10064 24-25 8.1 Introduction to analytical techniques I(1) 2

Introduction to Analytical Techniques

• Course: LSC 10064 Analytical Techniques I: Chromatography and Electrophoresis

• Institution: Keele University, School of Life Sciences

Types of Analytical Techniques

• Chromatography

  • Purpose: Separation of components of a mixture based on size, charge, hydrophobicity, composition, specificity.

  • Subtypes:

    • Gel Filtration

    • Ion Exchange

    • Affinity

    • High-Performance Liquid Chromatography (HPLC)

    • Reversed Phase

    • Gas Chromatography (GC)

• Electrophoresis

  • Purpose: Separation of macromolecules (DNA, RNA, proteins) based on size and/or charge.

  • Subtypes:

    • SDS PAGE

    • Isoelectric Focusing

    • 2D Gel Electrophoresis

• Spectroscopy

  • Purpose: Structure determination through absorption and transmission measurement of electromagnetic radiation.

  • Subtypes:

    • Infrared

    • UV-visible

    • Nuclear Magnetic Resonance (NMR)

• Mass Spectrometry

  • Purpose: Structural characterization by fragmenting molecules and measuring resultant masses.

  • Subtypes:

    • MALDI

    • ESI

    • MS-MS

    • GC-MS

• X-ray Crystallography

  • Purpose: Structure determination by analyzing the scattering pattern from a crystalline molecular array.

Chromatography Techniques

• General Mechanism

  • Based on interaction between the mobile and stationary phases for separation of target molecules.

• Types of Chromatography

  • Column Chromatography

  • Gel Filtration Chromatography

    • Separates based on size; larger proteins exit first, smaller proteins enter beads and exit last.

• Ion Exchange Chromatography

  • Utilized typically for protein purification.

  • Adjusts elution by modifying salt concentration or pH of the mobile phase.

• Affinity Chromatography

  • Exploits proteins' affinity for specific chemicals (ligands).

  • Proteins bound to the stationary phase are eluted with an enriched solution or breaking interaction conditions.

Detection Methods in Chromatography

• Types of Detectors Include:

  • UV

  • IR

  • Refractive Index

  • Fluorescence

  • Conductivity

  • Mass Spectrometry

  • Electrochemical Detectors

• Monitoring Protein Concentration

  • Typically by ultraviolet light absorption at a wavelength of 280nm.

Gas Chromatography (GC)

• Mechanism

  • Uses an inert gas as the mobile phase to carry vaporized substances into a column.

  • Interaction with a stationary phase affects retention time.

Gel Electrophoresis

• Applications

  • Commonly used for separating macromolecules like proteins, nucleic acids based on size and charge.

  • SDS-PAGE

    • Denatures proteins and allows separation based solely on mass.

    • Principle: Migration velocity correlates with electric field strength and protein charge inversely related to mass.

Isoelectric Focusing

• Mechanism

  • Allows separation of proteins based on their pI in a gel with a pH gradient under an electric field.

Conclusion**

• Analytical techniques such as chromatography and electrophoresis are essential in the biochemical separation of proteins and nucleic acids, providing insight into their properties and interactions.