Chromatography is a powerful analytical technique used to separate and analyze complex mixtures. This post will provide an overview of some common chromatography methods, their principles, steps, uses and examples.
What is Chromatography?
Chromatography is a laboratory technique in which a mixture is separated into its individual components. It relies on the differential distribution of the sample components between a moving fluid mobile phase and a stationary phase to achieve separation.
Chromatography Definition:
Chromatography is a laboratory technique for the separation of a mixture into its constituent parts.
Stationary Phase: The stationary phase is the immobile phase fixed in place in a chromatography column. It can be a solid or liquid.
Mobile Phase: The mobile phase is the solvent that moves through the chromatography column carrying the sample.
1. Affinity Chromatography
- Principle: Based on specific biological interactions between antibody and antigen, enzyme and substrate, etc.
- Steps: The sample is applied, target binds to stationary phase, impurities are washed away, target is eluted.
- Uses: Purification of biomolecules like proteins.
- Example: Purification of IgG antibodies using Protein A affinity column.
2. Anion Exchange Chromatography
- Principle: Based on interaction between positively charged stationary phase and negatively charged sample ions.
- Steps: The sample is applied, anions bind to positively charged sites, impurities are washed away, anions are eluted by increasing salt concentration or changing pH.
- Uses: Separation of anions and polar molecules.
- Example: Separation of proteins, nucleic acids, carbohydrates.
3. Cation Exchange Chromatography
- Principle: Based on interaction between negatively charged stationary phase and positively charged sample ions.
- Steps: The sample is applied, cations bind to negatively charged sites, impurities are washed away, cations are eluted by increasing salt concentration or changing pH.
- Uses: Separation of cations and polar molecules.
- Example: Separation of proteins, peptides, amines.
4. Column Chromatography
- Principle: Based on differential partitioning between stationary and mobile phase.
- Steps: The sample is applied, components separate as they travel down the column at different rates, fractions are collected.
- Uses: Analytical and preparative separation and purification of chemicals.
- Examples: Separation of plant pigments, lipids, drugs, etc.
5. Flash Chromatography
- Principle: A faster version of column chromatography by using pressurized gas to push the mobile phase through a short column.
- Steps: Sample is loaded, pressure pushes mobile phase, components separate quickly, fractions are collected.
- Uses: Quick analytical and preparative separation of organic compounds.
6. Gas Chromatography
- Principle: Based on partitioning between mobile gaseous phase and stationary liquid or solid phase.
- Steps: Sample is vaporized, carried by inert gas through the column, separates based on affinity for stationary phase, detected.
- Uses: Separate and analyze volatile mixtures.
- Examples: Analyze essential oils, detect air pollutants, etc.
7. Gel Filtration Chromatography
- Principle: Based on size-exclusion separation technique. Larger molecules cannot enter pores and elute first.
- Steps: Sample loaded, molecules separate based on size as they pass through column, smaller molecules elute later.
- Uses: Separate proteins and other biomolecules based on size.
- Example: Fractionate proteins and estimate their molecular weight.
8. High Performance Liquid Chromatography (HPLC)
- Principle: Improved column chromatography with optimized stationary phase, high pressure delivery of mobile phase, sensitive detectors.
- Steps: Sample injected, carried by mobile phase at high pressure through column, separates based on affinity, detected.
- Uses: Qualitative and quantitative analysis of compounds.
- Example: Analyze pharmaceuticals, foods, biomarkers, etc.
9. Hydrophobic Interaction Chromatography
- Principle: Based on interaction between hydrophobic sample and hydrophobic stationary phase.
- Steps: Sample applied in high salt buffer, hydrophobic molecules bind, changing to low salt buffer elutes sample.
- Uses: Separate proteins and biomolecules based on hydrophobicity.
- Example: Purify monoclonal antibodies, hormones, enzymes etc.
10. Ion Exchange Chromatography
- See Anion and Cation exchange chromatography.
11. Liquid Chromatography
- Principle: Separation based on differential partitioning between liquid mobile phase and solid or liquid stationary phase.
- Steps: Sample injected, carried through column by mobile phase, separates based on affinity for stationary phase.
- Uses: Separate and analyze non-volatile mixtures.
- Examples: Amino acid analysis, purification of drugs, vitamins, proteins etc.
12. Paper Chromatography
- Principle: Based on partition between water held in cellulose paper (stationary phase) and mobile solvent phase.
- Steps: Spot sample on paper, place in solvent, components separate as solvent moves up paper.
- Uses: Separation and identification of amino acids, carbohydrates, etc.
- Example: Identify amino acids in protein hydrolysate.
13. Reverse Phase Chromatography
- Principle: Based on hydrophobic interactions with a non-polar stationary phase and polar mobile phase.
- Steps: Polar sample injected, interacts weakly with non-polar stationary phase, elutes quickly. Less polar compounds elute more slowly.
- Uses: Commonly used HPLC method for separation of organic compounds.
- Example: Separate lipids, steroids, vitamins, etc.
14. Thin Layer Chromatography (TLC)
- Principle: Based on partition between a thin stationary phase immobilized on a plate and a mobile phase.
- Steps: Spot sample on plate, place in solvent tank, components separate as solvent moves up plate.
- Uses: Analytical separation and identification of organic and biomolecules.
References:
- Skoog, Holler and Crouch. Principles of Instrumental Analysis.
- Wilson and Walker. Principles and Techniques of Biochemistry and Molecular Biology.
- Mohrig et al. Techniques in Organic Chemistry.
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