Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on macroporous anion-exchange resin.
We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a macroporous anion-exchange resin of 10 microm in particle diameter. We studied effects of flow-rate, gradient time (steepness of salt concentration gradient) and column length on resolution in wide ranges. It was found that very high resolutions are attainable at long gradient times with long columns. The resolution continuously became higher as the gradient time and the column length became longer except in some special cases. The dependence of resolution on gradient time was particularly great when the column was long and the gradient time for the change of 0-0.5 M NaCl was longer than 2 h. On the other hand, the effect of flow-rate on resolution was very small. Although the separations at long gradient times with long columns have not been popular in high-performance IEC and it takes several hours for one separation, such separations should be advantageous when very high resolutions are required like in proteomics research.
Kato
Yoshio
Y
Nanyo Research Laboratory, Tosoh Corporation, Kaisei-cho 4560, Shinnanyo, Yamaguchi 746-8501, Japan. katoh_y@tosoh.co.jp
Nakamura
Koji
K
Kitamura
Takashi
T
Tsuda
Teruhiko
T
Hasegawa
Masazumi
M
Sasaki
Hiroo
H
D000837:Anion Exchange Resins; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D010316:Particle Size; D016062:Porosity; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D014361:Trypsin Inhibitors / Q000737:chemistry
D000837:Anion Exchange Resins; D011506:Proteins; D014361:Trypsin Inhibitors