Chromatography - it's just a box
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Chromatography Science Fair

If you have arrived here it may be because you have probably carried out a chromatography experiment and want to know what is happening or perhaps want to know what chromatography is. If you are writing a report, try to use some of the highlighted words - it should get you extra marks :-)

What is it?

Most things we encounter in our lives are mixtures of different substances. Very few things consist of a single substance.  For example, a perfume may consist of hundreds of different substances, each one of which can influence the overall smell of it.

Chromatography is a method used to separate mixtures. This is important because if we can separate a mixture then we can measure how much there is of each substance. It also allows us to identify those substances and to isolate them for further study.

It was developed for this last reason about a hundred years ago by a Russian botanist named Michael Tswett who used it to study plant pigments. tswett7.jpg (5292 bytes)

What is happening?

If you have carried out a paper chromatography experiment you may have applied some colour from a felt tipped pen or an extract from a plant leaf near the bottom of a strip of paper and suspended the end of the strip in water. As the water rose up the paper strip the mark you applied appeared to spread upwards and other colours appeared.

paper3.gif (5897 bytes) In this example I cut a strip about 10cm long and 1cm wide from a daily newspaper and drew a line across it with a black felt tip pen (Penol 300) about 2cm from the bottom. The top of the strip was attached to a pencil with some adhesive tape and the strip was lowered into a glass containing water to a depth of about 1cm. The line should stay above the level of the water. The pencil rests across the top of the glass with the strip hanging down into the water. This stops the paper touching the sides of the glass. I left it for about 20 minutes before removing the paper and hanging it up to dry.

In this ink there appears to be at least 4 colours - royal blue at the bottom, then a band of light blue followed by a band of orange and finally a narrow band of yellow at the top. I find that coffee filter paper and a black or brown pen work well.

Notice that you have three parts to the experiment.

1. One part that stays still - the paper. This is called the stationary phase
2. One part that moves - the water. This is called the mobile phase.
3. The pen colour. This is called the sample.

Nearly all chromatographic methods have a mobile and a stationary phase.

I sometimes think of the process like a family shopping trip:

You and your family set out down a road full of shops to do some shopping. It is a very modern shopping complex with a moving pavement and you all agree to meet at a restaurant at the end for lunch. 

Now different people have different preferences.  Everyone hops on and off the moving pavement to browse in the shops . The first to arrive at the restaurant will be the ones who only liked a few of the shops and didn't spend much time in them. After them will be the ones who browsed for longer. And, typically, some go into the first shop and like it so much they stay there! The family gets separated because each member is spending a different amount of time in the shops. They actually all spend the same amount of time on the moving pavement.

The black ink is actually made up of different colours. These different colours are different substances and, like the members of the family, they have different likes and dislikes. Some like to be in the water and the paper has very little attraction for them (like the yellow and orange on my paper). These will be carried along quickly by the water and move furthest up the strip. Some like to be in the water but also find the paper attractive and tend to stick to it (like the pale blue).

You will usually see that some colour remains where you put it. In my paper the darker blue has only moved up slightly. It may be because it doesn't like water so won't go into it very easily. Also it may be because it is sticking so strongly to the paper that the water just can't get it to shift very easily. Tomato sauce stains on clothing are a good example of this. If we want to get it to move we may have to use a liquid that it does like more than water. In some experiments you may see "rubbing alcohol" ( iso-propyl alcohol) being used.

This "sticking" is called adsorption and slows the colour up. Don't use the word absorption. What's the difference?

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Reference 1

Like the family, the colours are getting separated because they spend different times sticking to the paper. Again, they all spend the same time in the water.

What if it doesn't work?

There is really a competition going on between the paper and the water for the colours. By the way, the colour must be able to dissolve in the water - if it doesn't dissolve then it can't be moved up the paper.

If the paper wins the competition outright then the colour won't move, it will stay stuck where you placed it - result - no separation.
If the water wins the competition outright then all the colours will be carried up to the top of the paper - result - no separation.

What we do is to choose a paper and a liquid which suits the sample and gives us something in between.

Remember, you actually have three things in your experiment - the pen, the paper and the water (or solvent). So, if it doesn't work, try changing one or all of them e.g.

1. Try using a different make of pen. The colour might be made up of a different set of substances and so have different properties. These may work better in your experiment. You could also try a different colour of pen. You could even try extracting the colour from something like spinach or cabbage leaves. (See the links at the end)


2. Try using different paper.


paper3.gif (5897 bytes) In the example on the left I have run the  pen with water using a piece of newspaper.

In the example on the right I have run the same pen with water BUT I have used filter paper instead of newspaper. The colours have now changed the order in which they have moved up the paper. The blue colour has not stuck to the paper as much so has moved to the top.

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3. Some sites describe using a different liquid (solvent) such as rubbing alcohol or vinegar. Or a mixture of rubbing alcohol and water and vinegar. (Caution: rubbing alcohol contains iso propyl alcohol which is flammable and may harm you, so should be used only under supervision following any safety precautions on the container). Or try changing the properties of the water by adding salt. The salt "fills up" the water leaving less room for the colour. This has the effect of pushing the colour back onto the paper so that it can't be carried up as far. If we add enough salt you can even stop the colour from moving at all. It is a bit like being in the basement of a building and you want to get to the top using the elevator - you press the button - the doors open but it is full boxes with no room for you.


4. If it works but doesn't separate very well, try putting on less colour from the pen by just touching the paper lightly with it.

How do you know if you have got good chromatography?

Good chromatography is when there are spaces between the bands and the bands are narrow. This gives us a better chance to see how many different substances are in the sample.With the experiment you have carried out don't worry if the bands are close to each other - it is difficult to get them as well separated as in this example.

From left to right the ink separations are of Geha blue, Pelikan blue, Pentel red, Pelikan black, Pentel blue and Pentel green.

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How do we measure how much of a substance is present in our sample?

If we know what the substance is in the band then we can run different strengths of that substance on the paper and compare by eye to see which is closest to our sample in terms of how intense the band is.

With this type of chromatography we can also use an instrument which works like a scanner on your computer. Instead of looking at it with our eyes, a light beam is moved up the paper and where there is a band less light is reflected. The changes in reflected light are changed into an electrical signal. The size of the signal will be bigger the more material there is in the band. Again we can compare the size of the signal of our sample with those obtained from standards made up of different strengths and calculate how much is present in our sample.

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How do we identify the substances that we have separated?

We apply pure standards of what we suspect the substance to be and see if moves to the same place as the unknown.  In practice we would put the sample and the standards in different lanes on the same piece of paper so that they can all run at the same time under the same conditions. See examples of this in the next section on thin layer chromatography. Although it is not definite proof that it is the same it can give us a good indication. We often use other instruments to positively identify it.

What other types of chromatography are there?

All chromatography requires putting the sample on, separating it and detecting the separated parts. No matter how sophisticated the techniques or instruments they will use those same basic principles. Here are few of the other types.

1. Thin layer chromatography

This is similar to paper chromatography. A thin layer (not much thicker than a sheet of paper) of a fine powder is spread onto a small sheet of glass or plastic.  It is like a thin sheet of icing on a cake. This thin layer takes the place of the paper. This system has advantages over the paper in that we can use a wider range of materials for the layer and can produce much better separations. 


Unlike the pen colours, a lot of samples that we look at are not easy to see. In fact some don't have any colour and are practically invisible. One way around this is to spray the plate with chemicals which combine with the invisible spots to make them visible, as we have done in this example on the right. Notice how we have been able to run a lot of samples at the same time. tlc3.jpg (4376 bytes)



Another way is to look at the plate under ultra-violet light which can make some separated substances shine, as in this example on the right. This experiment is looking at the  fluorescer which is put into fabric washing powder to make your white clothes look whiter.

The fluorescer in lane 1 is a good sample. The main spot at the top is the fluorescer itself and the spots underneath it are impurities.

The sample in lane 2 is from another supplier and you can see that this has a lot more impurities. This means that the washing powder manufacturer is paying for impure material. Also, if there are are a lot of impurities then there will be less of the pure material, so your clothes won't look as white.

The sample in lane 3 is from another supplier who says that his fluorescer is the same as the one in lane 1 that we normally use. You tell me, is it the same?

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2. Column chromatography

I am going to divide this into two sections.

a) Liquid chromatography

You may have already tried paper chromatography but you could also use a tube packed with a fine powder just as Tswett did in his early work nearly 100 years ago. His tubes were only 25mm long with an internal diameter of 2mm. He sometimes used chalk as the powder. Below is a sketch taken from one of his papers. (Reference 2)

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I have used a longer glass tube about 30cm long and 1cm wide filled with icing sugar. Chromatographers call this tube a column. I extracted some cabbage leaves and put the extract onto the top of the column. Then the solvent is added to top of the column (in this case a liquid called petroleum ether)  to wash the extract down the column. What happens then is exactly the same as with the paper chromatography experiment.


liqcol1.jpg (6214 bytes) With this method it is easy to collect the separated substances as they emerge from the end of the column for further study. For example, imagine that the leaf from a particular plant in the rain forest is used by the local people to treat wounds to stop them getting infected. We can extract the leaf and separate the substances on the column. We collect each separated substance as it drips out of the end of the column and test to see which one has the healing properties.  We would use other instruments to identify what that substance was. We could even use our paper chromatography to do this except that we would cut the separated parts out with scissors and extract the paper. column2.jpg (7713 bytes)


Modern instruments use very fine powders and high pressure pumps to push the solvent through the column. A typical metal column and the instrument it is used in is shown here. Because a liquid is used to carry the sample through the column this is called liquid chromatography.


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As each separated separated substance leaves the end of the column a detector produces a signal which shows as a "peak". The series of peaks which are produced for the separated substances from the sample is called a chromatogram.


Below are two chromatograms of well known sugar containing products produced by this equipment (taken from Agilent Technologies catalogue). Remember, each peak in the signal is a different substance being detected as it leaves the end of the column. These chromatograms would be used in quality control - the amounts of the different sugars will affect flavour, texture and sweetness.


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b) Gas chromatography

Instead of using a liquid to carry the sample through the column we can use a gas - usually helium or nitrogen. This is called gas chromatography. It is usually used for separating substances which evaporate easily. This covers a wide range including gases, pesticides, perfumes and oils.

Here is a typical column and instrument used in gas chromatography. The column is a very fine tube about 30 metres long. To fit in the instrument it has to be coiled up.

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Here is a sample that I took of someone's breath who had volunteered not to clean his teeth! and separated the substances in it using gas chromatography. Remember, each peak in the signal is a different substance being detected as it leaves the end of the column. Usually, the bigger the peak the more of that substance there is. The substance I have marked is acetic acid, which you may know better in its dilute form as vinegar. It is this and other acids which are formed in the mouth by the action of bacteria on food that can cause damage and decay to teeth.

How much do they cost?

Chromatography equipment ranges in cost from a few pence (cents) to 500,000 (a million dollars). An instrument which tests athletes for illegal drugs would cost about 60,000


Yes, but what have the chromatographers ever done for us?

Rather than give a list of substances which can be separated I have tried to imagine some of the things that would happen if we didn't have chromatography. In its ability to separate gases, liquids and solids, these are just a few of the many ways in which chromatography touches all of our lives every day.

Planes would fall out of the sky - chromatography tests the purity of engine oils and fuel.

The bubbles in your cola could poison you - chromatography tests the carbon dioxide gas used in drinks for substances which could harm you.

Your smile wouldn't be as nice - chromatography is used to check the levels of fluoride in your toothpaste which helps to stop cavities.

You would cough more - chromatography checks the atmosphere for pollution and makes sure that medicines are pure, as well as helping to develop and find new medicines.

The transparent door of your washing machine would crack and flood the kitchen - chromatography checks that the clear plastic in the door of the machine is of the right type and grade.

Fish would die in the rivers - chromatography checks river water for pollutants.

Your fruit and vegetables would be either full of insects or covered in pesticides - unfortunately we have to use pesticides to stop insects destroying our crops but chromatography is used to check that the levels in our food are low. In addition chromatography is currently identifying the substances given off into the air by the crop which the insects use to home onto the crop. So, plants which produce more of the substances that the insects like are planted at the edge of the field which keeps them away from the crop. Also, chromatography has helped us to identify plants which give off substances that the insects don't like.  So, the plants they hate can be planted in the field with the crop which also helps to keep them away. This means that little or no pesticide needs to be used. We win and so do the birds and other creatures which rely on the insects for food.

Athletes could cheat - chromatography tests for banned performance enhancing drugs.

I could go on but I won't!

Links This is a really useful site. brief and good details using coffee filter paper. some chemistry introduced. some nice touches in modifying the mobile phase with salt or vinegar. good text lab description of spinach extract and the spots. nice student project-well laid out.