Lesson video

Hello, my name's Mrs. Nevin, and today we're going to be talking about how to interpret chromatograms as part of our unit on separating substances.

Now, you may have some experience of this from your previous learning, but what we learn in today's lesson will not only help us to answer those big questions of what are substances made of and how can we explain how substances behave, but also help us to better appreciate the wealth of knowledge that simple test of chromatography can provide to chemists.

So by the end of today's lesson, you should feel more comfortable being able to both qualitatively and quantitatively interpret a chromatogram.

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

Now, the definitions for these key terms are provided in sentence form on the next slide, and you may wish to pause the video here so you can read through them or to perhaps jot them down so you can refer to them later on in the lesson or later on in your learning.

Now, I mentioned that there is a wealth of knowledge that can be gleaned from a chromatogram.

So we'll start the lesson by looking at the qualitative analysis and finish up by looking at the quantitative analysis of chromatograms with the calculation of Rf values.

But let's get started by looking at how we can use chromatograms to identify unknowns.

Now, before we get started, we should remind ourselves what a chromatogram is.

It is simply the pattern that forms as a result of chromatography.

So we can see here we have a setup to analyse the components in four different coloured pen inks.

And once it has undergone chromatography, this is what is formed.

This pattern that's formed is called a chromatogram.

Now, the simplest analysis that we can get from a chromatogram is to determine whether or not a sample is pure or a mixture.

And this comes from a qualitative, that means visual, assessment of the chromatogram in a vertical manner.

So the first thing I'm going to do to help me with this is I'm going to draw some vertical lines.

So using a pencil and a ruler, just draw a line from the sample source along the bottom, all the way to the top of your chromatogram.

Now, what that does is it helps us just to keep things a little bit more clear in our head in how we analyse things.

Now, the thing to remember is that a pure sample will show only one colour or one spot.

So if we look at this chromatogram that is on the left, we can see that it's just sample B is the only sample that has simply one spot.

A mixture then or a sample that is a mixture will show multiple spots or multiple colours.

So in this case, again looking at the same chromatogram, sample A has three spots, so it has three components in its its mixture, and C has two spots and so therefore, has two components in its mixture.

Now, you can also qualitatively analyse a chromatogram horizontally to see whether or not samples are the same substance.

So if we draw a line in pencil and using our ruler across, so that's horizontally across these spots, then it makes that comparison a little bit more organised and a little bit easier to perform.

So continue drawing those lines across all of the spots, making sure that they're nice and straight.

Don't try and link them up in any way.

And then what we can do is see whether or not the spots match across those different chromatograms for the different samples, and if they do match, they're the same substance.

So if we look at this chromatogram on the left, we can see that the spot for sample A and sample D match.

As we read across from the left to the right, we can see that the sample spots in A and D both have a green spot and a yellow spot above it around about the same size and roundabout in the same location.

So this visual qualitative comparison has helped us to identify that A and D are most probably the same substance.

Now, if we stick with that horizontal visual comparison of chromatograms, if we're not able to identify the actual sample by having all of the spots between the chromatograms match, we might be able to determine at least the components within a sample.

So same as before, I'm going to use a pencil and ruler and draw those lines across the different spots so that I can keep my analysis a little bit more organised and same as before, I'm going to then compare those spots, do any of them match? Because if they do, it's quite possible the components are present in both of these samples.

So if I look at the sample on the right, I can see that sample A has a spot that also matches sample B, and it also has the two spots that match sample C.

So that tells me that even though sample A is not the same as sample B and C, I could say that sample A is composed of or made up, includes those components of B and C.

Let's stop here for a quick check to see how you're getting on.

Which statement about this chromatogram is correct? Now, to help you, I've drawn on those horizontal lines for you.

You may wish to pause the video here so you can consider your options, maybe discuss your ideas with the people nearest you and come back when you're ready to check your answer.

Well done if you said C.

Sample B and sample C match the spots in sample D and therefore, B and C are components of sample D.

Sample D does not just contain sample B only because it also has that blue spot from sample C, so that's why B is incorrect.

Sample A is a component of sample B is incorrect because sample A only has that one red spot and it's found in none of the other chromatograms, and therefore, sample A is not a component of any of the other ones on our chromatograms, but very, very well done if you managed to choose C.

Good job, great start.

So up to this point, we've been using a horizontal qualitative, that's visual, comparison of chromatograms to see if samples are the same sample, if known samples are components of other known samples.

But this horizontal comparison can also help us to identify whether or not an unknown sample is a specific known sample or if it maybe contains components of other known samples.

And we're gonna do this in the exact same way.

We're going to draw those horizontal lines using a pencil and a ruler.

Now, when we compare that in our chromatogram, same as before, we're looking for spots that match up along those horizontal lines.

And what we can easily say then is that the unknown does not contain sample A because sample A's spot does not match any of those that are in the unknown, that one that's marked with a question mark at the bottom.

So sample A is out.

What we can see when we look at the rest of these spots, when we look at B and C, we can see that the spot for B, that tall red one at the very top matches that in the unknown.

And we can also see that the yellow and the green spots from sample C also match the ones in our unknown.

And therefore, samples B and C are components of our unknown sample.

We've been able to identify components of our unknown mixture through this horizontal qualitative comparison.

Let's stop here for another quick check.

True or false? Vertical comparison of a chromatogram will determine if substances are components of an unknown sample.

Well done if you said false, but which of these statements best justifies your answer? Well done if you chose A, comparison of chromatograms is done horizontally, not vertically.

Vertical analysis can determine if a substance is pure, but it can also determine if a substance is a mixture and therefore, statement A was the best justification for our true or false statement being false.

So well done if you managed to get that correct.

That was a little bit of a tricky one, so well done if you managed to get it right.

We're on a roll.

So let's have a go at another quick check.

Horizontal comparison of chromatograms can determine what? Now, there are a lot of options here and you may wish to discuss them with the people nearest you.

So I would recommend pausing the video here and coming back when you're ready to check your answer.

Well done if you said all of them.

Now, you may remember at the beginning of this lesson, I said that there was a wealth of knowledge that could be gleaned from a chromatogram, and all of these can be done simply by drawing those horizontal lines with a ruler and looking at how you compare the spots that line up along those drawn lines.

Vertical analysis can also tell us if a substance is pure or a mixture and a little bit about the solubility of the components within the solvent that's used to develop that chromatogram.

So an amazing amount of information that can be gleaned from that chromatogram.

Really well done, guys.

Good job.

Time for the first task in our lesson, and we're gonna put some of these skills that we've been learning to the test.

What I'd first like you to do is to use this chromatogram to identify the colour components that are found in samples A, B, and C.

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

Okay, let's see how you got on.

Now, hopefully, you've been able to draw some of those horizontal lines that we talked about to try and keep your analysis clear.

And when we've done that, we can say then that sample A matches up with the blue colour only.

Sample B has spots for blue and the red, so it's composed of two different colours, and that sample C has spots that match both red and yellow, so therefore, also has two colours within it.

So very well done if you managed to get at least one of those colours and excellent work if you managed to identify both colours for samples B and C.

Great start, guys.

Let's keep it up.

Okay, for the next part of this task, I'd like you to use this chromatogram to identify which substance or substances the unknown question marked sample contains.

And I'd like you then to also explain your answers.

So I'm looking for a because clause.

Why have you made that identification? 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 same as before.

I would start this analysis by drawing that horizontal line using a ruler and a pencil.

And when I do that, I would say that sample A matches the unknown sample the best.

And the reason I've made that choice is because when you are horizontally comparing these chromatograms, the unknown sample's only spot matches sample A because it also has a spot in the same location, the same colour, and approximately the same size, so they match.

So well done if you managed to first of all, identify the correct sample that matches the unknown and very well done if you were able to explain why you've made that choice.

Great job, guys.

A fantastic start to this lesson.

Really, really pleased with you.

Now that we're feeling a little bit more comfortable being able to qualitatively analyse chromatograms in order to identify unknowns, let's look at how we can quantitatively analyse them by calculating Rf values.

So one thing to remember is that when the chromatogram is developing, what's actually happening is that that mobile phase or the solvent that's being used is moving along the stationary phase, carrying any of the dissolved components of that sample with it, okay? Now, some of those substances are more soluble than others and so they're retained or kept and carried along with that solvent a little bit longer than the other components of the sample.

Now, a chromatogram then is simply that visual representation of the components within a sample being separated out.

Okay? We can then take measurements from that chromatogram and calculate an Rf value for each of the components within that sample.

Now, an Rf value, Rf stands for retention factor.

So how long has it been retained within the solvent? And we can use these values to quantitatively compare how far then a substance or a component has travelled along that stationary phase in relation to the solvent that was carrying it along that stationary phase.

So these quantitative comparisons are considered a little bit more robust than our qualitative assessments because they're based on measurements, they're based on numbers, not your visual comparison, okay? Now that means there's a little bit of maths.

And the equation that we're going to use to calculate an Rf value is this.

The Rf value is equal to the distance moved by the substance, the spot or the component of this mixture divided by the distance that's moved by the solvent.

Now, under the same conditions, a calculated Rf value can then be compared to a database of known Rf values and that can help us to identify components in a sample even if we don't have to hand to visually compare a chromatogram of that particular component.

Let's stop here for a quick check.

Which of the following is the correct equation that would be used for calculating a retention or Rf value? Well done if you chose B.

It can be a little confusing when so much looks the same.

So what you want to be focusing in on is what is different between each of these equations.

And what I see that's different is actually just the last word on both the numerator and denominator.

So what's above and below the line.

And what we're looking for is that it's the substance that is dissolved divided by the solvent.

So how far the substance has moved compared to how far the solvent has moved.

Well done if you managed to get that correct.

Now, if we are gonna be taking some measurements, then we're going to need a reference point.

We need to know where we're starting and where we're ending our measurements on this chromatogram.

So the first thing we're gonna look at here is this top line.

It's called the solvent front.

Now, the solvent front represents how far that solvent was allowed to travel along the stationary phase before it was removed from our solvent reservoir.

So taken out of that container that had the solvent along the bottom.

Now, the solvent front is always found above the highest spot on our chromatogram, okay? And it is noted in pencil immediately upon removing that stationary phase from the solvent.

So remove the paper from the container, grab your pencil and mark that solvent front on your stationary phase almost immediately.

That's the one reference point.

The other reference point that we're gonna be needing is known as the sample line.

Now, the sample line represents where those samples have started, where they were placed on that stationary phase before chromatography was allowed to carry out.

Now, this is always found at the bottom of the chromatogram and it's also always noted in pencil before that stationary phase has actually been placed in the solvent.

So the sample line is at the bottom along with the samples and placed on there with pencil at the start of chromatography.

And the solvent front is found at the top of the stationary phase noted in pencil when we remove that stationary phase from the solvent.

Now, we've gone through a lot of information in a short space of time, so let's stop here for a quick check.

Which two of the following is true about a solvent front? You may wish to pause the video here so you can read and discuss your options before coming back to check your answers.

Well done if you chose A and D as true statements about a solvent front.

B is incorrect because it's talking about making a note in pencil before placing the stationary phase in the solvent.

The solvent front is actually noted after removing the stationary phase from the solvent.

And C is incorrect because the solvent front represents how far the solvent has travelled, not the sample.

So well done if you managed to choose at least one of the correct statements and very well done if you managed to choose both of the correct statements.

Great job, guys.

Let's keep cracking on.

Okay, so in order to actually carry out a calculation for the Rf values, you need to first have the values to put into your equation.

We need to find the distance moved by each of the substances in our chromatogram, and we also need to find the distance that moved by the solvent.

So the first thing we need to look at is the distance moved by the substance.

Now, the substance is always measured from your sample line.

So if I look here, I'm going to just create an extended line for my sample line out to the side and the same for one of the spots on my chromatogram.

Then I'm gonna place my ruler along those lines with the zero mark on the sample line.

And when I measure that out, that line for the yellow spot comes in at 2.

3 centimetres.

Now, if I do the same thing for the red spot, again placing the zero mark of my ruler on the sample line, measuring up, and that comes at 5.

5 centimetres cubed.

The next thing I need to do is I need to find the distance for the solvent, and how far that has moved.

So again, I'm going to extend that line for the sample line and do the same for my solvent front.

Again, place my ruler on the zero mark on the sample line measuring up to the solvent line and I get a value of 7.

0 centimetres.

Now, the main thing to remember here is when you are calculating the distance that substances have moved, you need to make sure that you are taking the measurements from the centre of the spot.

You can see these marked here on the yellow and red spots here, that those lines are extended from the centre of the spot, not the bottom or the top of them.

Now that I have the measurements from my chromatogram, I can use them to calculate the Rf value for each of the spots on it.

So I'm gonna focus mostly on the yellow spot for this example.

Now, because it's looking at just the yellow spot, I'm going to need the distance that's moved by that particular substance, that component, and I'm going to divide that value by the distance that the solvent has moved.

So I'm gonna be using these two values, 2.

3 and I'm gonna divide that by 7.

0.

Now, there's no marks for mental maths here.

You are going to put this into your calculator, and when you get that value, it's quite long.

Now, I know it's more precise to put more digits down from your calculator, but we need to consider something here.

The values that I am using in my calculation were limited by the values I could get from my ruler when I took those measurements in the first place.

And if I go back to those values, I can see that actually, they only had two significant figures.

And because of that, the answer for my Rf value must also be to two significant figures.

So when I look at this value of 0.

32857, I need to round this to two significant figures, and that means the Rf value for the yellow spot is 0.

33.

I've had to round the two upwards to a three because the number after it, the eight is above five.

So my answer is 0.

33 for the Rf value for the yellow spot.

The other thing to remember about Rf values is normally in science when we're calculating something, we expect there to be units involved and there are, but in this particular calculation, the units cancel out.

So if I look at simply the units for the measurements that I've taken and used in my calculation, they actually cancel out.

And because of this, they need to cancel out.

I need to make sure that all the measurements that I take and record for my different substances and for the solvent front are recorded in the same unit.

So you need to be consistent in how you are recording those measurements so that you can use them correctly within your calculation of the Rf value.

Now, there were two spots on my chromatogram, so I need to move now to the red spot.

This time I'm going to use the different values.

So the red spot moved 5.

5 centimetres and the solvent front is still 7.

0.

So I'll put that into my calculator.

5.

5 divided by 7.

0 gives me a value of 0.

7857.

But again, because the values that I used for my measurements were to two significant figures, my answer also needs to be to two significant figures.

So the Rf value for the red spot is not 0.

79.

Now, a good rule of thumb when calculating Rf values is that they should always have a value of less than one because the spots should never get past that solvent front.

So if for some reason you're doing your calculation and the Rf value is larger than one, what you've probably done is simply put your numbers in reversed in your calculation.

So just do the calculation again, reversing your numbers, and you're more than likely going to get the correct answer that should have a value of less than one.

Let's have another go at calculating some Rf values.

Now, what's interesting here is that the spots are actually part of the same sample.

So this particular sample was composed of two components.

So the spots are on top of each other rather than the previous chromatogram that showed two different samples having spots that were not along the same vertical line.

Now, does it make any difference? Actually, no, it doesn't.

I'm still gonna use the same equation of the Rf value is the distance move by the substance or component spot divided by the distance move by the solvent.

So I get my ruler out, put the zero mark on that sample line at the bottom, take my measurements and record them.

Now, this time, I've used millimetres rather than centimetres, but you can see that I've been consistent in the way that I've recorded those numbers.

And then for each of the spots there, I'm going to calculate the Rf value.

So for the top spot, I'm going to take 43 divided by 59.

I have two significant figures in these values and therefore, my Rf value is also to two significant figures of 0.

73.

I'm gonna do the same thing for the bottom spot, this green spot, but this time using the fact that it's moved only 10 millimetres divided by 59 millimetres, which is what the solvent front is.

Again, I only had two significant figures, and so my value is 0.

17 for the Rf value for the green spot.

What I'd like you to do now is to have a go at calculating your own Rf values.

Now, I've saved you the trouble of trying to do the measurements and provided you the values directly.

So use those and our calculation equation to calculate the Rf values for the spots provided.

You may wish to pause the video here, maybe grab a calculator, double check your calculations with the people nearest you and come back to me when you're ready to check your answers.

Okay, let's see how you got on.

So for the green spot, that larger, taller one, you are going to take the value of 3.

1 divided by the solvent front of 5.

9, and you should have a value to two significant figures of 0.

53.

For the grey, thinner lower spot, you'll take a value of 1.

1 divided by 5.

9, and again to two significant figures, you should have a value of 0.

19 for the Rf value.

Now, some of you maybe have put these values straight into your calculator and wrote your answer down, which is a very common thing.

But I would always, always recommend that you show your working out, write down the values as you would've put them into your calculator so if for some reason your final answer is incorrect, we can go back to double check and see if you were doing the calculation correctly in the first place.

We can find your errors more quickly, okay? It might just be that you had the right idea with the calculation you wanted to do, but for some reason, maybe it was put into the calculator incorrectly.

So you would maybe only lose one mark in your calculation rather than two.

So word to the wise, good practise is always to show your work.

Okay? Well done on this, guys.

Great job.

Let's move on to the last tasks then for this lesson.

What I'd like you to do for this first part is to add these labels to the chromatogram, the substance spots, the sample line, and the solvent front.

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

Okay, let's see how you got on.

Well, the very top, if you remember is where the solvent front should be located.

These spots along our chromatograms are the substance spots, and then the line at the very bottom of our chromatogram is the sample line.

And these are the most important things that we need to be able to identify in a chromatogram if we're to take any measurements and then calculate any Rf values.

So well done if you've managed to correctly label them on your chromatogram.

Good job, guys.

Okay, for this next part of the task, I'd like you to calculate the Rf value for each of the substance spots.

Now, again, I've saved you the trouble of actually measuring these out.

I'd simply like you to calculate them.

So you may wish to pause the video here.

Again, grab that calculator and double check your answers with the people nearest you.

Then come back when you're ready to check your answers.

Okay, let's see how you get on.

Now, whenever I'm doing a calculation, I always find it so much easier to have the equation I'm going to use written down so I don't have to try and remember it in my head.

I can just look at it and refer to it really quickly on my paper.

So the first thing I'm gonna do is write down the equation that I'm using.

So the Rf value is the distance moved by the substance or spot component and divide that by the distance moved by the solvent.

So I can just use that as my guide in terms of where I'm putting my values.

You needed to calculate the Rf value then for each component spot.

So you'll see here that I've actually labelled the spot in brackets that I am referring to in my calculation here.

It's a really good idea to do this again, good practise so that you can just keep track of which values are which.

So you don't have to do any kind of referencing later on if you need to go back and check it.

So we know that the Rf value for the yellow is going to be 7.

2 divided by 8.

8, and you should get an Rf value then of 0.

82.

The red spot Rf value then is going to use the values of 4.

5 divided by 8.

8, and you should get an Rf value of 0.

51, again to two significant figures.

And finally, for the blue spot's Rf value, you're going to be taking 1.

2, dividing it by 8.

8 and getting a value then of 0.

14.

Well done if you managed to get all of those correct.

Give yourself one mark if you managed to put down your working out and give yourself a second mark if you've managed the correct answer.

And a third mark if you managed to do them to the correct significant figures.

Really impressed here, guys with how you are working on this.

What a great job.

Okay, we've gone through a lot today.

So let's summarise what we've managed to learn.

Well, we've learned that chromatograms are very, very useful.

They provide a lot of information about a mixture and they can be analysed both qualitatively, which is a visual comparison, and then a quantitative analysis by comparing calculated Rf values that are based on the measurements taken from a chromatogram.

And that Rf calculation then is the distance moved by the substance divided by the distance moved by the solvent.

Now, in order to make these calculations, we need to be using appropriate equipment, and that does include using a pencil and especially a ruler in order to identify those substances in the mixture, either by that qualitative visual comparison, drawing our lines horizontally or vertically, and also when we are using our rulers and pencils to make those initial measurements needed for our Rf values.

A bit of a tongue twister there.

And then finally, we know that with our Rf values, we need to ensure that the values are provided using an appropriate number f significant figures, and I would only ever round to those significant figures in the very last part of your calculation.

So lots of stuff that we've done today.

It's a practise makes perfect kind of feeling here.

So if you were a little unsure about how to analyse these chromatograms, either qualitatively or quantitatively, I would highly recommend that you look for some other examples and keep practising it because practise really does make perfect.

The more experience you get, the more confident you feel and the better off you are as you go forward.

So hopefully you guys have had a good time learning with me.

I certainly had a really good time.

I love doing our chromatogram analysis, and I hope to see you again soon.

Bye for now.