Chromatography

Chromatography is a separation technique used to separate the components of a mixture. It relies on the differential distribution of the mixture's components between a mobile phase and a stationary phase.

Principles of Chromatography

The fundamental principle behind chromatography is the difference in the affinity of the various components of a mixture for the stationary and mobile phases. Components with a higher affinity for the stationary phase will move slower, while those with a higher affinity for the mobile phase will move faster. This difference in migration rates leads to the separation of the components.

The process involves:

1. Sample Introduction: The mixture to be separated is introduced as the sample.
2. Mobile Phase: A solvent or a mixture of solvents that carries the sample through the stationary phase.
3. Stationary Phase: A fixed material that interacts differently with the components of the sample.
4. Separation: The components of the mixture separate as they travel through the stationary phase at different rates.
5. Detection: The separated components are detected as they elute from the stationary phase.

Types of Chromatography

1. Paper Chromatography

Paper chromatography is a simple and inexpensive technique. It uses a sheet of paper as the stationary phase and a solvent as the mobile phase.

Stationary Phase: A thin layer of absorbent material (e.g., cellulose) coated onto paper.

Mobile Phase: A solvent that travels up the paper by capillary action.

Procedure:

1. A line is drawn lightly with a pencil near the bottom of the paper.
2. A small spot of the sample is applied to the line.
3. The bottom edge of the paper is placed in a shallow dish containing the solvent, ensuring the spot is above the solvent level.
4. As the solvent travels up the paper, it carries the sample components with it.
5. The different components separate based on their affinity for the paper and the solvent.
6. Once the solvent front has reached a suitable height, the paper is removed, and the separated components are visualized (e.g., by drying, using a UV lamp, or staining).

2. Thin Layer Chromatography (TLC)

TLC is a more sophisticated form of chromatography that uses a thin layer of adsorbent material coated on a solid support (e.g., glass, plastic, or aluminum). It offers better resolution than paper chromatography.

Stationary Phase: A thin layer of adsorbent material (e.g., silica gel, alumina) coated on a solid support.

Mobile Phase: A solvent or a mixture of solvents.

Procedure:

1. A thin layer of the stationary phase is coated onto the solid support.
2. The sample is spotted onto the TLC plate near the bottom.
3. The plate is placed in a developing chamber containing a shallow layer of the mobile phase.
4. The solvent travels up the plate by capillary action, carrying the sample components with it.
5. The separated components are visualized after the solvent front has reached a suitable height (e.g., by UV light, staining, or chemical reaction).
6. The Rf value (retardation factor) is calculated for each component: $Rf = \frac{distance\ travelled\ by\ component}{distance\ travelled\ by\ solvent}$

3. Column Chromatography

Column chromatography is used to separate larger quantities of material. It involves packing a column with a stationary phase and eluting the sample with a mobile phase.

Stationary Phase: A solid adsorbent material (e.g., silica gel, alumina) packed into a glass column.

Mobile Phase: A solvent or a mixture of solvents that flows through the column.

Procedure:

1. A glass column is packed with the stationary phase.
2. The sample is loaded onto the top of the column.
3. The mobile phase is allowed to flow through the column, carrying the sample components with it.
4. The separated components are collected as they elute from the bottom of the column.
5. The fractions are analyzed to identify the separated components.

4. Gas-Liquid Chromatography (GLC)

GLC is used to separate volatile compounds. The sample is vaporized and passed through a column containing a stationary phase. The separation is based on the differences in boiling points and affinities of the components for the stationary and mobile phases.

Stationary Phase: A liquid coated on a solid support inside a column.

Mobile Phase: An inert gas (e.g., helium, nitrogen) that carries the vaporized sample through the column.

Procedure:

1. The sample is vaporized and injected into the column.
2. The vaporized sample components travel through the column with the mobile phase.
3. The separation occurs based on the differences in boiling points and affinities for the stationary phase.
4. The separated components are detected as they elute from the column (e.g., using a flame ionization detector or a mass spectrometer).

Technique	Stationary Phase	Mobile Phase	Typical Use
Paper Chromatography	Cellulose	Solvent	Separating organic compounds, pigments
Thin Layer Chromatography (TLC)	Silica gel, Alumina	Solvent	Monitoring reaction progress, identifying compounds
Column Chromatography	Silica gel, Alumina	Solvent	Separating large quantities of compounds
Gas-Liquid Chromatography (GLC)	Liquid coated on a solid support	Inert gas (e.g., Helium, Nitrogen)	Separating volatile compounds

Applications of Chromatography

Chromatography is a versatile technique with numerous applications in various fields, including:

• Chemical Analysis: Identifying and quantifying the components of a mixture.
• Pharmaceutical Industry: Analyzing drug purity and identifying impurities.
• Environmental Monitoring: Detecting pollutants in water and air.
• Food Science: Analyzing food composition and detecting adulteration.
• Forensic Science: Identifying unknown substances in criminal investigations.