Chromatography as a Separation Technique
- This technique is used to separate substances that have different solubilities in a given solvent (e.g. different coloured inks that have been mixed to make black ink).
- It is also used to identify unknown substances.
- A pencil line is drawn on chromatography paper and spots of the sample are placed on it. Pencil is used for this as ink would run into the chromatogram along with the samples.
- The paper is then lowered into the solvent container, making sure that the pencil line sits above the level of the solvent so the samples don’t wash into the solvent container.
- The solvent travels up the paper by capillary action, taking some of the coloured substances with it.
- Different substances have different solubilities so will travel at different rates, causing the substances to spread apart. Those substances with higher solubility will travel further than the others.
- This is because they spend more time in the mobile phase and are thus carried further up the paper than the less soluble components.
The pigments in ink can be analysed using paper chromatography
- All chromatography techniques use two phases called the mobile phase and the stationary phase.
- In paper chromatography:
- The mobile phase is the solvent in which the sample molecules can move, which in paper chromatography is liquid e.g. water or ethanol.
- The stationary phase in paper chromatography is the actual chromatography paper itself.
Distinguishing Pure & Impure Substances
- Pure substances will produce only one spot on the chromatogram.
- If two or more substances are the same, they will produce identical chromatograms.
- If the substance is a mixture, it will separate on the paper to show all the different components as separate spots.
- An impure substance therefore will produce a chromatogram with more than one spot.
Diagram showing the analysis of a mixture and pure substances using chromatography
Identifying Substances Using Retention Factor (Rf) Values
- These values are used to identify the components of mixtures.
- The Rf value of a particular compound is always the same but it is dependent, however, on the solvent used.
- If the solvent is changed then the value changes.
- Calculating the Rf value allows chemists to identify unknown substances because it can be compared with Rf values of known substances under the same conditions.
- These values are known as reference values.
- Retention factor = distance moved by compound ÷ distance moved by solvent.
- The Rf value is a ratio and therefore has no units.
Using Rf values to identify components of a mixture
Required Practical 6: Investigating Food Coloring Using Paper Chromatography
Investigate how paper chromatography can be used to separate and identify a
mixture of food colourings.
Rf values can be used to identify the components of an unknown mixture by comparison with Rf values of known substances.
- a 250 cm3 beaker
- a wooden spill
- a rectangle of chromatography paper
- four known food colourings labelled A–D
- an unknown mixture of food colourings labelled U
- five glass capillary tubes
- paper clip
- ruler & pencil
Diagram of the apparatus neededPractical Tip:
The pencil line must never be below the level of the solvent as the samples will be washed away.
- Use a ruler to draw a horizontal pencil line 2 cm from the end of the chromatography paper.
- Use a different capillary tube to put a tiny spot of each colouring A, B, C and D on the line.
- Use the fifth tube to put a small spot of the unknown mixture U on the line.
- Make sure each spot is no more than 2-3 mm in diameter and abel each spot in pencil.
- Pour water into the beaker to a depth of no more than 1 cm and clip the top of the chromatography paper to the wooden spill. The top end is the furthest from the spots.
- Carefully rest the wooden spill on the top edge of the beaker. The bottom edge of the paper
should dip into the water solvent.
- Allow the solvent to travel undisturbed at least three quarters of the way up the paper.
- Remove the paper and draw another pencil line on the dry part of the paper as close to the wet edge as possible. This is called the solvent front line.
- Measure the distance in mm between the two pencil lines. This is the distance travelled by the water solvent.
- For each of food colour A, B, C and D measure the distance in mm from the start line to the
middle of the spot.
Record your results in a suitable table, eg:
The Rf values of food colours A, B, C and D should be compared to that for the unknown sample as well as a visual comparison being made.
The use of chromatography and Rf values is a viable method of identifying unknown mixtures given reference material.
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Morgan’s passion for the Periodic Table begun on his 10th birthday when he received his first Chemistry set. After studying the subject at university he went on to become a fully fledged Chemistry teacher, and now works in an international school in Madrid! In his spare time he helps create our fantastic resources to help you ace your exams.