Chromatography and Instrumentation

What is Chromatography?

Chromatography is a powerful separation technique used to analyze complex mixtures by isolating their individual components.

It works by moving a mobile phase (liquid or gas) containing the mixture across a stationary phase (solid or liquid on a solid support). Different components move at different rates depending on their interactions with the two phases, leading to their separation.

Principle of Chromatography

Chromatography separates substances based on their differing affinities for the mobile and stationary phases:

• Mobile phase: The solvent or gas that carries the mixture.
• Stationary phase: The material (such as silica gel or paper) that retains some components more strongly than others. The separation occurs because substances that interact strongly with the stationary phase move more slowly, while those that interact weakly with the stationary phase move faster with the mobile phase.

Thin-Layer Chromatography (TLC)

Thin-layer chromatography (TLC) is a simple, inexpensive method for separating small amounts of substances.

It is commonly used in forensic science and other fields for rapid separation and identification.

• Principle: A thin layer of silica or alumina on a plate serves as the stationary phase. The sample is spotted onto the plate, and a solvent (mobile phase) carries the components up the plate, separating them.

Advanced Chromatographic Techniques

Gas Chromatography (GC)

Gas chromatography (GC) is a technique used to separate volatile compounds. It is particularly useful for environmental analysis and testing in the food and pharmaceutical industries.

• Principle: A gaseous mobile phase (usually helium or nitrogen) carries the sample through a column containing a liquid stationary phase. The components separate based on their boiling points and interactions with the stationary phase.

High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) is used to separate and quantify non-volatile compounds in liquid samples. It is highly precise and widely used in food testing, pharmaceuticals, and agriculture.

• Principle: The mobile phase is a liquid under high pressure, and the stationary phase is a packed column. Separation is based on differences in solubility and interactions with the stationary phase.

Instrumental Methods of Separation and Analysis

Chromatography is often coupled with other analytical techniques to provide more detailed information about the separated compounds.

Mass Spectrometry (MS)

Mass spectrometry (MS) is a technique used to identify compounds by measuring the mass-to-charge ratio of ionized molecules. It is often combined with chromatography (GC-MS or HPLC-MS) for high-precision analysis.

• Principle: After separation by chromatography, compounds are ionized and separated based on their mass-to-charge ratio, producing a unique spectrum for each substance.

Infra-red Absorption Spectrometry (IR)

Infra-red (IR) spectrometry identifies organic compounds based on their absorption of infrared light, which causes molecular vibrations. Each type of bond absorbs at a specific wavelength, providing a "fingerprint" for the compound.

• Principle: Organic molecules absorb specific wavelengths of infrared radiation, resulting in a characteristic absorption spectrum.

Ultraviolet Absorption Spectrometry (UV)

Ultraviolet (UV) spectrometry is a quantitative technique used to measure the concentration of compounds that absorb UV light, often in the analysis of plant pigments or drug metabolites.

• Principle: Compounds absorb ultraviolet light at specific wavelengths, and the amount of absorption is directly proportional to the concentration of the compound.