Column chromatography

FDA History - Column Chromatography

Fraction collector - sampler LAMBDA OMNICOLL

Column chromatography sequence

Cromatografia su colonna

Metrohm 850

Column chromatography is a laboratory technique used to separate the different components of a mixture based on their chemical properties. This method involves the passage of a mixture dissolved in a solvent (known as the mobile phase) through a column filled with a solid material (known as the stationary phase). The individual components of the mixture move through the column at different rates, allowing them to be separated and collected individually.

Principles of Column Chromatography

The separation of components in column chromatography relies on differential adsorption to the stationary phase. Components that interact more strongly with the stationary phase move more slowly through the column than components that interact less strongly. The choice of stationary phase and solvent can be adjusted depending on the specific properties of the components to be separated.

Types of Column Chromatography

There are several types of column chromatography, each tailored for specific applications. These include:

• Size-exclusion chromatography (SEC), which separates molecules based on size.
• Ion-exchange chromatography (IEC), which separates molecules based on charge.
• Affinity chromatography, which separates molecules based on specific binding interactions.
• Reverse-phase chromatography (RPC), which is commonly used for the separation of small, non-polar molecules.

Applications

Column chromatography is widely used in biochemistry and organic chemistry for purifying proteins, nucleic acids, and small organic compounds. It is a fundamental technique in the development of pharmaceuticals, allowing for the purification of active compounds from complex mixtures.

Procedure

The procedure for column chromatography involves several steps: 1. Preparation of the column: The column is packed with the chosen stationary phase. 2. Application of the sample: The mixture to be separated is dissolved in a suitable solvent and applied to the top of the column. 3. Elution: Solvent is passed through the column, moving the components of the mixture through the stationary phase at different rates. 4. Collection of fractions: The eluate (solvent and separated components) is collected in fractions. The composition of these fractions can be analyzed to identify the separated components.

Advantages and Limitations

Column chromatography offers several advantages, including the ability to separate complex mixtures and the flexibility to adjust the separation process by changing the stationary phase or solvent. However, it can be time-consuming and requires optimization to achieve the best separation.

See Also

• Chromatography
• High-performance liquid chromatography (HPLC)
• Gas chromatography (GC)

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