Following work presents basic principles of affinity chromatography on which this separation technique is based and its application. This sepparation approach is based on physical and chemical properties of bioactive molecules, which specifically bind to target ligand. Based on this principle, a number of affinity separations procedures have been developed. Different separational matrices and ligands are reviewed in this seminar work.. Therefore, a number of affinity techniques are described such as protein separation approaches based on methylation post-translational modifications, separation of non-coding RNA, separation of plasmid isoforms and separation based on stereochemistry. Various combinations of matices, ligands and conditions like pH value, ionic strenght, etc. that can be used during separations of molecules are making this technique applicable to structurally diverse target molecules. Therefore, this technique presents very important step in purification at accademic as well as industrial environment.