Muhammad Nawaz Sharif University of Agriculture, Multan
Assignment Title: High-Performance Liquid Chromatography
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INTRODUCTION
The analytical technique of High-Performance Liquid Chromatography (HPLC) is used
extensively throughout the pharmaceutical industry. It is used to provide information on
the composition of drug-related samples. It can separate, identify, and quantify the
compounds that are present in any sample that can dissolve in liquid. HPLC is the most
accurate analytical method widely used for the quantitative as well as qualitative analysis of
drug products and the determination of drug product stability. High pressure is applied to
allow the eluent to flow through the column due to the physical properties of HPLC. HPLC is
distinguished from traditional liquid chromatography by its fundamentally higher
operational pressures (50 bar to 350 bar).
DEFINITION
High-performance liquid chromatography (HPLC) is a chromatographic technique used to
separate a mixture of compounds in analytical chemistry, biochemistry, and industrial
applications. The main purposes of using HPLC are identifying, quantifying, and purifying a
mixture of individual compounds.
PRINCIPLE
HPLC works by injecting a sample into a column filled with a stationary phase (porous
material), while a mobile phase (liquid solvent) is pumped through at high pressure. The
separation process depends on different interactions between the sample components and
the stationary phase. There are different modes of separation, including adsorption
chromatography, partition chromatography, ion-exchange chromatography, and size
exclusion chromatography.
TYPES OF HPLC
1. **Normal Phase Chromatography:** Uses a polar stationary phase and a non-polar mobile
phase.
2. **Reversed-Phase Chromatography:** Uses a non-polar stationary phase and a polar
mobile phase.
3. **Size Exclusion Chromatography:** Separates molecules based on size.
4. **Ion-Exchange Chromatography:** Separates based on charge interactions.
5. **Bio-Affinity Chromatography:** Uses biological interactions for high-purity separation.
ADVANTAGES OF HPLC
- High sensitivity and accuracy
- Good repeatability
- Continuous monitoring of column effluent
- Suitable for complex mixtures
- No need to vaporize samples like in gas chromatography
DISADVANTAGES OF HPLC
- Expensive equipment and maintenance
- Complex operation
- Requires a power supply and large amounts of organic solvents
APPLICATIONS OF HPLC
**Pharmaceutical Applications:** Used in drug stability control, quality control, and active
ingredient identification.
**Food Applications:** Analysis of sugars, fats, proteins, and contaminants.
**Forensics Applications:** Detection of drugs in biological samples.
**Clinical Applications:** Monitoring glucose control in diabetics, identifying poisoning
causes, and studying drug metabolism.
CONCLUSION
HPLC is a highly efficient and reproducible technique used in various fields such as
pharmaceuticals, food science, forensics, and clinical research. Its ability to separate,
identify, and quantify compounds makes it one of the most essential analytical techniques
available today.
