High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique used for the separation, identification, and quantification of components in complex mixtures. Its principle is based on the differential interactions of analyte molecules with a stationary phase and a mobile phase, allowing separation based on chemical properties such as polarity, charge, and molecular size. The stationary phase is typically a finely divided solid or a chemically bonded phase packed into a column, while the mobile phase is a liquid solvent or a mixture of solvents that carries the sample through the column. Separation occurs because different compounds in the mixture interact with the stationary phase and the mobile phase to different extents, resulting in distinct retention times for each component as they elute from the column. The fundamental principle underlying HPLC is chromatographic partitioning, which may involve adsorption, partitioning, ion exchange, or size exclusion, depending on the column type. In reversed-phase HPLC, the stationary phase is non-polar, usually a C18-silica bonded phase, and the mobile phase is relatively polar, which causes polar compounds to elute faster while non-polar compounds are retained longer. In normal-phase HPLC, the stationary phase is polar, and non-polar compounds elute faster than polar compounds. Ion-exchange HPLC relies on electrostatic interactions between charged analytes and the charged stationary phase, while size-exclusion HPLC separates molecules based on their hydrodynamic volume, with larger molecules eluting earlier as they cannot enter the pores of the stationary phase. The instrumentation of HPLC comprises several key components that work together to achieve accurate separation and detection of analytes. The primary components include the solvent reservoir, pump, injector, column, detector, and data acquisition system. The solvent reservoir holds the mobile phase, which is degassed to prevent the formation of bubbles that could interfere with the flow and detection. The pump provides a high-pressure flow of the mobile phase through the column, maintaining a steady and controlled flow rate, which is essential for reproducible retention times. Modern HPLC systems often use high-precision pumps capable of delivering pressures up to several hundred bars to overcome the resistance of densely packed columns. The injector introduces the sample into the mobile phase stream in a precise and reproducible manner. Manual injection can be performed using a loop injector, while automated systems allow for high-throughput sample introduction. The column, which is the heart of HPLC, is