Chromatography

Chromatography is a family of separation techniques used to separate and identify components of mixtures. All forms of chromatography work on the same principle: components of a mixture distribute themselves between a stationary phase and a mobile phase at different rates, causing them to separate. This lesson covers thin-layer chromatography (TLC), column chromatography, gas chromatography (GC), and HPLC.

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Principles of Chromatography

Stationary Phase and Mobile Phase

• The stationary phase is a fixed material that does not move. It may be a solid (e.g., silica gel, alumina) or a liquid coated on a solid support.
• The mobile phase is a fluid (liquid or gas) that moves through or over the stationary phase, carrying the sample components with it.

Separation depends on the relative affinities of each component for the two phases:

• A component with a strong affinity for the stationary phase moves slowly (it spends more time adsorbed or dissolved in the stationary phase).
• A component with a strong affinity for the mobile phase moves quickly.

Key Definition: Adsorption is the process by which molecules stick to the surface of a solid. Absorption is the process by which molecules dissolve into a liquid. Both mechanisms can operate in chromatography.

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Thin-Layer Chromatography (TLC)

How TLC Works

1. A thin layer of silica gel (SiO₂) or alumina (Al₂O₃) is coated onto a glass, plastic, or aluminium plate (the stationary phase).
2. A small spot of the sample (dissolved in a volatile solvent) is applied near the bottom of the plate using a capillary tube.
3. The plate is placed upright in a shallow layer of solvent (the mobile phase, also called the eluent) in a sealed chamber. The solvent level must be below the sample spot.
4. The solvent rises up the plate by capillary action, carrying the sample components with it.
5. Components with greater affinity for the mobile phase travel further; those with greater affinity for the silica (stationary phase) lag behind.
6. When the solvent front nears the top of the plate, the plate is removed and the solvent front is marked immediately with a pencil.
7. If the spots are coloured, they can be seen directly. If they are colourless, the plate may be viewed under UV light (if a UV-fluorescent indicator is incorporated in the silica) or developed using a locating agent such as iodine vapour or ninhydrin (for amino acids).

Rf Values

The retention factor (Rf) is calculated as:

Rf = distance travelled by the spot / distance travelled by the solvent front

• Rf values are always between 0 and 1.
• Rf values are characteristic of a substance under specific conditions (same stationary phase, same mobile phase, same temperature).
• Rf values can be used to identify a substance by comparison with known standards run on the same plate.

Exam Tip: Rf values are only valid for comparison if the conditions (solvent, temperature, stationary phase) are identical. Always run a known standard alongside the unknown on the same TLC plate.

Applications of TLC

• Monitoring the progress of a chemical reaction (checking if starting material has been consumed).
• Identifying components of a mixture.
• Checking the purity of a product (a pure compound gives one spot; impurities give additional spots).

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Column Chromatography

Column chromatography uses the same principles as TLC but on a larger scale, allowing preparative separation (collecting separated fractions).

1. A glass column is packed with the stationary phase (typically silica gel or alumina).
2. The mixture is loaded onto the top of the column.
3. Solvent (eluent) is continuously added to the top and allowed to flow through the column by gravity.
4. Components separate as they travel down the column at different rates.
5. The eluent dripping out of the bottom of the column is collected in fractions (small portions).
6. Different fractions contain different components of the mixture.

The polarity of the eluent can be gradually increased during the separation to elute more strongly adsorbed components.

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Gas Chromatography (GC)

How GC Works

In gas chromatography, the mobile phase is an inert carrier gas (typically helium or nitrogen), and the stationary phase is a high-boiling-point liquid coated on the inside of a long, narrow capillary column (or on an inert solid support in a packed column).