Higher and foundation tier
Chromatography is another method used to separate mixtures and also identify substances in the mixture. The method shown below is called ascending paper chromatography. Chromatography uses a mobile phase and a stationary phase to separate out a mixture of substances. In the method shown below chromatography is used to separte out the mixture of substances in ink. Here the mobile phase is a liquid solvent, water or ethanol are commonly used. This solvent soaks into the chromatography paper and dissolves the mixture of substances in the coloured dots. Since the solvent moves it is the mobile phase. The chromatography paper is called the stationary phase, it obviously does not move.
The image below shows a typical paper chromatography set-up. Here a pencil line, not a pen as the ink would run and spoil the chromatogram, is drawn approximately 2-3cm from the bottom of the chromatography paper. Capillary tubes are used to spot the samples onto the paper which is placed in the chromatography tank. The paper should dip into the solvent but it is important that the solvent is below the pencil line. A lid is placed on the tank to keep the solvent vapours in. The solvent is allowed to rise approximately 75% of the way up the paper, the paper is then removed and allowed to dry.
In the example above three dots of coloured ink, red, yellow and green dots are placed on a pencil line. As the solvent rises up the paper each of the substances present in the coloured dots will dissolve by a slightly different amount. If any of the substances in the dots are insoluble they will stay on the pencil baseline. The more soluble the substance the further up the chromatography paper the solvent will carry it. However the substances in the mixture will also be attracted by intermolecular forces to the paper, each of the substances in the dot will be attracted by slightly different amounts. So we have a combination of two factors at work that will determine how far up paper the substance rises. The more it is attracted to the paper (stationary phase) and the less soluble it is the less it will travel up the paper. So the opposite would be true, highly soluble substances which are weakly attracted to the paper will travel further up the paper. Another way of stating this is: the more time the substance spends in the mobile phase and the less time it spends in the stationary phase the further up the paper it is carried. When the solvent has risen close to the top of the paper it should be taken out of the tank and left to dry.
Once the chromatogram has dried out it is possible to measure how far each of the substance has moved through the stationary phase (travelled up the paper!). All measurements are taken from the pencil line or baseline. This is shown in the diagram below.
The retention factor or Rf value can be calculated for each substance. The Rf value is simply the ratio of how far the substance or solute moves divided by how far the solvent (mobile phase moved). It is calculated using the formula:
The red ink produced two dots on the chromatogram, one dot was magenta and one yellow.
The fact that there were two dots tells us that the red food coloring is not a pure substance but that
it is a mixture of two other chemicals. The Rf value for the magenta dot is:
Rf= 3cm/9cm =0.33
The green ink also produced two dots on the chromatogram, so like the red ink it also is not a pure substance but it consists of two other coloured chemicals, one blue and one yellow. The Rf value for the blue dot/coloring would be:
Rf = 5cm/9cm=0.55
It should be noted that if the solvent, temperature or type of chromatography paper used in a chromatography experiment is changed then the Rf values will change. The Rf values are ONLY true if the same conditions are used for repeated experiments. However if two substances have the same Rf values under the same conditions it is highly likely they are the same substances. This is how chromatography can be used to identify unknown substances.
Chromatography can also be used to identify substances. You can compare the Rf values for known substances with your unknown e.g. The chromatogram below shows 5 known substances A,B,C,D and E. Each of these known substances are pure as only one "dot" or "smudge" was produced on the chromatography paper. The unknown substance is composed of 3 separate substances. Two of the three substances in the unknown match up with substances B and D and a third substance in the unknown mixture is still unidentified as it matched up with none of the other known substances.