Influence of surface modification on protein retention in ion-exchange chromatography. Evaluation using different retention models.
A large number of different stationary phases for ion-exchange chromatography (IEC) from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (alpha-lactalbumin, beta-lactoglobulin A, bovine serum albumin and alcohol dehydrogenase) were determined for three anion-exchange adsorbents based on synthetic copolymer beads with differences in the functional group chemistry. Fractogel EMD DEAE and Fractoprep DEAE consist of functional groups bound to the surface via "tentacles", ToyopearlDEAE by a short linker. Three models which describe chromatographic retention were used to analyse the characteristic parameters of the protein/stationary-phase interactions. The number of electrostatic interaction between the stationary phase and the model proteins, the protein specific surface charge densities and the interacting surface of the proteins with the adsorptive layer of the chromatographic media depend on the surface modification as well as on the molecular mass of the model proteins. In general, protein retention of the model proteins on the weak anion exchangers was found to be greater if the stationary phase carries tentacles and protein mass is above 60 kDa.
Bruch
Thomas
T
Institut für Biochemie, Hochschule Mannheim, University of Applied Sciences, Paul-Wittsack-Strasse 10, 68163 Mannheim, Germany.
Graalfs
Heiner
H
Jacob
Lothar
L
Frech
Christian
C
D000327:Adsorption; D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000465:Algorithms; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004983:Ethanolamines / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007475:Ion Exchange Resins / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry; D007782:Lactoglobulins / Q000737:chemistry; D016014:Linear Models; D008956:Models, Chemical; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013499:Surface Properties
D004983:Ethanolamines; D007475:Ion Exchange Resins; D007782:Lactoglobulins; D011108:Polymers; D011506:Proteins; D012710:Serum Albumin, Bovine; D007768:Lactalbumin; D000426:Alcohol Dehydrogenase