Displacement chromatography of proteins using a self-sharpening pH front formed by adsorbed buffering species as the displacer.
The preparative-scale separation of two proteins into adjoined, pure bands was accomplished using a novel, hybrid chromatography method which employs chromatofocusing using a self-sharpening pH front and displacement development. The method eliminates the use of a traditional displacer for accomplishing displacement chromatography, and was used to separate the A and B forms of beta-lactoglobulin using a strong-base anion-exchange column packing and a buffer system composed of acetic acid and either 3-(N-morpholino)propane-sulfonic acid (MOPS) or 2-(N-morpholino)ethanesulfonic acid (MES). Sample loads up to 150 mg of protein were applied to a 75 x 7.5 mm column to produce a displacement train composed of highly pure protein bands with greater than 90% recovery of protein. A discussion is given of the chromatographic behavior of proteins under concentration overloaded conditions for the case where a self-sharpening pH front formed using adsorbed buffering species is used to desorb proteins from an anion-exchange column packing.
Narahari
C R
CR
Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore 21250, USA.
Strong
J C
JC
Frey
D D
DD
D000327:Adsorption; D002845:Chromatography / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet
D007782:Lactoglobulins; D011506:Proteins