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.
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.
Notice that you have three parts to the experiment.
1. One part that stays still - the paper. This is called
the stationary phase
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?
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.
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.
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.
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
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)
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.
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.
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.
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!
http://home.att.net/~gcresource/kidschromatography.html This is a really useful site.
http://chemlearn.chem.indiana.edu/demos/PaperChr.htm brief and good details using coffee filter paper.
http://wwwchem.csustan.edu/chem2000/Exp1/Bkg1.htm some chemistry introduced.
http://antoine.frostburg.edu/chem/senese/101/matter/faq/separating-artists-pigments.shtml some nice touches in modifying the mobile phase with salt or vinegar.
http://www.iit.edu/~smile/bi8605.html good text lab description of spinach extract and the spots.
http://www.doggedresearch.com/chromo/ nice student project-well laid out.