Chromatography Study Notes

Introduction to Chromatography

  • Instructor: Dr. Nigel Brissett

Reading Assignments

  • Chapters Required:
    • Chapters 44 & 45 (4th Edition): Online via library
    • Chapters 50 & 51 (5th Edition): Online via library
    • Chapters 48 & 49 (6th Edition): Available as a physical book in the library

Learning Outcomes

  • LO1: Understand the theory of chromatography and apply it to:
    • Gel Filtration Chromatography
    • Ion-Exchange Chromatography
    • Affinity Chromatography
  • LO2: Explain how Thin Layer Chromatography (TLC) separates molecules and calculate the Retention factor (Rf).
  • LO3: Learn how different types of electrophoresis separate biomolecules.

Purpose of Chromatography

  • Primary Uses:
    • Remove contaminants
    • Identify molecules
    • Estimate concentration
  • Explanation:
    • Chromatography is a technique used in analytical chemistry to separate a mixture of molecules.

Etymology

  • Origin of Term:
    • Chroma: Greek word for "color"
    • Graphos: Greek word for "to draw"
  • Example: Different chlorophyll pigments in green leaves demonstrate various colors in chromatographic separation.

Common Molecules Encountered in Chromatography

  • Types of molecules often separated include:
    • Proteins
    • Amino Acids
    • Lipids
    • Nucleic Acids
    • Enzymes
    • Sugars

Defining Phases in Chromatography

  • Chromatography:
    • Process that separates molecules between a stationary phase and a mobile phase.
    • Once separated, molecules can be quantified.
    • Understand factors influencing this process.

Process of Separation

  • Sample Introduction:
    • Sample is placed into the mobile phase.
  • Movement and Partitioning:
    • As the sample moves with the mobile phase, components partition themselves between the mobile phase and the stationary phase.

Types of Chromatography

Planar Chromatography

  • Surface types:
    • Layer of paper or gel particles (e.g., silica) supported by a glass plate.
  • Examples:
    • Paper Chromatography
    • Thin Layer Chromatography (TLC)

Column Chromatography

  • Type:
    • Tightly packed bead matrix gel.
  • Examples:
    • Gel Filtration
    • Ion-Exchange
    • Affinity

Key Concepts in Chromatography

Stationary Phase

  • Definition: Fixed phase acting as an adsorbent.
  • Adsorption Process:
    • Interaction of adsorbent molecules with the surface of an adsorbent.
  • Adsorbent Material Properties:
    • Usually porous.
    • Selection criteria includes:
    • Selectivity
    • Capacity
    • Chemical and thermal stability
    • Cost

Mobile Phase

  • Definition: Acts as a carrier of solutes.
  • Composition: Can be liquid or gas.
  • Function: Carries biomolecules that have varying degrees of affinities to the stationary phase.

Phase Characteristics in Different Chromatography Methods

TypeStationary PhaseMobile Phase
Thin LayerSilica, AluminaSolvent
Gel FiltrationSepharose, Sephadex, various polyacrylamide gelsBuffers
Ion-ExchangeGels with charged resinsBuffers, salts
AffinityGels with covalently-bound ligandsBuffers, salts
High PerformanceSilica, C8, C18Buffer plus solvent
Gas-LiquidVarious greases, gums, resinsGases (e.g., helium and nitrogen)

Theoretical Principles of Chromatography

  • Separation Principle:
    • Based on the difference in solute concentration across the two phases.
    • Expressed by the Partition Coefficient.
  • Roles:
    • Stationary Phase: Acts as an adsorbent and is fixed.
    • Mobile Phase: Acts as a carrier for solutes.

Gel Filtration Chromatography (GF)

Fundamental Concepts

  • Exclusion Limit:
    • Fixed pore size in the stationary phase bead.
    • If the solute molecular weight is greater than the exclusion limit, it will elute first.
    • If the solute molecular weight is less than the exclusion limit, it will elute last.

GF Resins


  • Resin Selection: Based on the size of molecules being separated.

  • Molecular Weight Definitions:
  • 1 kilodalton (kD) = 1000 Daltons.


  • Examples of Resins and Their Exclusion Limits:

    • ResinMolecular Weight Range
    Sephadex G-503000 – 20000 Da
    Sephadex G-754000 – 50000 Da
    Superdex 753000 – 70000 Da
    Superdex 20010000 – 600000 Da

    GF – Key Theoretical Constants

    • Important Constants:
      • Void Volume (Vₒ): Mobile phase volume excluded from stationary phase.
      • Total Bed Volume (V_c): Total volume of stationary phase and mobile phase.
      • Pore Volume (Vᵢ): Volume of pores that solute can exchange into.
    • Equation for Partitioning:
      K_d = rac{ ext{Concentration of solute in stationary phase}}{ ext{Concentration of solute in mobile phase}}

    Ion-Exchange Chromatography (IEX)

    Fundamental Concept

    • Types of Ion Exchangers:
      • Anion Exchanger
      • Cation Exchanger

    IEX - Stationary Phases

    • Examples:
      • DEAE (Diethylaminoethyl-cellulose) – used for anion exchange
      • CM (Carboxymethyl) – used for cation exchange

    IEX Process Details

    • Sample and Wash Processes:
      • Hydrated positively charged beads with negative counter-ions.
      • Negative ions in solute adsorb onto the anion exchange resin.
    • Elution Process:
      • A salt gradient (e.g., by adding NaCl) increases ionic strength, causing the displacement of proteins.

    Key Facts about Ion Exchange Chromatography

    • pH Impact:
      • Acidic or basic pH alters the charge density of IEC functional groups.
    • Protein behavior:
      • Proteins with high density charge require buffers of high ionic strength for desorption.

    Affinity Chromatography (AC)

    Definition and Process

    • Matrix for Ligand Attachment:
      • Should be chemically and physically inert.
    • Binding Mechanism:
      • The ligand is covalently bound to the stationary phase, binding reversibly to a specific molecule or group of target molecules.

    AC Process Steps

    1. Equilibration:
      • Medium equilibrated in binding buffer.
    2. Sample Application/Wash:
      • Binding under specific conditions; unbound material is washed away.
    3. Elution:
      • Recover target protein by changing conditions.
    4. Re-equilibration:
      • Medium is re-equilibrated with binding buffer.

    Key Facts about Affinity Chromatography

    • Mobile Phase pH: Maintains the active form of the bound molecule.
    • Specific Applications:
      • Exemplified in HbA1c assay with phenylboronic acid as ligand for purification of monoclonal antibodies.

    Hydrophobic Interaction Chromatography (HIC)

    Mechanism

    • Water Interaction: Highly ordered water drives hydrogen bonding resulting in unique properties such as high boiling point.
    • Role of Hydrophobicity: Hydrophobic substances immersed in water modulate interaction based on water molecule activities.

    Reversed Phase Chromatography (RPC)

    Definition

    • Hydrophobic Phase Characteristics:
      • Stationary phase has more hydrophobicity than HIC leading to stronger interactions.
    • Solvent Use:
      • Reversed interactions using non-polar solvents such as methanol or acetonitrile.

    Multimodal Chromatography (MMC)

    • Characteristics of Ligands:
      • Possess mixed physical characteristics, allowing for varied mobile phase conditions.
    • Benefits:
      • Broader selectivity profile enables interactions from IEX to HIC.

    Summary of Learning Outcomes

    • Chromatography Techniques:
      • Gel Filtration Chromatography
      • Ion Exchange Chromatography
      • Affinity Chromatography
      • Hydrophobic Interaction Chromatography
      • Reversed Phase Chromatography
      • Multimodal Chromatography
    • Core Principle: All rely on intrinsic properties of biomolecules that are separated.