C01-centrifugation

Centrifugation Overview

  • Basic Principle: Centrifugation is used to separate components in a mixture based on density, with high-speed spinning creating centrifugal force.

  • Types of Rotors:

    • Fixed Angle Rotor: Holds tubes at a fixed angle, efficient for pelleting.

    • Vertical Rotor: Tubes remain vertical; suitable for certain applications.

    • Swing-out Rotor: Tubes swing out horizontally during operation, allowing sedimentation of larger particles.

    • Zonal Rotors: Used for continuous separation of particles based on density gradients.

Types of Centrifugation

  • Preparative Centrifugation: Separates components for further analysis.

  • Density Gradient Centrifugation: Uses a gradient medium to separate based on density.

  • Density Gradient Preparations: Common media include sucrose and cesium chloride.

  • Analytical Ultracentrifuges: High-speed devices for detailed analysis of biomolecules.

Bioanalytical Techniques

Spectroscopy

  • Basic Concepts: Study of interaction between matter and electromagnetic radiation.

  • Beer-Lambert Law: Relates absorbance to concentration and path length.

  • Visible & UV Spectroscopy: Measures absorbance in visible and ultraviolet ranges.

  • Fluorescence Spectroscopy: Studies emitted light from fluorescent compounds.

  • Atomic Absorption Spectrophotometer: Detects the concentration of elements.

  • Infrared & FT-IR Spectroscopy: Analyzes molecular vibrations and bonds.

  • Mass Spectroscopy: Identifies compounds based on mass-to-charge ratio.

  • Applications of Radioisotopes: Uses in tracing and imaging in biological research.

Chromatography

  • Basic Principle: Separation technique based on distribution between stationary and mobile phases.

  • Modes & Types:

    • Paper Chromatography: Used for separating mixtures on a paper substrate.

    • Thin Layer Chromatography (TLC): Similar to paper, uses a thin layer of adsorbent.

    • Column Chromatography: Separates compounds through a column filled with stationary phase.

    • Gel Permeation: Separates based on size (molecular weight).

    • Ion Exchange: Based on charge affinities of molecules.

    • Affinity Chromatography: Selective separation based on specific interactions.

    • Gas-Liquid Chromatography (GLC): Uses gas as mobile phase for volatile compounds.

    • High-Performance Liquid Chromatography (HPLC): High resolution liquid phase separation.

Electrophoresis

  • Principle: Movement of charged particles in an electric field.

  • Types:

    • Agarose Gels: Common for nucleic acids.

    • SDS-PAGE: Denaturing polyacrylamide gel for proteins.

    • PFGE: Pulsed-field gel electrophoresis for large DNA fragments.

    • 2-D Gel Electrophoresis: Combines isoelectric focusing and SDS-PAGE for protein separation.

Cell Disruption Techniques

  • Importance: Critical process for releasing biomolecules like DNA, RNA, proteins.

  • Mechanical Methods:

    • Mortar & Pestle: Simple grinding for small samples.

    • Sonication: Uses ultrasound to disrupt membranes.

    • Bead Beating: Beads agitate samples for effective disruption.

    • Homogenizers: Mix samples mechanically to break cells.

  • Non-Mechanical Methods:

    • Chemical Disruption: Use of solvents (alcohols, ethers) to disrupt cell walls.

    • Enzymatic Methods: Enzymes like lysozyme selectively degrade cell walls.

Centrifuge Mechanics

  • Centrifuge Function: Spins samples to create centrifugal force, separating components.

  • Components: Includes rotors, tubes, and chambers for sample containment.

  • Applications:

    • Isolating Cell Organelles: Nuclei, mitochondria, and viruses.

    • Isolating Nucleic Acids: In DNA/RNA extraction processes.

    • Removing Impurities: In protein purification methods.