Chromatographic & Electrophoretic Methods
Key Points
- Chromatography and electrophoresis are two main separation techniques.
- Chromatography separates substances based on their physical and chemical properties.
- Electrophoresis separates substances based on their charge.
- Chromatography is used in a wide variety of applications, including drug discovery, environmental analysis, and food safety.
- Electrophoresis is used in a wide variety of applications, including DNA fingerprinting and protein analysis.
Main Concepts
ChromatographyChromatography is a separation technique that separates substances based on their physical and chemical properties. The most common type of chromatography is HPLC (high-performance liquid chromatography). In HPLC, a liquid sample is injected into a column that is packed with a stationary phase. The stationary phase is typically made of a porous material, such as silica gel or alumina. The sample components are separated as they pass through the column. The components that are most strongly attracted to the stationary phase will move through the column more slowly than the components that are less strongly attracted to the stationary phase.
ElectrophoresisElectrophoresis is a separation technique that separates substances based on their charge. The most common type of electrophoresis is gel electrophoresis. In gel electrophoresis, a sample is placed in a gel that is made of a polymer, such as agarose or polyacrylamide. The gel is then placed in an electric field. The components of the sample move through the gel at different rates, depending on their charge. The components that are most strongly charged will move through the gel more quickly than the components that are less strongly charged.
Applications of Chromatography and ElectrophoresisChromatography and electrophoresis are used in a wide variety of applications. Some of the most common applications include:
- Drug discovery
- Environmental analysis
- Food safety
- DNA fingerprinting
- Protein analysis
Chromatographic & Electrophoretic Methods Experiment
Chromatography
Materials:
Paper chromatography paper Glass beaker
Mobile phase (e.g., water, ethanol) Stationary phase (e.g., sand, activated carbon)
* Sample containing mixture of compounds
Procedure:
1. Draw a line near the bottom of the chromatography paper.
2. Spot the sample onto the line.
3. Dip the bottom of the paper into the mobile phase.
4. Allow the mobile phase to slowly rise up the paper.
5. Remove the paper when the mobile phase reaches the top.
6. Observe the separation of compounds based on their relative affinity for the mobile and stationary phases.
Electrophoresis
Materials:
Electrophoresis apparatus Agarose gel
Loading buffer Gel electrophoresis buffer
DNA samples DNA size markers
Procedure:
1. Prepare an agarose gel by mixing agar powder with electrophoresis buffer.
2. Pour the gel into the electrophoresis apparatus and allow it to solidify.
3. Mix the DNA samples with loading buffer.
4. Load the samples into the wells in the gel.
5. Apply an electric current across the gel.
6. Allow the DNA molecules to migrate through the gel based on their size and charge.
7. Stain the gel with a fluorescent dye to visualize the DNA bands.
Significance
Chromatography: Used to separate and analyze mixtures of compounds based on their different physical and chemical properties. It has applications in many fields, including analytical chemistry, biochemistry, and forensic science. Electrophoresis: Used to separate and analyze DNA molecules based on their size and charge. It is a fundamental technique in molecular biology and biotechnology, and is used for purposes such as DNA fingerprinting, genetic diagnosis, and gene cloning.