Chromatography separates using stationary and mobile phases
Definition
Stationary phase
The part of a chromatography system that stays in place while the solvent moves through it.
Definition
Mobile phase
The part of a chromatography system that moves, carrying the substances being separated.
- Paper chromatography uses paper as the stationary phase and a solvent as the mobile phase.
- Thin layer chromatography uses a thin layer of silica gel on a glass, metal or plastic plate as its stationary phase.
- The substances in a sample start on the stationary phase and begin to move once the solvent reaches them.
- Each substance is distributed between the two phases, spending part of its time dissolved in the solvent and part held on the stationary phase.
- A substance that spends more time in the mobile phase travels further, so the substances separate.
Key Idea
Separation depends on how each substance is shared between the two phases, so two substances that are shared in the same way cannot be separated by that solvent.
Paper and thin layer chromatography follow the same method
- Draw a start line in pencil about 1 cm1\,\text{cm}1cm from the bottom of the paper or plate.
- Pencil is used because it does not dissolve in the solvent, whereas ink would run and add its own spots.
- Put a small, concentrated spot of each sample on the line.
- Stand the paper in a beaker of solvent with the solvent level below the start line, then cover the beaker.
- If the solvent were above the line, the spots would dissolve into the solvent in the beaker instead of moving up the paper.
- Remove the paper when the solvent is near the top, mark how far the solvent reached and leave the paper to dry.
Practical
Separating the dyes in food colourings
- Aim: find out which dyes are present in several food colourings.
- Apparatus: chromatography paper, a pencil and ruler, capillary tubes, food colourings, samples of known dyes, a beaker with a lid or a cover, water as the solvent, a glass rod or splint, sticky tape and a hairdryer or warm place for drying.
Method
- Draw a pencil start line about 2 cm2\,\text{cm}2cm from the bottom of the chromatography paper.
- Use a capillary tube to put a small spot of each food colouring and each known dye on the line, at least 1 cm1\,\text{cm}1cm apart.
- Build up each spot by adding a drop, letting it dry and adding another, so the spot stays small but holds enough dye to see.
- Pour water into the beaker to a depth of about 1 cm1\,\text{cm}1cm, so that the solvent will be below the start line.
- Tape the top of the paper to a glass rod resting across the beaker, so that the bottom edge dips into the water, and cover the beaker.
- Remove the paper when the solvent is about 1 cm1\,\text{cm}1cm from the top, and draw a pencil line at the solvent front straight away.
- Let the paper dry, then measure from the start line to the centre of each spot and to the solvent front.
- Calculate each Rf value using Rf=distance moved by spotdistance moved by solvent\text{R}_\text{f} = \dfrac{\text{distance moved by spot}}{\text{distance moved by solvent}}Rf=distance moved by solventdistance moved by spot.
Accuracy and safety
- Draw the start line in pencil, because ink would dissolve in the solvent and run up the paper with the dyes.
- Keep the solvent below the start line, or the spots will dissolve into the solvent in the beaker.
- Cover the beaker, so that the air inside stays full of solvent vapour and the solvent does not evaporate from the paper.
- A colouring that matches a known dye in colour and Rf value contains that dye, and one that gives more than one spot is a mixture.
- Thin layer chromatography follows the same steps on a plate coated with silica, and it runs faster and gives sharper spots.
A chromatogram shows how many substances a mixture contains
Definition
Chromatogram
The pattern of separated spots produced after a mixture has been analysed by chromatography.
Definition
Solvent front
The furthest point reached by the solvent as it moves through the stationary phase.
- Each separate spot is a different substance.
- A mixture of three dyes gives three spots, while a pure dye gives only one.
- A spot that stays on the start line is insoluble in that solvent.
- If two samples produce spots at the same heights in the same solvent, they are likely to be the same substance.
- Repeating the run in a second solvent makes the match more certain, because two different substances rarely match in both.
Example
Forensic scientists compare the ink on a forged document with inks from suspects' pens, and a match in several solvents points to the pen that was used.
The Rf value compares substance and solvent distances
Definition
Rf value
A value that compares how far a substance travels in chromatography with how far the solvent travels.
- Measure from the start line to the centre of the spot, and from the start line to the solvent front.
- Calculate the Rf value by dividing the two distances: Rf=distance moved by substancedistance moved by solventR_f = \frac{\text{distance moved by substance}}{\text{distance moved by solvent}}Rf=distance moved by solventdistance moved by substance
- An Rf value is always between 0 and 1, because a substance cannot travel further than the solvent.
- A substance has the same Rf value in the same solvent and stationary phase, so comparing Rf values with known values identifies it.
- Rf values have no units, because they are a ratio of two lengths.
Example
- A dye moves 3.6 cm3.6\,\text{cm}3.6cm from the start line while the solvent front moves 8.0 cm8.0\,\text{cm}8.0cm; calculate its Rf value.
- Write the ratio: Rf=3.6 cm8.0 cmR_f = \dfrac{3.6\,\text{cm}}{8.0\,\text{cm}}Rf=8.0cm3.6cm.
- Divide: Rf=0.45R_f = 0.45Rf=0.45.
Chromatography can show whether a substance is pure
- A pure substance gives a single spot in every solvent tried.
- An impure substance gives two or more spots, showing the extra substances present.
- Running the sample alongside a pure reference sample shows whether its main spot matches.
- A single spot in one solvent is not conclusive, because two substances might move the same distance in that solvent.
- Testing in different solvents gives stronger evidence that the sample is pure.
Exam technique
When a question asks you to identify substances from a chromatogram, compare the height or Rf value of each spot with the reference spots and name each match.
Self review
- What are the stationary and mobile phases in paper chromatography?
- Why is the start line drawn in pencil?
- What does a chromatogram with four spots tell you about a sample?
- A spot moves 2.4 cm2.4\,\text{cm}2.4cm and the solvent moves 6.0 cm6.0\,\text{cm}6.0cm; what is its Rf value?
- How can chromatography show that a substance is pure?
