Introduction High Performance Liquid Chromatography (HPLC) is one of the most powerful and widely used analytical techniques in modern laboratories. It is employed for the separation, identification, and quantification of chemical compounds in complex mixtures. Due to its high accuracy, sensitivity, and reproducibility, HPLC is extensively applied in pharmaceutical analysis, environmental monitoring, food analysis, and biochemical research. Principle of HPLC HPLC is based on the differential distribution of sample components between two phases: Mobile phase (liquid solvent) Stationary phase (solid adsorbent packed inside the column) When a sample is injected into the system, it is carried by the mobile phase through the column. Each compound interacts differently with the stationary phase, causing separation according to polarity, molecular size, or chemical affinity. The separated components are detected as they elute from the column. Main Components of the HPLC System 1. Solvent Reservoir Contains the mobile phase such as water, methanol, acetonitrile, or buffer solutions. 2. Pump Provides high pressure to push the mobile phase through the column at a constant flow rate. 3. Injector Introduces a precise volume of the sample into the mobile phase. 4. Column The heart of the HPLC system, packed with stationary phase (commonly C18 silica). Separation occurs inside the column. 5. Detector Detects the separated compounds. Common detectors include: UV–Vis detector Diode Array Detector (DAD) Fluorescence detector 6. Data Processing System Records chromatograms and calculates peak areas, retention times, and concentrations. Types of HPLC Normal Phase HPLC Uses polar stationary phase and non-polar mobile phase. Reverse Phase HPLC (RP-HPLC) Uses non-polar stationary phase and polar mobile phase. It is the most commonly used type. Ion-Exchange HPLC Separates ions based on charge differences. Size Exclusion Chromatography (SEC) Separates compounds based on molecular size. Applications of HPLC in Analytical Chemistry Pharmaceutical Analysis Determination of drug purity Quantification of active ingredients Stability studies Environmental Analysis Detection of pesticides and pollutants in water and soil Food Analysis Determination of additives, preservatives, and contaminants Biochemical Analysis Separation of proteins, amino acids, and nucleic acids Examples of HPLC Analysis Determination of caffeine in beverages Analysis of antibiotics in pharmaceutical formulations Detection of organic pollutants in water samples Calibration Curve Analysis A series of standard solutions with known concentrations is prepared and injected into the HPLC system. The peak area is plotted against concentration to generate a calibration curve. This curve is then used to determine the concentration of unknown samples. Advantages of HPLC High resolution and accuracy Excellent sensitivity Ability to analyze complex mixtures Applicable to non-volatile and thermally unstable compounds Disadvantages of HPLC High operational cost Requires skilled operators Large consumption of organic solvents Conclusion High Performance Liquid Chromatography (HPLC) is an essential analytical technique in modern chemistry laboratories. Its versatility, precision, and wide range of applications make it indispensable in analytical chemistry, pharmaceutical quality control, and scientific research.