Selective separation of a target biomolecule from a highly complex biological matrix is based on the useof an affinity ligandthat has been immobilized on a stationary phase packed in a short column. The ligand can be specific so that the antibody binds to only one epitope, or a group type ligand such as nucleotide analogues or Protein A or Protein G that will capture a group of selected compounds. Thus affinity ligands may be selected to serve a specific purpose, such as when immobilized antibody is used for antigen purification, or conversely when immobilized antigen is used for antibody purification.

Affinity Chromatography on Renewable Microcolumn

Separation of mouse IgG on Protein Afrom bovine serum albuminon Sepharose microcolumn.

Sample: IgG 2g/L, BSA 4.6g/L. Injected volume 75L. Mobile phase PBS (0.14MNaCl, 3.8mM KCl, EDTA 1mM, phosphate 18mM, pH=7.4. Eluant 1M HCl, volume 5L. Sample on column flow rate 5mL/sec, elution flow rate 2L/sec. Column capacity 20mg/mL, column volume 10L. Flow cell volume 6L, optical path 3mm.

H. Erxleben and J. Ruzicka, Analyst, 469, vol. 130, (2005)