How paper chromatography works
Definition
Paper chromatography
A method that separates the soluble substances in a mixture as a solvent carries them different distances through paper.
Definition
Solvent front
The furthest point reached by the solvent as it moves up the paper during chromatography.
- Paper chromatography separates a mixture of soluble substances, such as the different dyes in an ink or a food colouring.
- A small spot of the mixture is placed on a pencil baseline drawn near the bottom of a strip of chromatography paper.
- The bottom edge of the paper is dipped in a suitable solvent, which soaks up through the paper and passes through the spot.
- Each substance in the spot is pulled in two directions: it dissolves in the moving solvent and it is attracted to the paper.
- A substance that is more soluble in the solvent spends more time moving with it, so it travels further up the paper.
- A substance that is less soluble in the solvent, or more strongly attracted to the paper, travels a shorter distance.
- Because the substances move at different speeds, they separate into a column of individual spots.
- A substance that is insoluble in the solvent does not move at all and stays on the baseline.
- A suitable solvent is one in which the substances dissolve to different extents, so water works for many food colourings, while ethanol is used for inks that do not dissolve in water.
- The paper is removed before the solvent reaches the top, and the solvent front is marked in pencil straight away.
Practical
Separating the dyes in food colourings
- Aim: to separate the dyes in an unknown food colouring and compare them with known dyes.
- Apparatus: a strip of chromatography paper, a 250 cm3250\ \text{cm}^3250 cm3 beaker, a lid or watch glass, a ruler, a pencil, fine capillary tubes, a paper clip, a glass rod, water, an unknown food colouring and samples of four known dyes.
- Method, setting up the paper:
- Draw a pencil line about 2 cm2\ \text{cm}2 cm from the bottom of the paper.
- Mark five points along the line, at least 1 cm1\ \text{cm}1 cm apart, and label each one in pencil below the line.
- Use a clean capillary tube to put a small, concentrated spot of each sample on its own point.
- Let each spot dry, then add a second drop on top to make it more concentrated without making it wider.
- Method, running the chromatogram:
- Pour water into the beaker to a depth of about 1 cm1\ \text{cm}1 cm, so that the solvent level is below the baseline.
- Clip the top of the paper to the glass rod and rest the rod across the beaker, so that the bottom edge dips into the water without the paper touching the sides.
- Cover the beaker so that the solvent does not evaporate from the paper.
- Leave the paper until the solvent has risen about three quarters of the way up.
- Remove the paper, mark the solvent front in pencil immediately and hang the paper up to dry.
- Results: the dried paper is the chromatogram, with a column of coloured spots above each starting point.
- Conclusion: the unknown food colouring contains each known dye that has a spot at the same height as one of its own spots.
- Watch out: large spots spread into each other and blur the separation, so keep every spot small.
- Safety: if ethanol is used as the solvent instead of water, keep it away from flames, because it is flammable.
Interpreting chromatograms
Definition
Chromatogram
The pattern of separated spots left on the paper after chromatography.
Definition
Pure substance
A single substance, either one element or one compound, that has no other substances mixed with it.
- A chromatogram is read by counting the spots above each starting point and comparing how far up the paper they have travelled.
- Identifying an unknown substance: the unknown is run on the same paper as known substances, using the same solvent.
- If a spot from the unknown is at the same height as the spot from a known substance, the unknown contains that substance.
- If the unknown has a spot at a height that matches none of the known substances, it contains a substance that was not among those tested.
- Pure substances: a pure substance contains only one substance, so it gives one spot.
- Impure substances: an impure substance or a mixture gives two or more spots, one for each soluble substance present.
- A spot that stays on the baseline belongs to a substance that is insoluble in the solvent used.
- Running the sample again in a different solvent can make such a substance move, which shows whether it is one substance or more.
- Spots can only be compared fairly when the samples were run together on one sheet, because a different solvent or paper changes how far each substance travels.
Example
Reading a food colouring chromatogram
- Four known dyes, A, B, C and D, and an unknown colouring X are spotted on the same baseline and run in water.
- Dyes A, B, C and D each give a single spot, so each of them is a pure substance.
- X gives three spots, so X is a mixture.
- The three spots from X are level with the spots from A, B and C.
- X therefore contains dyes A, B and C, and it does not contain dye D.
Exam technique
Explaining the method
- If asked why the baseline is drawn in pencil, say that pencil does not dissolve in the solvent, whereas ink would dissolve and move up the paper as extra spots.
- If asked why the solvent level must be below the baseline, say that otherwise the samples would dissolve into the solvent in the beaker instead of travelling up the paper.
- When you identify a substance, refer to spots at the same height, not to spots of the same colour.
Common Mistake
- Do not judge purity from the colour of a sample, because a single colour can be made of several dyes, so count the spots instead.
- Do not let the solvent cover the spots on the baseline.
- Do not compare spots from chromatograms that were run in different solvents.
Self review
- Why does a more soluble substance travel further up the paper?
- Why is the baseline drawn in pencil?
- What does a single spot on a chromatogram show about a sample?
- How can you tell that an unknown ink contains a particular known dye?
- What does a spot that stays on the baseline tell you?
