Reversible conversion of octadecyl-bonded silica to ion-exchange surfaces for protein separations.
Octadecyl-bonded silica, commonly used for reverse-phase high-pressure liquid chromatography, was modified using surfactants bearing ionizable groups and the modified packing used in ion-exchange chromatography of proteins. The surfactants 2-(n-hexadecylheptaethoxy)acetic acid, 1-(n-hexadecyloctaethoxy)ethylene-diamine, and N-(n-hexadecyloctaethoxy)pyridinium were adsorbed onto test columns packed with octadecyl-bonded silica particles. The proteins lysozyme, bovine serum albumin, trypsin, horse serum cholinesterase, and bovine liver carboxylesterase were used to study the ion-exchange characteristics of the modified packings. The retention order of the proteins on the surfactant-modified stationary phases were as predicted by the isoelectric point of each protein. In addition, the interaction of enzymes with the packings did not result in significant loss of enzymatic activity. Surfactant removal was possible with the use of organic solvents and this allowed the octadecyl-bonded surface to be used again in the reverse-phase mode. During the course of the experiments, no degradation in the packing's performance was observed due to loss of adsorbed surfactant, even after over 85,000 column volumes of sodium chloride and Tris-HCl buffers were circulated through the column.
Keller
D E
DE
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905.
Torres
J L
JL
Carbonell
R G
RG
Kilpatrick
P K
PK
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D002417:Cattle; D002802:Cholinesterases / Q000097:blood; D002852:Chromatography, Ion Exchange / Q000379:methods; D006736:Horses; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology; D009113:Muramidase / Q000032:analysis; D011506:Proteins / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000032:analysis; D012822:Silicon Dioxide; D013501:Surface-Active Agents / Q000138:chemical synthesis; D014357:Trypsin / Q000032:analysis
D007202:Indicators and Reagents; D011506:Proteins; D013501:Surface-Active Agents; C026679:octadecylsilica; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide; D002265:Carboxylic Ester Hydrolases; D002802:Cholinesterases; D009113:Muramidase; D014357:Trypsin