Chromatography

Definition of Chromatography

• Chromatography: A separation technique used to separate and sometimes identify the components of a mixture based on their different physical and chemical properties.
• Works because different substances have different solubilities in a solvent and different attractions to a stationary phase.
• Common forms include paper chromatography, thin-layer chromatography, and column chromatography (only paper chromatography is in the syllabus here).

1. Paper Chromatography to Separate Mixtures of Soluble Substances

Principle

• Involves two phases:
  • Stationary phase: The chromatography paper (often made of cellulose fibres).
  • Mobile phase: The solvent that moves up the paper by capillary action.
• Components of the mixture travel at different speeds because:
  • They have different solubilities in the mobile phase.
  • They have different attractions (adsorption) to the stationary phase.

Method

1. Preparation
   • Draw a pencil baseline near the bottom of the paper (pencil is used because it is insoluble in the solvent).
   • Place small spots of the mixture(s) to be tested on the baseline using a capillary tube.
   • If comparing with known substances, place their spots alongside the unknown.
2. Setting up
   • Pour a small amount of solvent into a beaker (depth should be below the baseline so the spots are not submerged).
   • Suspend the chromatography paper so that the bottom edge dips into the solvent but the spots are above the solvent level.
3. Separation
   • The solvent travels up the paper by capillary action.
   • As the solvent front passes the spots, the substances dissolve and are carried upwards at different rates.
4. Completion
   • Remove the paper before the solvent reaches the top.
   • Mark the solvent front immediately with a pencil before it evaporates.
   • Allow the paper to dry.

2. Use of Locating Agents for Colourless Substances

Purpose

• Many substances (e.g., amino acids, sugars) are colourless and cannot be seen after separation.
• Locating agents make them visible by producing a coloured compound.

Procedure

• After the chromatogram has dried, spray or dip it into a locating agent.
• Examples (not required by syllabus to memorise, but for understanding):
  • Ninhydrin for amino acids (gives purple spots).
  • Iodine vapour for certain organic compounds.
• This step allows the separated spots to be seen and measured.

3. Interpretation of Simple Chromatograms

Identifying Unknown Substances

• Compare the position (and sometimes colour) of an unknown spot with spots from known substances under the same conditions.
• Same Rf value and same colour → likely to be the same substance.

Pure vs Impure Substances

• Pure substance → Produces a single spot on the chromatogram.
• Impure substance (mixture) → Produces two or more spots, each representing a different component.

4. Retention Factor (Rf) Equation

Definition

• Retention factor (Rf): A ratio that represents how far a substance travels compared to the solvent.

Equation

Rf=distance travelled by substancedistance travelled by solventRf = \frac{\text{distance travelled by substance}}{\text{distance travelled by solvent}}

How to Measure

1. Measure from the baseline (where the spot started) to the centre of the spot (distance travelled by substance).
2. Measure from the baseline to the solvent front (distance travelled by solvent).
3. Divide the two distances to calculate the Rf.

Example

• Distance travelled by substance: 3.2 cm
• Distance travelled by solvent: 5.0 cm

Rf = \frac{3.2}{5.0} = 0.64
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Key Points

• Rf is always between 0 and 1.
• Same substance under the same conditions will have the same Rf.
• Changing the solvent changes Rf values.

Additional Notes

Factors Affecting Chromatography Results

• Type of solvent: Different solvents dissolve substances to different extents.
• Temperature: Higher temperature can increase solubility and migration rates.
• Quality of stationary phase: Different paper types can affect retention.

Applications

• Identifying food dyes.
• Separating plant pigments.
• Checking the purity of pharmaceuticals.
• Analysing amino acids in proteins.