High-performance liquid chromatography (HPLC) is a chromatographic technique in analytical chemistry used to separate a complex mixture of analytes, in order to identify and quantify of each component in the sample. For the identification and quantification of compounds, the HPLC technology is used. There are many applications, both in normal and reverse phase HPLC, RP-HPLC is not useful for the separation of polysaccharides, inorganic ions, and polynucleotides. Inorganic ions separated by ion exchange chromatography, whereas polysaccharides are hydrophilic in nature, so they are rapidly absorbed in the solid phase. Reverse phase HPLC can be used for the separation of different compounds such as amino acids, aromatic hydrocarbons, peptides, proteins, lipids, and sugars. High-performance liquid chromatography is also used in the medical, analysis of pharmaceuticals, forensic, food industries, clinical tests, environment, and Pharma manufacturing units.