Chromatography is one of the most important analytical separation techniques used in pharmaceutical analysis. It is widely employed for the purification, identification, and quantitative determination of chemical substances. The principle of chromatography is based on the differential migration of components between two phases: a stationary phase and a mobile phase. Components of a mixture distribute themselves between these phases depending on their chemical nature, leading to separation.
Chromatography is essential in drug analysis, impurity profiling, stability testing, forensic science, biotechnology, food analysis, and environmental monitoring. Its sensitivity, selectivity, and versatility make it indispensable in quality control laboratories.
Terminologies Used in Chromatography
- Stationary Phase: The fixed phase (solid or liquid) on which components are adsorbed or partitioned.
- Mobile Phase: A liquid or gas that moves over the stationary phase and carries components along.
- Adsorption: Binding of molecules to a solid surface.
- Partition: Distribution of solutes between two immiscible phases.
- Elution: Process of washing solutes off the stationary phase with mobile phase.
- Retention Time (Rt): Time taken for a component to pass through the system.
- Rf Value (in planar chromatography): Ratio of distance moved by solute to distance moved by solvent.
- Resolution: Efficiency of separation between two components.
- Chromatogram: Visual output of the separation process.
Steps of Chromatographic Separation
- Sample introduction onto the stationary phase
- Mobile phase begins migration through the stationary phase
- Components interact differently based on their affinity
- Separated components travel at different speeds
- Detection and recording of separated components
Classification of Chromatography
Chromatography can be classified based on the physical state of mobile phase, mechanism of separation, or layout of the stationary phase. Common classifications include:
- Based on mechanism: Adsorption, partition, ion-exchange, affinity, size-exclusion
- Based on mobile phase: Gas chromatography, liquid chromatography
- Based on method: Column and planar chromatography
(a) Column Chromatography
Column chromatography utilizes a vertical column filled with a stationary phase such as silica or alumina. A liquid mobile phase percolates through the column, carrying components based on their adsorption affinity. This is one of the most traditional and versatile separation techniques.
Procedure
- Packing the column with stationary phase
- Loading the sample
- Adding mobile phase to begin elution
- Collection of separated fractions
- Analysis of collected eluents
Applications
- Purification of natural products
- Isolation of synthetic intermediates
- Separation of colored compounds
(b) Paper Chromatography
Paper chromatography uses filter paper as the stationary phase, with water molecules trapped inside the paper fiber acting as the partitioning medium. Components move at different rates depending on their solubility in mobile phase.
Advantages
- Simple and inexpensive
- Useful for separating amino acids and dyes
(c) Thin Layer Chromatography (TLC)
TLC uses a thin layer of adsorbent (silica, alumina) coated on a glass, plastic, or metal plate. Components rise through capillary action when the mobile phase travels upward.
Advantages
- Fast and easy visualization
- Suitable for identification and purity checks
(d) High-Performance Thin-Layer Chromatography (HPTLC)
HPTLC is an advanced form of TLC that uses high-quality plates, improved sample application, and densitometric scanning. It provides better resolution and quantitative analysis compared to conventional TLC.
(e) Gas Chromatography (GC)
GC uses an inert gas (helium, nitrogen, hydrogen) as the mobile phase and a liquid or polymer-coated column as stationary phase. Compounds must be volatile and thermally stable.
Key Features
- High resolution and sensitivity
- Used for volatile oils, solvents, and environmental pollutants
- Coupled with detectors like FID, ECD, TCD, or MS
(f) High Performance Liquid Chromatography (HPLC)
HPLC is one of the most widely used chromatographic techniques in pharmaceutical analysis. It uses high pressure to push liquid mobile phase through tightly packed columns.
Advantages
- High accuracy and reproducibility
- Suitable for heat-sensitive drugs
- Compatible with UV, PDA, RI, and MS detectors
Applications
- Assay of drugs
- Impurity profiling
- Stability testing
- Bioanalytical studies
(g) Supercritical Fluid Chromatography (SFC)
SFC uses supercritical fluids (commonly CO₂) as the mobile phase. These fluids possess both liquid-like solvating power and gas-like diffusivity, offering fast and efficient separations.
Applications
- Separation of chiral compounds
- Purification of pharmaceuticals
- Natural product analysis
(h) Electrophoresis and Capillary Electrophoresis (CE)
Electrophoresis separates charged molecules under the influence of an electric field. Capillary electrophoresis uses narrow capillaries that allow rapid, high-resolution separations with minimal sample volumes.
Uses
- Peptide and protein analysis
- Nucleic acid separation
- Pharmaceutical impurity profiling
Modes of Chromatography
- Adsorption Chromatography: Separation based on different adsorption affinities.
- Partition Chromatography: Based on distribution between two liquid phases.
- Ion-Exchange Chromatography: Based on ionic interactions.
- Size-Exclusion Chromatography: Based on molecular size and pore exclusion.
- Affinity Chromatography: Based on specific biological interactions.
Difference Between Normal Phase and Reverse Phase Chromatography
Normal Phase Chromatography
- Stationary phase: Polar (e.g., silica)
- Mobile phase: Non-polar solvents (hexane, chloroform)
- Polar compounds have higher retention
Reverse Phase Chromatography
- Stationary phase: Non-polar (C18, C8)
- Mobile phase: Polar solvents (water, methanol, acetonitrile)
- Non-polar compounds have higher retention
Choice of Method
The selection of chromatographic method depends on several factors:
- Nature of analyte (polarity, volatility, stability)
- Required sensitivity and specificity
- Availability of instruments
- Matrix complexity
- Regulatory and pharmacopeial requirements
Detailed Notes:
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