CHEM 3650 Lecture Notes - Lecture 4: Affinity Chromatography, Column Chromatography, Ionic Strength
Document Summary
Advantages: uses specific binding properties of molecules/proteins, produces very pure proteins. A change of ph or ionic strength will disrupt the ligand-protein interaction- Use this after getting partially pure protein to get pure protein. Problems: ligand doesn"t bind properly to polymer, ligand binds too strongly; can"t remove, expensive. Ion exchange of peptides a biochemist wants to separate two peptides by ion-exchange chromatography. At the ph of the mobile phase to be used on the column: Peptide a has a net charge of 3, due to the presence of more glu and asp residues than arg, Peptide b has a net charge of +1. A cation-exchange resin has negative charges and binds positively charged molecules, retarding their progress through the column. Peptide b, (+1) with its net positive charge, will interact more strongly with the cation-exchange resin than peptide a, (-3) and thus peptide a will elute first. On the anion-exchange resin, peptide b (+1) will elute first.
