Electrophoresis is a powerful analytical technique used for separating charged molecules under the influence of an electric field. It is widely used in biochemistry, molecular biology, pharmaceutical analysis, and forensic science. Molecules such as proteins, nucleic acids, amino acids, and peptides migrate at different speeds depending on their charge, size, shape, and interaction with the supporting medium.
The technique is essential for analyzing biomolecules, monitoring drug purity, identifying genetic mutations, and performing clinical diagnostics. It offers high resolution, reproducibility, and flexibility across several variations, including gel electrophoresis and paper electrophoresis.
Principle of Electrophoresis
Electrophoresis operates on the principle that charged molecules migrate in an electric field toward the electrode of opposite charge. The rate of migration depends on:
- Net charge of the molecule
- Molecular size and shape
- Strength of the electric field
- Nature of the supporting medium
- Buffer pH and ionic strength
Positively charged molecules move toward the cathode, while negatively charged molecules move toward the anode. The difference in mobility forms the basis for analytical separation.
Factors Affecting Electrophoretic Mobility
1) Electric Field
The applied voltage directly influences the migration speed. A higher electric field increases mobility but excessive voltage may cause overheating, band distortion, or degradation of samples.
2) The Sample
A molecule’s charge, size, conformation, and purity significantly affect its mobility. Highly charged molecules move faster, while large or compact molecules move slower through the medium.
3) The Buffer
Buffers maintain constant pH and ionic strength, ensuring stable migration patterns. A high ionic strength buffer reduces migration speed due to increased resistance and heat generation.
4) The Supporting Medium
The medium (gel or paper) provides mechanical support and influences separation efficiency. Porosity, composition, and interaction with analytes determine separation characteristics.
Gel Electrophoresis
Gel electrophoresis is the most widely used form of electrophoresis. It uses a semi-solid gel matrix to separate molecules based on charge and size. The gel acts as a molecular sieve, allowing smaller molecules to move faster than larger ones.
Separation Based on Charge
At a constant gel concentration, molecules with greater charge migrate faster toward the oppositely charged electrode. Highly charged proteins or nucleic acids move rapidly, producing distinct, sharp bands.
Separation Based on Size
In gels, smaller molecules pass easily through pores and migrate faster, while larger molecules are slowed. This is especially important in nucleic acid and protein analysis, where differences in molecular weight guide interpretation.
Types of Gels
- Agarose Gel: Used for nucleic acids; moderate resolution; larger pore size
- Polyacrylamide Gel (PAGE): Used for proteins; high resolution; smaller pore size
- Starch Gel: Used for isoenzyme analysis; good resolving power
Gel Concentration
Gel concentration controls pore size. Higher concentration = smaller pores, suitable for small molecules. Lower concentration = larger pores, suitable for large molecules. Proper selection of gel concentration ensures optimal band separation.
Instrumentation for Gel Electrophoresis
A typical gel electrophoresis setup includes:
- Power supply: Provides stable voltage
- Gel casting tray: Molds the gel
- Combs: Create wells for sample loading
- Electrophoresis tank: Holds buffer and gel
- Electrodes: Deliver current across the gel
- Documentation system: UV/visible imaging for visualization
Modern systems are automated with temperature control and integrated imaging software.
Quantification
Quantification is performed by analyzing band intensity using densitometric methods. Peak area correlates with concentration. Digital imaging enhances accuracy and enables comparison with standards for precise quantification.
Starch Gel Electrophoresis
Starch gel electrophoresis is a specialized technique used primarily for separating isoenzymes and proteins. The gel is prepared from hydrolyzed starch, which forms a dense, stable matrix.
Advantages
- Good resolution for isoenzymes
- Stable gel structure allowing long runs
- Suitable for biological samples
Disadvantages
- Complex gel preparation
- Brittle and difficult to handle
- Longer run times compared to agarose or PAGE
Applications of Gel Electrophoresis
- DNA and RNA analysis
- Protein separation and purity testing
- Isoenzyme studies
- Forensic fingerprinting and genetic identification
- Drug and metabolite analysis
- Quality control in biotechnology
Paper Electrophoresis
Paper electrophoresis uses cellulose paper strips as the supporting medium. It is simple, cost-effective, and suitable for separating amino acids, peptides, and small ions.
Principle of Paper Electrophoresis
Charged molecules migrate across moistened paper under an electric field. Separation occurs based on the molecule’s net charge and interaction with cellulose fibers.
Components of Paper Electrophoresis
- Paper strip (Whatman No. 1 is commonly used)
- Buffer reservoir
- Power supply
- Electrophoresis chamber
- Sample applicator
Types of Paper Electrophoresis
- Zone electrophoresis
- Moving boundary electrophoresis
- Ionophoresis
Instrumentation of Paper Electrophoresis
A horizontal chamber holds the buffer, with paper strips placed between electrodes. Temperature is carefully regulated to prevent drying and maintain consistent migration.
Detection and Quantification
After electrophoresis, the separated bands are visualized using staining techniques such as ninhydrin (for amino acids), silver nitrate, or fluorescent dyes. Quantification can be performed by densitometry or by measuring spot intensities.
Applications of Paper Electrophoresis
- Separation of amino acids and peptides
- Analysis of serum proteins
- Clinical diagnostics
- Detection of impurities
Advantages
- Inexpensive and simple setup
- Low sample requirement
- Suitable for routine qualitative analysis
Disadvantages
- Low resolution compared to gel electrophoresis
- Not suitable for high molecular weight biomolecules
- Slow and less sensitive
Detailed Notes:
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