is extremely polar.1036

It is an extremely polar stationary phase.1042

If you have a polar compound like A, it is going to grab onto that stationary phase and have a very strong affinity to it.1045

How easily does it migrate up the plate, not easily at all.1052

We expect it to travel very slowly and we are going to have a low RF.1057

If we compare that to a compound that is less polar, if it is less polar, it does not stick as much to that silica gel.1062

It is a lot easier for it to partition into the mobile phase.1069

Therefore, it is going to travel faster.1074

If it travels faster then it is higher at the plate when you are done and it has a higher RF.1076

In comparing these two compounds in this sample, I had two compounds.1081

I would say that A is the more polar compound because it has a lower RF.1087

Sometimes, we can very often predict if we have two things that we are expecting to see in the TLC.1097

We can predict which one should be higher, which one should be lower.1102

You can make miscible predictions but sometimes it varies by the solvent.1105

Sometimes it is not as straightforward.1109

But this is a general trend that we see for TLC.1110

The more polar compound, sticks better to the polar stationery phase.1114

Again, I wanted to make sure I have silica gel as the stationary phase.1120

This is the most common one for TLC.1123

Therefore, it has a lower RF.1125

Another thing that we can consider though is the polarity of the solvent.1129

Very often, we use a mixed solvent system.1134

We mix a non-polar solvent with a polar solvent.1137

Therefore, by varying the composition, we can find how polar or non polar the solvent is that we are using.1140

Now what happens if we increase the polarity of the solvent?1150

If we have a more polar solvent then that is going to compete better with the stationary phase, that polar stationary phase.1153

Compounds are now going to have an easier time transferring into the mobile phase.1162

If they are spending more time on the mobile phase,1167

that means they are moving faster and they are going to move higher up the plates.1169

We are going to expect a higher RF with a more polar solvent.1172

Let us see some examples of this.1176

Let us say we did this initial mixture with 10% ethyl acetate hexane.1179

Ethyl acetate is an ether, it is like that.1184

This is a polar solvent.1189

Hexane is just 6 carbons, it is a hydrocarbon that is non polar.1191

This is ethyl acetate and this is hexane.1199

We have mostly non polar solvent, hexane.1202

90% of it and we have added 10% ethyl acetate.1205

This is the TLC plate we get.1209

Now what happens if we lower the amount of ethyl acetate to just 5%?1211

This is now less polar.1217

It is not as effective at pulling the polar components or any of the components off of the stationary phase.1219

The last polar solvent, we are going to see our RF go lower.1229

They are going to be lower now.1235

What if we went from 10% up to 20%?1237

Now we have a more polar solvent.1241

It is going to compete better with that silica gel.1243

It is going to spend more time in the mobile phase.1246

It is going to have higher RF.1249

It is going to be up the plate more.1251

What if we use a 100% ethyl acetate, no hexane at all?1254

This is now an extremely polar solvent.1258

It is very likely you are going to take every spot we had down here and just move it completely along with the mobile phase.1260

We are going to get an RF of 1.1267

The opposite would be true if we had an extremely non polar solvent like 100% hexane,1268

we would expect nothing to move.1274

RF equal 0.1276

This is kind of assuming that these two spots have some kind of polarity.1279

Most functional groups have some kind of polarity.1282

Most organic compounds are not completely non polar.1286

If you had a totally non polar compound, that would still travel.1290

You could use hexane on a cone or on a TLC to move something that is a completely non polar compound.1302

What we are looking at is relative polarities and relative RF.1310

Assuming that we have some kind of polarity,1315

we are going to see that our RF are going down with the less polar solvent and our RF are going up.1316

An increase in solvent polarity, increases all RF.1321

It does not really matter whether your compound is more polar or less polar, increasing,1333

because what we are doing is we are competing with the stationary phase.1338

Everything is sticking to the stationary phase initially.1340

The more polar solvent is going to compete with that better and they are all going to get moving.1344

Let us talk about it briefly about some other forms of chromatography.1350

We have talked about TLC, thin layer chromatography, where we have is a thin layer of a stationary phase.1354

Silica gel is what you are going to encounter, most often, but you can also have a lumina,1362

another white powder that you can use for that.1365

The mobile phase is typically going to be an organic solvent.1369

That is what is going to move through there.1374

If we do column chromatography, column chromatography takes advantage of the same theory that TLC does.1377

Except now instead of doing analysis, TLC is for analysis.1383

But if you were to fill this column with silica gel, and then load your sample to the top here1389

and fill those reservoir and pass the solvent through, your components would separate out.1394

And then, you can collect down here in different test tubes.1402

You could start to collect your less polar compound that travels more quickly, you could collect that.1405

And then eventually, your more polar compound, eventually, makes it down and you can collect that.1412

You can actually use that to purify a mixture of compounds.1416

You would use a combination, you would typically use TLC to explore your different solvent combinations1421

and see which is the one that gives you good separation of your spots.1427

And then once you decide on a good solvent combination,1432

then you can move to a column to separate your components of your larger mixture.1435

HPLC is a high performance liquid chromatography.1441

Usually it is like a column but it is very small, very thin, and it is really long.1446

They kind of coil it up inside of your instrument.1453

It can also use silica gel as the stationary phase or it can have some other hybrid silica gel, something similar to that.1457

Typically, you have aqueous solutions that you are injecting in there.1465

If you want to find out how much caffeine you have in a sample or something like that,1469

you could inject some of that into the HPLC and that is going to a separate it out.1474

And then, GC is gas chromatography.1481

That is where you use gas as your mobile phase.1483

Again, it is a very long thin column that is kind of coiled up.1487

You have gas shooting through there and you either have a liquid, some kind of like oil, or a solid, that it is packed with.1493

You inject your sample and it gets loaded onto that stationary phase and the gas passes it through and separate.1501

Where GC is really useful is when you have GC-MS, that is where you add mass spectrometry, doing a mass spec analysis.1508

After your components can come off of the GC, they pass through a mass spec1520

and then they get analyzed to see what is the molecular mass and how does it fragment.1527

That tells us something about the structure.1533

That is a really powerful analytical tool where you not only see how many components you have,1535

but you see precisely how much you have.1543

It tells you the relative amounts.1545

And then, you can maybe even identify the compounds when you are done.1546

GC is great for analysis, HPLC is great for analysis.1551

Again, telling you how much you have and how many components you have.1555

TC is analysis but it is not quantitative.1559

It is not quantitative, in other words, I see that I have two spots but I cannot really see how much of each component I have.1563

The best you can do is if the spot is really faint, you can make the assumption that you do not have very much of that.1569

If you see a spot fading over the course of our reaction, you can follow a reaction by TLC, very useful tool.1575

You start out with your reaction and you have your starting material.1585

And then 30 minutes later, you run a TLC again and all of the sudden you start to see the appearance of your product.1588

And then maybe an hour later, you start to see the starting material spot fading in intensity.1595

Your product maybe increasing intensity.1603

But really, is it there or it is not out there.1607

Ideally, at the end of the reaction, you starting materials are all gone and all you have left is product.1610

That is kind of in a perfect world, you did not see that happen that way.1616

TLC is a really great analytical tool for checking that.1618

Where to purify mixtures, you would typically not use TLC for that, you could use column.1624

But it is possible to do a TLC and that is possible if you have a big TLC plates, that are kind of thicker layer of silica gel.1633

And then, you would layer the bottom, you would spread out your sample all the way along the bottom.1648

As the solvent rose, then you would have a band of your compound separating.1653

And then, after developing, after looking in the UV lamp,1658

you can literally scrape off the silica gel for the bond that you are interested in and wash your sample off there.1661

Same thing is true with the HPLC and GC, is that you can also have preparatory versions of that1667

where you collect it as it comes off the column.1676

You can cool it on the GC and you can recollect the samples as they come off.1680

We call that preparative GC or TLC, when you can actually isolate the components.1687

But for the most part, the column chromatography is the one that1694

for sure we are using to separate and purify and isolate compounds.1698

Rather than just testing a small portion of it to see what is in there,1702

we are taking a gram of this mixture and separating it into 12 ml of this and 600 ml of this,1705

and recovering them and in the end isolating them.1713

Where the others are primarily used for analysis.1714

Hopefully, you got to see what chromatography is all about.1718

There are some tips and tricks for TLC that we will cover in the video.1723

I hope you have successful TLC experiences in the future.1727