Harpers

22 / CHAPTER 4RCFigure 4–1. Components of a simple liquid chromatography apparatus. R: Reservoirof mobile phase liquid, delivered either by gravity or using a pump. C: Glass orplastic column containing stationary phase. F: Fraction collector for collecting portions,called fractions, of the eluant liquid in separate test tubes.FProteins thus emerge from a gel filtration column in descendingorder of their Stokes radii.Absorption ChromatographyFor absorption chromatography, the protein mixture isapplied to a column under conditions where the proteinof interest associates with the stationary phase sotightly that its partition coefficient is essentially unity.Nonadhering molecules are first eluted and discarded.Proteins are then sequentially released by disrupting theforces that stabilize the protein-stationary phase complex,most often by using a gradient of increasing saltconcentration. The composition of the mobile phase isaltered gradually so that molecules are selectively releasedin descending order of their affinity for the stationaryphase.Ion Exchange ChromatographyIn ion exchange chromatography, proteins interact withthe stationary phase by charge-charge interactions. Proteinswith a net positive charge at a given pH adhere tobeads with negatively charged functional groups such ascarboxylates or sulfates (cation exchangers). Similarly,proteins with a net negative charge adhere to beads withpositively charged functional groups, typically tertiary orquaternary amines (anion exchangers). Proteins, whichare polyanions, compete against monovalent ions forbinding to the support—thus the term “ion exchange.”For example, proteins bind to diethylaminoethyl(DEAE) cellulose by replacing the counter-ions (generallyCl − or CH 3 COO − ) that neutralize the protonatedamine. Bound proteins are selectively displaced by graduallyraising the concentration of monovalent ions in