Chromatography

Section 1 of 12Principle of paper chromatography

Use

• Use separates substances with different solubilities in a solvent, such as the dyes in black ink.

Spots

• Spots a pencil line is drawn and spots of sample placed on it.
• Pencil
  • used because ink would run with the samples.
• Solvent
  • the paper is lowered into solvent with the pencil line above the surface.
• Movement
  • the solvent rises by capillary action, carrying substances with it.
• Separation
  • more soluble substances travel further.

Section 2 of 12Method

1

Draw a pencil line near the bottom of the chromatography paper.

2

Place small spots of each sample on the line, with a known reference alongside.

3

Lower the paper into a container with solvent below the line.

4

Let the solvent rise through the paper.

5

Remove the paper before the solvent reaches the top and mark the solvent front.

6

Dry the paper and examine the chromatogram.

Section 3 of 12Interpreting a chromatogram

Terms

• Terms paper chromatography is the technique and a chromatogram is the paper showing the result.

Same substance

• Same substance gives the same pattern and identical spots.
• Reference spot
  • a known compound used for comparison with unknown spots.
• Mixture
  • separates into several spots, one for each component.
• Pure substance
  • gives one spot.
• Impure sample
  • shows more than one spot.

Section 4 of 12Locating agents

Problem

• Problem colourless substances such as amino acids and sugars cannot be seen on the paper.

Locating agent

• Locating agent a chemical that reacts with the sample to give a coloured product.
• Use
  • apply after the run so the spots become visible to the naked eye.

Section 5 of 12Retention factor (Rf)

Definition

• Definition the distance moved by a substance divided by the distance moved by the solvent.

Equation

• Rf = distance travelled by substance ÷ distance travelled by solvent.

  What the symbols mean: R_f has no unit and is always between 0 and 1 · both distances are measured from the baseline
• Units
  • none, as it is a ratio.
• Always between 0 and 1
  • a substance cannot move further than the solvent.
• Constant
  • the Rf of a compound is the same under the same conditions.
• Identification
  • compare the Rf of an unknown with known values.

Section 6 of 12Worked example of Rf

1

Measure the distance from the baseline to the centre of the spot, e.g. 3.6 cm.

2

Measure the distance from the baseline to the solvent front, e.g. 9.0 cm.

3

Divide: 3.6 ÷ 9.0 = 0.40.

4

Compare 0.40 with the table of known Rf values.

• Measurement tip
  • always measure from the pencil line, not from the bottom of the paper.

Section 7 of 12Reasons for a good chromatogram

Small spots

• Small spots large spots overlap and separate poorly.

Solvent level

• Solvent level keep it below the baseline.
• Lid
  • cover the container so solvent does not evaporate from the paper.
• Stop
  • remove the paper before the solvent front reaches the top so the distance can be measured.
• Drying
  • dry in air before using a locating agent.

Section 8 of 12Identifying components from a chromatogram

Observation	Conclusion
Spot level with a reference spot	Sample contains the reference substance
One spot only	Sample is probably pure
Several spots	Sample is a mixture
Spot not matching any reference	Unknown substance present

• Rf use
  • compare calculated Rf values with a data table for the same solvent.

Section 9 of 12Uses of chromatography

Food

• Food testing whether a colouring is permitted by comparing with known dyes.

Forensics

• Forensics matching an ink or dye from a sample to a suspect source.
• Medicine
  • detecting drugs and checking that a product is pure.
• Colourless samples
  • amino acids from a protein can be separated and sprayed with a locating agent.

Section 10 of 12Worked examples

1. Explain why the starting line is drawn in pencil. [2]2 marks

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2. A spot travels 4.2 cm while the solvent travels 6.0 cm. Calculate the Rf. [2]2 marks

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3. A chromatogram of a food dye shows two spots. What does this show? [1]1 mark

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4. Why must a locating agent be used for amino acids? [2]2 marks

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Section 11 of 12Key terms

Term	Meaning
Chromatography	A method to separate substances based on their different solubilities.
Chromatogram	The paper showing the separated spots.
Solvent front	The furthest point reached by the solvent.
Locating agent	A reagent that makes colourless spots visible.
Rf value	Distance moved by a substance divided by distance moved by solvent.

Section 12 of 12Exam technique

1

Having the baseline below the solvent level — samples would wash away.

2

Calculating Rf with the wrong distances.

3

Giving Rf units — it has none.

4

Saying more soluble substances move less far.