Chromatography

What is the fundamental principle upon which all chromatography techniques rely?

• The separation depends on the balance between solubility in the mobile phase and retention (adsorption) by the stationary phase.

Define the stationary phase and the mobile phase in chromatography.

• Stationary Phase: The fixed, non-moving phase (e.g., solid silica/alumina in TLC).

• Mobile Phase: The phase that moves through the stationary phase (e.g., a liquid solvent).

What type of substance (polar or non-polar) is typically used for the stationary phase in TLC and Column Chromatography?

• A polar solid, such as silica gel or alumina (Al₂O₃).

On a polar silica plate, which type of compound moves the slowest and why?

• Polar compounds move the slowest. They are strongly adsorbed (retained) by the highly polar silica surface, meaning they spend less time moving with the mobile phase.

Write the equation for the Retardation Factor (R_f) and state what R_f is used for.

R_f = \frac{\text{Distance moved by component}}{\text{Distance moved by solvent front}}

Describe how a Thin-Layer Chromatography (TLC) plate is developed to locate colourless components like amino acids.

A locating agent (or developing agent) is used. Examples:

1. Ninhydrin (sprayed on and heated, reacts to give coloured spots).

2. UV Light (causes fluorescent spots to show up).

Describe the stationary phase and mobile phase, and the mechanism of separation, in Column Chromatography (CC).

• Stationary Phase: A polar solid powder (silica or alumina) packed into a vertical glass column.

• Mobile Phase: A liquid solvent (eluent).

• Mechanism: The solvent is passed through the column. Non-polar components move quickly (elute first), while polar components bind strongly to the silica and elute last.

In Column Chromatography, how can a chemist change the order in which components elute?

• By changing the polarity of the mobile phase (solvent). To push a strongly bound (polar) component through, the chemist must switch to a more polar solvent.

What are the stationary phase and the mobile phase in Gas Chromatography (GC)?

• Stationary Phase: A non-volatile liquid coated on the inner surface of a long, coiled capillary tube (the column).

• Mobile Phase: An inert gas (e.g., Helium or Nitrogen), often called the carrier gas.

How are components identified in GC?

• By measuring the retention time. This is the time taken for a component to travel from the injection port through the column to the detector. (Retention time is compared against known standards under identical conditions).

What property determines the separation of components in GC?

• Primarily the volatility (or boiling point) of the sample components, and their relative solubility in the liquid stationary phase.

  • Highly volatile components pass through the column fastest.

⚠ SAFETY What are the main safety hazards associated with the mobile phase solvents used in organic chromatography (e.g., petroleum ether, propanone)?

• Most organic solvents are highly flammable and/or volatile and toxic (harmful if inhaled or absorbed through the skin). Safety: Work in a fume cupboard and ensure there are no naked flames.

⚠ PRACTICAL In Column Chromatography, why might a student choose a solvent (mobile phase) that is less polar to separate two components that are very similar?

• To achieve better separation. If the components are too similar, they will move too quickly (or too slowly) together. Using a less polar solvent increases the difference in their adsorption rates to the polar stationary phase, giving a better resolution.

⚠ PRACTICAL Why is the R_f value always less than 1?

• Because the solvent front (the distance moved by the solvent) is the maximum distance a component can possibly travel; no component can move further than the solvent itself.

Question 1 (1 mark) What is the main property of the stationary phase in TLC and Column Chromatography?

Answer: It is a polar solid (e.g., silica or alumina). (1)

Question 2 (2 marks) On a TLC plate, a spot travelled 4.2 cm. The solvent front travelled 6.0 cm. Calculate the R_f value of the spot and state its units.

Answer:

• R_f = 4.2 / 6.0 = 0.70 (1)

• Units: R_f has no units, as it is a ratio of two distances. (1)

Question 3 (3 marks) A student performs TLC on a silica plate. Compound A is non-polar and compound B is highly polar. Explain which compound will have the larger R_f value.

• Compound A will have the larger R_f value. (1)

• The stationary phase (silica) is polar. (1)

• Non-polar compound A is less strongly attracted/adsorbed to the polar silica and spends more time moving with the mobile phase. (1)

Question 4 (4 marks) Describe the full experimental procedure a student would use to measure the R_f value for a sample using Thin-Layer Chromatography.

Answer: (4 marks)

1. Draw a pencil line (the baseline/origin) near the bottom of the plate and spot the sample onto it. (1)

2. Place the plate into a container with the solvent (mobile phase) such that the solvent level is below the pencil line. (1)

3. Allow the solvent to run up the plate (ascend) until it is near the top; then remove and mark the solvent front immediately. (1)

4. Measure the distance from the baseline to the centre of the spot  and the distance from the baseline to the solvent front . (1)

(4 marks) Describe how an organic component is separated and identified using Gas Chromatography followed by the analysis stage.

• The sample is injected, vaporised, and carried through the column by the inert carrier gas (mobile phase). (1)

• Components separate based on their volatility/boiling point and their solubility in the liquid stationary phase. (1)

• Components exit the column one by one at specific times (retention times). (1)

• Identification is achieved by comparing the retention time to known standards run under the same conditions (or by coupling to a Mass Spectrometer). (1)