Hello, my name's Mrs. Niven.

And today, we're going to be looking at how we can use chromatography to separate and analyse different samples of ink.

Now, you may have some experience of this from your previous learning, but what we do in today's lesson will allow us to apply our understanding of chromatography and how the products of chromatography are analyse to help us answer those big questions of, what are substances made of and how do substances behave? By the end of today's lesson then, you should be able to use paper chromatography to identify the composition of different inks.

Throughout the lesson, I'll be referring to some key terms, and these include chromatogram, sample line, solvent front, and Rf value.

The definitions for these key terms are given in sentence form on the next slide, and you may wish to pause the video here so you can read through them, perhaps make a note of them so you can refer to them back later in your lesson or for you to refer back to later in your learning.

Now, today's lesson is going to be separated into two sections, how we can prepare for chromatography, and then how we can use chromatography to analyse different inks.

So let's get started by looking at how we can prepare for carrying out chromatography.

Now, chromatography as a separation technique is incredibly useful because it uses or requires only very small volumes of the samples in order to separate them out into the different components.

But how well those samples are separated into its components depends on how they interact with both the stationary and mobile phases that are used in the chromatography process.

Now, because of that, the quality of the chromatogram that's actually produced as a result of carrying out this separation technique is gonna depend very much on how those materials are chosen, and then prepared before chromatography is allowed to take place.

Now, the two main materials that we're talking about here then is that stationary phase.

So what material are you going to be using? In paper chromatography, it tends to be some form of absorbent paper, like filter paper.

The other material choice we're talking about is that what you use for your mobile phase, for your solvent, what reservoir of solvent are you gonna have in the bottom of your container that will hopefully be able to dissolve the samples you put on the stationary phase, and then be able to carry them up that stationary phase to create that chromatogram that we can use.

Now, most frequently, we tend to use water.

And if, for some reason, that doesn't work, we might try a different solvent, for instance, ethanol or propanone.

Now, while we might be able to choose different solvents for our mobile phase because the chromatogram actually develops on the stationary phase, that's gonna take some very specific careful preparation.

So the first thing we need to remember to do is to draw a straight line, approximately one to two centimetres from the bottom of your stationary phase and that must be done in pencil so that it's not interacting with the solvent that's been chosen.

Now, depending on how many different samples you are going to try to separate on this stationary phase, you need to put a small X for each one and you'd like to have that evenly spaced on the sample line.

So one for each one that you are testing.

And the other thing we want to make sure is that these Xs are not placed too close to the edges of our paper just to make sure that any chromatogram that we are able to produce is more easily able to be interpreted.

Then what you're going to do is put in the centre of your X a small sample of that substance that you are testing.

Now, we can see here that no Xs have been placed on our stationary phase.

So if you have forgotten to draw the Xs, fine, okay? But it's absolutely important that you are ensuring that the samples you are placing on that stationary phase are evenly spaced even if you have forgotten to put those Xs on.

So it's not the end of the world if you've forgotten to put the X on there, but it is absolutely essential that you are ensuring the samples are evenly spaced on that stationary phase so that those chromatograms don't accidentally overlap and interfere with each other as they develop for each sample.

Then once you have your stationary phase prepared, you're gonna carefully place it in a container that has a very small volume of solvent in the bottom.

Now, we wanna make sure that the solvent reservoir stays below the sample line so that it doesn't interfere with the chromatogram that develops, and you're gonna need to wait quite patiently for this chromatogram to develop.

I have seen sadly in the past, some students shaking the container to try and force the solvent up a little bit more or carrying it around the room to show others because they're so excited about the chromatogram that is being developed, and that I can understand.

But by moving it around like that, you potentially force that solvent reservoir over the sample line and that can essentially destroy the chromatogram that you've developed and you need to start again.

So be patient, leave it still, and just enjoy it as it develops.

Let's stop here for a quick check.

Which of the following shows a correctly prepared stationary phase? Well done if you said B.

Some of you may have been tempted to say that D was a correctly prepared stationary phase, but those outside samples are a little too close to the edge and there is the possibility then that a chromatogram won't develop as well as we'd like to.

So we want to have them evenly spaced, our samples evenly spaced between each other, but also between the edges.

A and C are inappropriately prepared because as you can see, some of the sample spots are far too large and they're inconsistent.

We want our sample sizes to be approximately the same as well, so that we can have that fair comparison when the chromatogram has developed.

So you've created your stationary phase and you've put it into your solvent.

But remember, we have a choice of solvents for our mobile phase and that might affect the quality of the separation, and then the chromatogram that develops.

And you might also need to use a lid on your setup if you've used any volatile solvents like propanol or ethanol.

So if we have two different solvents here, we've got water on the left acting as our mobile phase and alcohol that is on the right.

Now, you can see with the, sorry, the alcohol, it is ethanol, but it's an example of alcohol.

The alcohol that is on the right actually has a lid on top, and what that does is prevent that alcohol from evaporating into the atmosphere 'cause it has quite a low boiling point and it keeps it within there.

But you can see already that we have two very different chromatograms that are developing as a result of this.

The thing that stands out to me as we compare them is that the right most sample doesn't appear to be developing in the water solvent on the left, and we definitely see that it is soluble and developing in the alcohol solvent that is on the right.

When you see that the chromatogram has reached towards the top of your stationary phase, you want to remove it from the solvent and you want to then use a pencil and being very careful not to tear your stationary phase because it's still damp.

You want to then mark that solvent front.

You can see that it's being marked here just at the very top.

Doesn't need to be a whole line across, just one little mark will do.

And then please to draw a circle around or very carefully outline where the spots are that have developed for each of your samples.

Now, it is absolute essential that you're being really careful with the pencil because with this damp paper, this stationary phase is very easy to tear and it's important that you're doing it immediately because sometimes that solvent is still travelling through the paper even though you've removed it from the solvent reservoir because there's some residual solvent still travelling through that stationary phase.

So make sure that you are marking these immediately upon removal from your container.

Let's stop for another quick check then.

True or false, the volume of solvent in the container should reach above the sample line.

Well done if you said false.

But which of these statements justifies that answer? Well done if you said A.

The solvent should be below the sample line to avoid the sample dissolving into the solvent.

We do not want that sample line or any of the samples on that sample line to be dissolving into the solvent, okay? That would completely destroy our chromatogram and very little would develop on that stationary phase.

So we're avoiding the sample dissolving into the solvent by making sure the volume of solvent in the container stays below that sample line.

Well done if you manage to get that correct.

Okay, time for today's first task.

What I'd like you to do is to read through each of these steps, and then place them into the correct order that would allow us to create a method for carrying out the chromatography of three inks.

You may wish to pause the video here so that you can read through them carefully, put them in an order, and maybe compare them with the people next to you before you come back to check your answers.

Okay, let's see how you got on.

So the very first thing we want to do if we're going to be carrying out chromatography is we're going to use a pencil and a ruler.

I'm gonna draw a straight line about one centimetre from the bottom of our chromatography paper.

So that was statement C.

The next thing we're gonna do is try to place three evenly placed X marks on that line and add a small sample of each substance to each of those Xs.

So one sample on one X.

Now, I need to have at least three spaced marks here because I'm testing three different inks.

So that was statement E.

The next statement I needed was statement A, because this tells me I should hang now my prepared paper in a container with that small volume of solvent in the bottom and to make sure that that solvent is staying below the sample line.

The next statement in my method should be statement B, which is simply to allow that chromatogram to develop.

It is an exercise in patience, but trust me, it is well worth the effort of waiting patiently.

And final step then would be statement D, where we're going to remove the paper from the container as those spots reach the top of the chromatography paper and mark that solvent front in pencil.

And don't forget to also outline those sample spots as well.

So well done if you've managed to get all of those in the correct order.

Great job, guys, fantastic start.

Now that we're feeling a little bit better prepared for carrying out chromatography, let's have a go at using it to analyse different ink samples.

Now, I said earlier that chromatography is an incredibly useful separation technique because it requires only very small volumes of their samples in order for the technique to be used.

But also, chromatograms are incredibly useful.

So the product of chromatography, because they can tell us really quickly if a sample is pure or a mixture, but we can also compare chromatograms then to identify the unknown sample or the components of an unknown sample.

So chromatography is quite frequently used in forensics for things like analysing different ink samples, detecting drugs within blood samples, for instance, for sporting events, things like that for athletes, and also for being able to identify different bits of debris.

But here's the rub.

For the identification of an unknown sample or the components within that unknown sample, we must have a fair comparison of the chromatograms that are being used.

It's absolutely essential.

Now, the development of a chromatogram can be affected by several variables.

And we've already mentioned earlier in today's lesson about how the stationary phase material could be a different choice.

The mobile phase, the solvent that you use could affect the development of your chromatogram, but there are a few other variables as well that might affect how your chromatogram develops.

One of them is how concentrated that sample is.

So how much of you actually put on? How concentrated is that sample in that sample line? The other thing is the temperature.

And this is a really big one because this can affect the solubility of the sample in the solvent as it is developing on that stationary phase.

And I'd like to take a closer look at how temperature might affect the development of a chromatogram.

The thing to remember is that some substances are actually more soluble at a higher temperature and it's the solubility of a substance that affects its movement along the stationary phase as that chromatogram develops.

So if chromatography for some samples takes place at a lower temperature, it's possible that the sample or its components would have a lower solubility, and therefore create a chromatogram that looks like the one on the left.

If chromatography takes place for those same samples at a higher temperature, it's possible that the sample or its components have higher solubility, and therefore would create a chromatogram like the one on the right.

So why should we clear? Well, this is going to affect our quantitative analysis of these chromatograms. The chromatogram on the left at a lower temperature would result in lower Rf values, whereas the quantitative analysis of the chromatogram that took place at a higher temperature would result in higher Rf values.

Now remember, it's the comparison of Rf values that helps us to identify a sample or its components.

Whew, so it's absolutely essential for a fair comparison of chromatograms to take place that all of the conditions are kept the same, the same stationary phase, the same solvent use, the same temperature, and the same concentration of the samples that have been placed on that stationary phase.

It is a tricky thing to get right, but what we need to aim for for a fair comparison of our samples.

So the most robust evidence that we can gather from a chromatogram comes from quantitative analysis of that chromatogram.

That means calculating Rf values for each of the components in a sample.

And we need to make sure that those Rf values are provided to the appropriate number of significant figures.

So I have here an example of chromatogram that developed from one sample.

And I can see two spots.

So that means that I have two different components for this particular sample and I'm going to need to calculate the Rf value for each of these spots.

And to do that, I'm going to use this equation.

So I'm going to need to find the distance moved by each of those substances, so each of the spots.

And I'm also going to need to know the distance that the solvent moved.

So I'm going to be using a ruler and I'm going to put the zero point of my ruler on the sample line at the bottom, and then measure the distance for each of these things, the spots and the solvent.

And you can see, remember that the spot is being measured from the centre of the spot, not the top or the bottom, but from the centre as best we can.

And also that I have measured them all in the same units.

Using then these values that I've measured and my equation, I can see that the Rf value for the top purple spot is going to use the values of 8.

1 divided by 10.

3.

And so I get an Rf value here of not 0.

79.

Now, I've given that answer as not 0.

79, which is two significant figures because when I'm looking at these two values, 8.

1 and 10.

3, 8.

1 has two significant figures where 10.

3 has three significant figures.

You always need to give your answer to the lowest number of significant figures here.

For the bottom spot then, I'm gonna do the same process.

The distance moved by the substance is 1.

7, the distance moved by the solvent is 10.

3.

When I divide those, I get a value of not 0.

17 to two significant figures.

So I've done one example here.

What I'd like you to do now is to calculate the Rf values for the spots on this chromatogram.

This may take you a little bit of time.

So what I'd like you to do is to grab yourself a calculator, pause the video to make your calculations, possibly check your answers with the people nearest you, and then come back when you are ready to check your work.

Okay, let's see how you got on.

So for the larger top spot, you should have taken the value of 41 divided it by 74, and you would got a value to two significant figures of not 0.

55.

For the bottom spot, you should have taken 19 and divided it by 74 and, again, to two significant figures got a value of 0.

26.

So very well done if you managed to get those correct.

Remember as well, it's very important that you are writing out your working so that if, for some reason, your final answer doesn't match the correct answer, we can identify where you've gone wrong.

Is it that you've put the numbers in incorrectly or is it that you have used your calculator incorrectly? Okay, so just make sure that you're writing down your workings out 'cause that will help us to identify how you can best improve.

So very well done though if you manage to get this correct.

Great job, guys.

So you've carried out chromatography and managed to quantitatively analyse your chromatogram to calculate the Rf values for each of the spots that developed on it.

The key here then is that you can take those Rf values and compare them against a database of values that were calculated when those chromatograms were carried out under the same conditions to identify an unknown substance.

So I have here a chromatogram that was developed using ethanol, a form of alcohol as its solvent.

And when I calculated out the Rf value for this particular spot, I got an answer of 0.

74.

I'd take that Rf value then and compare it against some other substances with Rf values that were calculated when chromatography took place under the same conditions.

So the same stationary phase, same temperature, same mobile phase, so ethanol and also the same concentration of sample.

And when I do that, I can see that not 0.

74 matches the Rf value for ibuprofen, and therefore this sample spot is ibuprofen.

Let's stop here for another quick check.

Which two of the following are essential when comparing chromatograms? Well done if you said A and D.

We definitely need to use a ruler so that we can correctly and accurately measure the distance that we need for the values to use in our Rf calculations.

And we need to make sure that the same conditions were used to develop those chromatograms so they're going to be developed in the same solvent.

The size of the container shouldn't make any difference.

And definitely, we are not using a pen because that might interact with our chromatogram and destroy our results.

So well done if you managed to get at least one of those answers.

And very well done if you got both A and D for your answers.

Great job, guys.

Let's keep it going and try another quick check.

Which chromatograms may contain the same substance? And I'd like you to try to justify your answer.

Now, this might take a little bit more time to analyse these appropriately and for you to be able to justify your answers.

So you may wish to pause the video here and come back when you're ready to check your answer.

Let's see how you got on.

So you may have said that samples A and D contain the same substance.

And that's because when you look at the Rf values between them, they both contain a spot with the same Rf value.

And because of that, it doesn't matter where they are in terms of the number of spot, what matters is that Rf value.

If they're the same and they were prepared under the same conditions, then it's possible they are the same component.

Well done if you manage to get them correctly identified and even better if you manage to justify your answer.

Great job, guys.

Time for the last task of today's lesson.

What I'd like you to do is to use the method that we developed in task A to produce chromatograms of three different inks on the same stationary phase.

Now, that's gonna take a little bit of time to develop, so you might wanna consider how you might answer or tackle the questions of part two.

You're going to then use the chromatograms that you develop in part one to decide, was a suitable solvent used for the mobile phase, and why or why not? You're going to use your chromatograms to calculate the Rf values for each of the substance spots.

And then I want you to use your qualitative and quantitative analysis of your chromatogram.

So visually and using Rf values, what can you conclude when you compare the chromatograms for your three different inks? This is gonna take some time, so pause the video here and come back when you're ready to check your answers.

Okay, let's see how you got on.

So for the first part, I asked you to use the method that was developed in task A to produce some chromatograms of three different inks on the same stationary phase.

Now, how yours looks is going to depend entirely on the stationary phase that you used, the solvent that you chose, and things like that.

So I have an example here of a particular chromatogram that was developed.

What I'm looking for are these things.

Do you have three different samples on the same piece of stationary phase? Are these samples evenly spaced between themselves and the edges of that paper? Has this chromatogram the solvent front at the top marked? Have the spots also been carefully marked with pencil? So I'm looking most specifically at how your chromatogram was developed, not the answers.

How was it developed, okay? So take a quick look at it now.

How does yours compare? Are they evenly spaced? Do you have everything marked, okay? So well done though if you managed to produce any chromatograms, but we're really looking for those three so that we can use comparisons on there, okay? Well done, 'cause I'm sure that took a little bit of time and care in order to develop them appropriately.

Let's move on then to look at your answers for part two of Task B.

So using your chromatograms produced in part one, you were asked to decide if a suitable solvent was used for the mobile phase, and to justify your answer.

Now again, this is gonna depend on the chromatogram that you actually developed.

So my answers here are going to be based on the chromatogram that I developed.

And what I've done to help in my answer, I've actually labelled each of my samples as A, B and C.

You may have used three different inks, so red, green, and yellow, something like that.

So make sure that you're referring to your different samples as we go through this.

Now, was a suitable solvent used? Well, looking at my chromatogram, I'd say, yeah, possibly because two out of the three inks appear to have separated well, okay? So I would say yes, a suitable solvent was used.

However, because one of my samples doesn't seem to have dissolved, I might wanna look at using a different solvent to see if it will dissolve all three inks to give me a better comparison.

But as a starting point, yes, I think it worked.

The second part that you were asked to do was to calculate the Rf value for each substance spot that developed on your chromatogram.

Now again, this is gonna depend on the chromatogram that you developed.

So I'm going to use my example here and I've put the ruler here.

Now, you can see the zero mark, not the bottom of the rule, but the zero mark on the ruler has been put onto the sample line at the bottom.

And I can use that now to measure out my substance spots and my solvent front in order to calculate the Rf value.

And I need to do this for every spot on my chromatogram.

So for sample A, I have two spots, a pink spot and a blue spot.

And when I take those measurements and calculate the Rf values, I have 0.

08 and 0.

98.

For sample B, again, I have two spots, so I need two Rf values.

Again, this also has a pink and a blue spot, which is interesting.

And when I calculate those values, I get 0.

07 and 0.

98.

Now, sample C hasn't moved.

So its Rf value is going to be zero.

It has not moved at all.

So zero divided by 4.

5, which is the solvent front distance is zero.

Now, again, these are Rf values that are calculated specifically for this particular chromatogram that was developed, but the thing I'm looking for in your answers is have you clarified in your calculation which spot you're talking about? So have you put something in brackets to clarify if it's the top first, second, third? What colour spot it is? Have you shown your working out? So what values are you using to calculate that Rf value? And have you given your final answer then to the correct number of significant figures if you want to challenge yourself up to that as well, okay? Well done though, guys.

You're doing supremely well.

Okay, so for the final part of this task, you were asked to write some conclusions upon comparing your chromatograms. Now, your conclusions will be wholly individual because they are based on your own chromatograms. So I'm gonna give you a guide of some of the things that you could have said about your own chromatogram using mine as an example.

So the first thing I'm gonna say is that sample C is insoluble in this particular solvent, and that's because the Rf value is zero, meaning it hasn't moved from that sample line.

The other thing I could say is that samples A and B contain similar components.

So they are made up of similar substances, and that's because their Rf values nearly match.

They both have an Rf value of 0.

97 and they have Rf values that are within 0.

01 of each other.

So they're very close.

They're very similar components.

Now, because of that, I can make a further conclusion.

I could say that potentially, samples A and B could be the same ink, and that's because both spots in each of their chromatograms are nearly identical.

So I've said something about solubility.

I've said something about the components individually, and then I've said something about the samples and those comparisons.

Are they the same or not? So have a look at your own conclusions.

Have you said something similar? Could you add anything additionally to it? This is not an easy task and it does take practise to get it right, so work with other people to compare your ideas of answers and see if you agree.

But very, very well done for having it go at this task.

Guys, I'm so impressed.

Now, we've done a lot in today's lesson, but let's summarise what we've learned.

Well, we've reminded ourselves that chromatograms can be used to identify if a substance is pure or a mixture.

We've also learned that a fair comparison of chromatograms is essential and it depends on several factors.

You need to make sure that if you're going to compare chromatograms, they have the same stationary and mobile phases, that samples are having the same concentration on that stationary phase, and that chromatography is carried out at the same temperature.

We've also learned that chromatograms can identify unknown substances through a visual comparison or also, and more robustly using calculated Rf values.

And the calculation we use for Rf values is the distance moved by each substance divided by the distance moved by the solvent.

We've also learned that appropriate equipment really needs to be used in order to do these quantitative assessments.

So we need to use a ruler and we need to use a pencil so that the measurements that we're taking are robust and that our calculations also are done to an appropriate number of significant figures for when we are comparing them.

A lot done today, guys.

You have worked so well.

I'm really, really pleased.

I hope you had a good time in today's lesson.

I certainly did, and I hope to see you again here soon.

Bye for now.