Chromatography is a chemical technique for separating mixtures of coloured chemicals. This technique is important in biology as well as chemistry; it is also used by forensic scientists.


We all know that green plants are green because they contain chlorophyll; we know that chlorophyll is green. Hang on; why are plants different shades of green if they all contain the same green pigment? Well, perhaps it is not quite as simple as you were told by your biology teacher. In fact there are several different kinds of chlorophyll and some other photosynthetic pigments in green plants. So exactly what colour they are depends upon which types of chlorophyll and other pigments their leaves contain.

What has all this to do with chromatography?

What is chromatography and what can we do with it?

Well, for a start we could separate the different pigments in leaves and find out a bit more about how photosynthesis works. Leaves contain a mixture of two or more of the following pigments: chlorophyll a, chlorophyll b, chlorophyll c, xanthophyll, carotene, phaeophytin and other pigments. The leaves of different plants contain different pigments, hence their different colours. We could also use chromatography to separate the different pigment in writing ink: a forensic scientist might do this to find out if all the writing on a cheque was written with the same pen.

We can even use chromatography to separate mixtures of colourless chemicals: it is possible to use the technique to separate the amino-acids produced when a protein is digested. Once we have separated the mixture it is necessary to stain the amino-acids using a coloured chemical. Ninhydrin is used; unfortunately this is not a very nice chemical. Smoke cigarettes if you must but don’t use carcinogenic chemicals without taking the proper precautions.

Here is an experiment to try for yourself at home (mum won’t mind):

  1. Get a square of blotting paper (you could use paper towel).
  2. Draw a line parallel to one edge about 2cms onto the paper.
  3. Put a series of small crosses in pencil along the line.
  4. Make a circular mark over one cross with one of your felt-tip pens (water soluble).
  5. Do the same to the other crosses with different colours.
  6. Now hang the piece of blotting paper in a dish of water so that it is just touching the water.

You should see the water soak up into the blotting paper. As the water rises up the blotting paper it will carry the ink from the felt-tip pens with it. When the water gets near to the top you can take it out of the water and leave it to dry. You should see that the ink marks have separated out into their different colours. Maybe some of your felt-tip pens contain inks made of only one coloured chemical, but most of them will contain mixtures.

This experiment will not work with non-washable felt-tip pens unless you use alcohol or acetone instead of water. You could use some of your parents’ gin or vodka if there is any and they will let you have it, otherwise you will have to do the experiment at school.Don’t be surprised if you cannot get the experiment to work with water and the chlorophyll in leaves, again you will have to use an organic solent.

The Secret:

How does chromatography work?

The solvent rises up the chromatography paper (blotting paper) by capillarity. When the solvent reaches the “spot” it dissolves the mixture of coloured chemicals. There is now a solution; this is a mixture of solutes dissolved in the solvent. The molecules of these different chemicals are all different sizes. The simple explanation is that the smallest solute molecules travel almost as quickly as the solvent molecules and so get carried to the top of the chromatogram. The largest solute molecules travel very slowly and stay near the bottom. So some of the coloured chemical travel further than others. If you are doing this with amino-acids you will not see anything happen until the end of the experiment when you stain it with ninhydrin.

Here are some more terms for you to look up in your textbook or library:

Thin Layer Chromatography (TLC). My wife uses TLC to mean “tender loving care”.

Electrophoresis (this is not getting rid of the hairs on your legs with electric currents).