Chromatography Overview and Principles

Chromatography Overview

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

Physical Basis of Separation

• Separation occurs based on differences in physical characteristics of the compounds, including:
  • Molecular size
  • Shape
  • Charge
  • Volatility
  • Solubility
  • Adsorptivity

Essential Components of Chromatographic Systems

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

General Chromatography Principle

• All forms of chromatography rely on the same principle.
  • A stationary phase
  • A mobile phase
• The mobile phase moves through the stationary phase, carrying the mixture's components.
• Components separate due to differing interactions with the stationary and mobile phases (redistribution).

Elution Process

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

Gel Exclusion Chromatography

• Separates molecules based on size.
• Molecules are separated by sieving through a porous matrix.
• Larger molecules are eluted faster (bypass the matrix).
• Smaller molecules are eluted slower (trapped in the matrix).

Ion Exchange Chromatography

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

Thin Layer Chromatography (TLC)

• Stationary Phase: A thin layer of silica gel, cellulose, or alumina coated onto a plate (e.g., glass or plastic).
• Mobile Phase: A liquid solvent.
• Components separate based on their relative affinities for the stationary and mobile phases.
• Compounds with higher affinity for the stationary phase move slower; those with higher affinity for the mobile phase move faster.
• The resulting spots on the plate are identified and quantified.

Measuring R_f Values (TLC)

• Rf value (Retention Factor): A measure of how far a compound travels relative to the solvent front on the TLC plate.
• R_f = Distance travelled by the compound/ Distance travelled by the solvent.
• R_f values are used to identify compounds.

Paper Chromatography

• Stationary Phase: Cellulose paper.
• Mobile Phase: A liquid solvent.
• Components separate based on their polarity towards the mobile and stationary phases.
• Separation is based on the relative affinities of compounds for the solvents and the paper fibers.
• R_f values can be used to identify substances separated.

Paper Chromatography Process

• Sample spotted onto the paper.
• Paper placed in a developing chamber with the mobile phase.
• Mobile phase moves up the paper via capillary action.
• Components separate based on their interactions with the paper and solvent.

Applications of TLC in Specific Conditions

• TLC is used to detect and measure amino acid concentrations in various conditions like phenylketonuria, alkaptonuria, and maple syrup disease.
  • These conditions involve specific amino acid abnormalities detectable via TLC.

This quiz covers the fundamental concepts of chromatography, an analytical technique vital for separating substances in various mixtures. It explores the physical basis of separation, essential components of chromatographic systems, and the general principles involved in the process.