Chromatography is a system that helps distinct, purify, and recognize mixture components for both quantitative as well as qualitative study [1]. Proteins can be separated and purified depends on their dimensions, shape, net charge, aquaphobic structures on the outermost layer, and binding capability with the immobile phase. The exchange of ions, superficial adsorption, separation, and size exclusion are examples of molecular property and interaction-based separation techniques. Stationary bed chromatography procedures, such as paper chromatography technique, Column Chromatography technique as well as Thin Layer Chromatography technique. Column chromatography technique serves as one of the most common protein purification methods. This review comprises an insight the separation techniques such as Column Chromatography technique, Gas Chromatography technique, Paper Chromatography technique, Thin Layer Chromatography technique, Hydrophobic Interaction Chromatography technique (HIC), used for separation of biomolecules.