In modern pharmaceutical sciences, the ability to separate, identify, and analyze complex mixtures is essential for drug development, quality control, and research. Chromatography and electrophoresis are among the most powerful analytical techniques used to achieve this goal. Unit 3 introduces the fundamental principles, methodologies, advantages, limitations, and applications of column chromatography, thin layer chromatography, paper chromatography, and electrophoresis. This article presents these techniques in a clear, news-style educational format suited for pharmacy students.
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Introduction to Chromatography: The Science of Separation
Basic Principle of Chromatography
Chromatography is a separation technique based on the differential distribution of components between two phases: a stationary phase and a mobile phase. When a mixture is carried by the mobile phase over the stationary phase, different components migrate at different rates depending on their affinity for each phase. This difference in migration forms the basis of separation.
Importance in Pharmaceutical Analysis
Chromatographic methods are widely used to separate drug substances, identify impurities, estimate purity, and analyze formulations. Their flexibility and precision make them indispensable in pharmaceutical research and industry.
Adsorption and Partition Column Chromatography
Adsorption Column Chromatography
In adsorption chromatography, the stationary phase is a solid adsorbent such as silica gel or alumina, while the mobile phase is a liquid solvent. Separation occurs because components adsorb onto the solid surface with varying strengths.
Methodology:
A column is packed with adsorbent, and the sample mixture is applied at the top. As the solvent flows downward, components with weaker adsorption move faster and elute first, while strongly adsorbed compounds move slowly.
Advantages and Disadvantages:
Adsorption chromatography is simple and effective for separating non-polar to moderately polar compounds. However, it may cause irreversible adsorption of sensitive substances and requires careful solvent selection.
Applications:
It is commonly used for purification of alkaloids, glycosides, steroids, and synthetic intermediates.
Partition Column Chromatography
Partition chromatography uses a liquid stationary phase immobilized on a solid support, with another immiscible liquid acting as the mobile phase. Separation depends on the partition coefficient of components between the two liquids.
Methodology:
Compounds distribute themselves between stationary and mobile phases based on solubility differences, resulting in separation as the mobile phase flows through the column.
Advantages and Disadvantages:
This method offers better reproducibility and gentler separation for delicate compounds, but preparation of the stationary phase can be complex.
Applications:
Partition chromatography is useful for separating polar compounds such as antibiotics, vitamins, and amino acids.
Thin Layer Chromatography (TLC): A Rapid Analytical Tool
Principle of TLC
Thin layer chromatography is based on adsorption or partition principles. The stationary phase is a thin layer of adsorbent coated on a glass, plastic, or aluminum plate, while the mobile phase moves upward by capillary action.
Methodology and Rf Value
A small sample spot is applied near the base of the plate, which is then placed in a developing chamber containing solvent. After development, separated spots are visualized using UV light or chemical reagents.
The Rf value (retardation factor) is calculated as the ratio of the distance traveled by the compound to the distance traveled by the solvent front and serves as a characteristic identification parameter.
Advantages, Disadvantages, and Applications
TLC is quick, economical, and requires minimal sample. However, it provides limited quantitative accuracy compared to advanced techniques.
It is widely used for identification of drugs, detection of impurities, monitoring reactions, and fingerprinting of herbal formulations.
Paper Chromatography: A Classical Separation Technique
Principle and Methodology
Paper chromatography employs cellulose paper as the stationary phase, with water bound to cellulose fibers acting as the partition medium. The mobile phase is an organic solvent or solvent mixture.
Samples are applied on the paper, which is then developed using techniques such as ascending, descending, or radial development. Separation occurs due to differential partitioning between stationary and mobile phases.
Advantages and Disadvantages
Paper chromatography is simple and inexpensive, making it suitable for educational and preliminary studies. However, it has lower resolution, longer development time, and limited sensitivity.
Applications
It is commonly used for separation of amino acids, sugars, alkaloids, and inorganic ions in basic analytical work.
Electrophoresis: Separation Based on Electrical Charge
Introduction to Electrophoresis
Electrophoresis is a technique used to separate charged particles under the influence of an electric field. Molecules migrate toward the oppositely charged electrode, and their movement depends on charge, size, and shape.
Factors Affecting Electrophoretic Mobility
Key factors include pH of the buffer, electric field strength, molecular size, charge, and nature of the supporting medium. Proper control of these factors ensures accurate separation.
Techniques of Electrophoresis
Paper and Gel Electrophoresis
Paper electrophoresis uses filter paper as support, while gel electrophoresis employs agarose or polyacrylamide gels. Gels provide better resolution and are widely used for proteins and nucleic acids.
Capillary Electrophoresis
Capillary electrophoresis uses narrow capillaries and high voltage, offering high efficiency, speed, and automation. It requires very small sample volumes and provides excellent resolution.
Applications
Electrophoresis is extensively used for separation of proteins, enzymes, nucleic acids, peptides, and pharmaceutical biomolecules. It plays a crucial role in biotechnology, clinical diagnostics, and quality control.