High Pressure Liquid Chromatography is becoming important tool for the separation proteins and peptides, insulin purification and similar biotechnology process. Chromatography is now gaining wide acceptability in the field of biotechnology for characterization of proteins, peptides for product purification and impurities isolation. Reverse Phase, Normal Phase and Supercritical Phase chromatography technique are now very commonly used. Among NP and SFC, the Reverse phase stationary phase plays a important role in the separation of key target peptides compounds from crude. RP stationary particles are mechanically stronger and easy to handle while packing and unpacking of DAC Column. It is found that Reverse Phase is useful to separate proteins of nearly identical structure. Dynamic Axial Compression column or column packer also popularly known as DAC Column are used for pilot scale and production scale chromatography. DAC Column allow user flexibility to pack and unpack with user friendly operation, making plant scale chromatography a feasible process. DAC Column used in chromatography purification step of insulin API production, Consider an example of bovine, human and porcine, variant of insulin, very similar protein that are easily separated by reverse phase chromatography. The human insulin and bovine differs only by three amino acids achieve good separation. Porcine and human insulin differ by just one amino acid. Bovine insulin has residue alanine, a valine alanine in tis chain. Human insulin has a threonine, isoleucine residue on its chain, still a good baseline separation can be achieved on reverse phase chromatography on production scale HPLC and DAC Column. The reverse phase chromatography show high resolving capability for peptides as well while purification using DAC Column. It is the ease of operation and user-friendly advancements in the DAC Columns technology, the high resolving capability along with its economics that is helping the API manufacturer to choose chromatography for purification for the separation of proteins and peptides. The column is packed with stationary phase with particle 10u, 20 u or as per the method of application, reverse phase chromatography remains the most popular technique with DAC Column. Inside the column, The hydrophobic surface of particles adsorb the protein on its surface. The mobile phase will flow the impurities and protein out of the column, the protein desorbs from the surface and elutes out of the column. The separation will depend on the concentration of the organic solvent. Once the protein is desorbed, the further interaction of stationary phase and protein will be minimum or negligible, not affecting the separation pattern.