Introduction
Paper electrophoresis is one of the earliest forms of electrophoretic techniques.
It employs cellulose paper as the medium through which molecules migrate under an electric field.
Principle
Molecules dissolve in a buffer solution and migrate through the porous structure of the paper based on their charge and size when an electric field is applied.
Methodology
Preparation of the Paper:
Cut the filter paper strip to the desired size and soak it in a buffer solution to maintain stable pH conditions.
Sample Application:
Apply a small, precise spot of the sample at a designated point on the paper to minimize spreading and maintain resolution.
Electrophoresis Chamber Setup:
Position the paper in a chamber containing a buffer solution.
Ensure both ends of the paper strip are in contact with the buffer for proper conductivity.
Place electrodes (anode and cathode) at opposite ends of the chamber.
Application of Electric Field:
Connect the chamber to a power supply to establish an electric field across the paper.
Charged molecules in the sample migrate toward the electrode with the opposite charge based on their charge-to-mass ratio.
Detection and Analysis:
After electrophoresis, remove the paper and detect separated molecules using staining or other visualization methods.
Analyze the migration distances to identify and characterize the sample components.
Types of Paper Electrophoresis
1) High Voltage Paper Electrophoresis:
Voltage: Above 2000V.
Duration: Faster separation due to higher voltage.
Resolution: Better resolution with sharper bands.
Applications: Ideal for rapid separations and smaller molecules.
2) Low Voltage Paper Electrophoresis:
Voltage: Below 300V.
Duration: Slower separation due to lower voltage.
Resolution: Gentler process but may result in less sharp bands.
Applications: Suitable for larger molecules or sensitive samples.
Advantages
Simplicity: Easy to perform with minimal equipment.
Cost-Effective: Inexpensive materials and setup.
Portability: Suitable for field applications and educational purposes.
Visualization: Clear band formation facilitates qualitative analysis.
Versatility: Can separate a variety of small to moderately sized molecules.
Disadvantages
Low Resolution: Limited ability to separate complex or closely related molecules.
Quantitative Limitations: Primarily qualitative with limited accuracy for quantification.
Reproducibility Issues: Variability due to manual handling and environmental factors.
Sensitivity: Lower sensitivity compared to more advanced electrophoretic techniques.
Sample Capacity: Limited sample loading capacity, restricting the analysis of large quantities.
Applications of Paper Electrophoresis
Amino Acid Separation: Differentiating amino acids based on charge and size.
Clinical Diagnostics: Analyzing blood proteins and other biomarkers.
Pharmaceutical Analysis: Quality control of drugs and active ingredients.
Forensic Science: Identification of substances in forensic samples.
Educational Demonstrations: Teaching fundamental electrophoretic principles in laboratories.