Introduction
Paper chromatography is an analytical technique used to separate and identify mixtures of soluble substances.
It is one of the simplest forms of chromatography, utilizing paper as the stationary phase and a solvent as the mobile phase.
Principle
The separation in paper chromatography is based on differential solubility and partitioning of components between the stationary phase (paper) and the mobile phase (solvent).
Components move at different rates depending on their affinity for the solvent versus the paper.
Methodology
Paper Selection:
Chromatographic paper (usually cellulose-based) serves as the stationary phase.
Sample Preparation:
The sample is dissolved in a suitable solvent for easy application.
Spotting the Sample:
A small, concentrated spot of the sample is applied near the paper's bottom edge (origin) using a capillary tube or micro-pipette.
Choice of Solvent (Mobile Phase):
The solvent (developing solution) is chosen based on the sample and desired separation.
Chamber Saturation:
The developing chamber is saturated with solvent vapor for consistency.
Development:
The paper is placed in the chamber with the solvent below the sample spot.
The solvent rises via capillary action, separating the sample components based on their affinities for the mobile and stationary phases.
Termination:
The paper is removed once the solvent has traveled a set distance. The solvent front is marked immediately.
Visualization:
For colorless compounds, methods include:
UV Light: Visualizes fluorescent compounds.
Staining: Reacts with compounds to produce color.
Iodine Vapors: Useful for certain organic compounds.
Analysis:
Separation is analyzed by calculating Rf values:
Rf values are compared for compound identification.
Development Techniques
Ascending (Vertical) Development: The solvent moves upwards from the bottom of the paper.
Descending (Horizontal) Development: The solvent moves downward from the top, though less common.
Two-Dimensional Development: The paper is developed first in one solvent direction and then rotated 90 degrees for a second development, enhancing separation.
Isocratic Development: Using a single solvent system throughout the development process.
Gradient Development: Gradually changing the solvent composition during development to improve separation.
Advantages
Simplicity: Requires minimal equipment and is easy to perform.
Cost-Effective: Low-cost materials and solvents.
Portability: Can be performed without sophisticated instruments.
Visualization: Easily visualized with common reagents or UV light.
Educational Use: Ideal for teaching fundamental chromatographic principles.
Disadvantages
Low Resolution: Limited ability to separate complex mixtures compared to advanced techniques.
Quantitative Analysis: Primarily qualitative with limited quantitative capabilities.
Reproducibility: Variability due to manual handling and environmental factors.
Scalability: Not suitable for preparative purposes or large sample sizes.
Sensitivity: Lower sensitivity compared to methods like TLC or HPLC, making it difficult to detect trace components.
Applications of paper Chromatography
Amino Acid Separation: Differentiating amino acids based on their charge and solubility.
Pigment Analysis: Separating pigments in plants, inks, or dyes.
Food Industry: Detecting additives, contaminants, or verifying the composition of food products.
Forensic Science: Analyzing substances such as drugs, inks, and toxins.
Pharmaceuticals: Monitoring the purity and composition of pharmaceutical compounds.