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Questions and Answers
Which type of chromatography exploits specific biological interactions for separation?
Which type of chromatography exploits specific biological interactions for separation?
Gradient elution is primarily used to increase the binding of analytes to the stationary phase.
Gradient elution is primarily used to increase the binding of analytes to the stationary phase.
False
What is one advantage of using a linear gradient in elution?
What is one advantage of using a linear gradient in elution?
It helps remove non-specific bands.
In affinity chromatography, the stationary phase is often immobilized with a ______ that specifically binds the analyte.
In affinity chromatography, the stationary phase is often immobilized with a ______ that specifically binds the analyte.
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Match the following chromatographic methods with their primary characteristics:
Match the following chromatographic methods with their primary characteristics:
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In which type of chromatography do solutes separate based on their charge?
In which type of chromatography do solutes separate based on their charge?
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What is the primary method by which gel permeation chromatography separates solutes?
What is the primary method by which gel permeation chromatography separates solutes?
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In gas chromatography, all components of the sample must be non-volatile.
In gas chromatography, all components of the sample must be non-volatile.
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The process of separating proteins by their specific binding affinity is known as __________ chromatography.
The process of separating proteins by their specific binding affinity is known as __________ chromatography.
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Match the following types of chromatography with their characteristics:
Match the following types of chromatography with their characteristics:
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Study Notes
Gradient Elution
- Elute with a linear gradient of solvent A to 60% solvent B
- Flow rate is 1mL/min
- Total volume of elution is 20 mL
- Advantage of gradient elution is to remove non-specific bands
Chromatography
- Chromatography separates component molecules (solutes) in a sample mixture, transported by a mobile phase over a stationary phase
- Interaction occurs between the solutes and the stationary phase, the solute is distributed between the stationary and mobile phase
- Different solutes move at different rates because of their varying affinity for the stationary phase with respect to the mobile phase
Chromatographic Theory
- Gas and liquid chromatography rely on different interactions
- Separation process is described by the same general theory
- Individual species are retarded by the stationary phase based on various interactions:
- surface adsorption
- relative solubility
- charge
Molecular Characteristics for Separation Techniques
- Polarity:
- Gas-liquid chromatography
- Liquid-liquid chromatography
- Liquid-solid chromatography
- Ionic:
- Ion-exchange chromatography
- Electrophoresis
- Size (mass) :
- Gel-permeation chromatography
- Dialysis
- Ultracentrifugation
- Shape:
- Affinity chromatography
Optimizing Flow Rate
- Too low a flow rate results in a longer analysis time
- Optimal flow rate considers the following:
- Small and uniformly packed particles
- Particle support and mobile phase
- Thin coating of the stationary phase
- Less viscous stationary/mobile phase
- Minimize flow rate
Chromatography Types
- Adsorption chromatography: Separation by polarity, used in Thin Layer Chromatography (TLC) or column
- Exclusion (permeation) chromatography: Separation by size, used in gel filtration
- Ion exchange chromatography: Separation by charge, two types: positive and negative
- Affinity chromatography: Utilizes an antibody or metal ion to bind the protein
- Partition chromatography:
- Liquid-liquid chromatography
- Gas-liquid chromatography
Affinity Chromatography
- Exploits the unique property of extremely specific biological interactions
- Relies on detailed knowledge of the structure and biological specificity of the analyte
- Immobilized ligand (substrate/inhibitor/co-enzyme) on the matrix can be used to purify an enzyme that binds the ligand specifically
- Elution of the enzyme can be done by excess ligand
Required Reading
- Biochromatography: Theory and Practice
- Chapter 2: Gel permeation chromatography
- Chapter 3: Ion-exchange chromatography
- Chapter 6: Affinity chromatography
- Chapter 10: Immobilized metal affinity chromatography (IMAC)
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