Chromatography Overview and Principles

Chromatography Overview

• Chromatography is an analytical technique separating closely related compounds in a mixture.
• It is used to separate proteins, peptides, amino acids, lipids, carbohydrates, vitamins, and drugs.

Physical Basis of Separation

• Compounds are separated based on differences in physical characteristics.
• Examples include molecular size, shape, charge, volatility, solubility, and absorptivity.

Essential Components of Chromatographic Systems

• Stationary Phase: A porous solid matrix, where the sample dissolved in the mobile phase travels through.
• Mobile Phase: The mixture of substances (to be separated) dissolved in a liquid or gas.

General Principle of Chromatography

• All chromatography types involve a stationary and a mobile phase.
• The mobile phase moves through the stationary phase, carrying the sample components.
• Separation happens through continuous redistribution between the mobile and stationary phases.

Definitions (Chromatography)

• Eluent: The fluid entering the column.
• Eluate: The fluid exiting the column.
• Elution: The process of passing the mobile phase through the column.

Gel Exclusion Chromatography

• Separates molecules based on size (sieving).
• Smaller molecules travel longer paths through the gel matrix.

Ion Exchange Chromatography

• Separates ions based on their charges.
• Anions or cations are attracted to oppositely charged groups on the stationary phase.

Thin Layer Chromatography (TLC)

• Stationary Phase: A uniform layer of silica gel, cellulose, or alumina coated on a surface (e.g., glass).
• Separation occurs based on the relative affinity of compounds to the stationary and mobile phases.
• Compounds with higher affinity for the stationary phase move slower.
• Rf value: The retention factor, calculated as distance travelled by a spot divided by distance travelled by the solvent.

Measuring Rf Values

• Each spot on a TLC plate has a unique Rf value.
• Rf = Distance travelled by compound / Distance travelled by solvent.

Paper Chromatography

• Stationary Phase: Cellulose filter paper.
• Mobile Phase: Liquid solvent.
• Separation happens based on the polarity of compounds towards both the mobile and stationary phases.

Paper Chromatography: Process

• Cellulose paper is placed in a developing chamber.
• The sample is spotted on the paper.
• The solvent moves up the paper, carrying components with it.
• Compounds with high affinity to the stationary phase move slower.
• The Rf value can be used to identify compounds.

Paper Chromatography: Specific Applications

• Used in identifying amino acids (e.g., using ninhydrin to visualize them).
• Unknown mixtures can be compared to known standards for identification.

TLC and Specific Applications

• Used in Biochemical analysis, such as separating or isolating biochemical metabolites from blood plasma, urine, body fluids, and serum.
• Used in testing for conditions like phenylketonuria, alkaptonuria, and maple syrup disease.

Explore the fundamental concepts of chromatography, an essential analytical technique used to separate mixtures of compounds. Learn about its physical basis, key components such as stationary and mobile phases, and the general principles that govern the separation process.