Visualizing Mass Transfer Kinetics In Ion Exchange Displacement Chromatography By Confocal Laser Scanning Microscopy

Compared with elution chromatography, displacement chromatography (DC)shows higher capacity, and better resolution. Due to the complexity of mechanisms ofDC, the application of DC in the purification of proteins is limited. Up to now, themechanism and protein displacement behavior have been investigated in the columnscale. Howver, few studies have been reported on the kinetics of the displacementprocess in microscopic scale.CLSM was introduced to visualize particle-scale binary component proteindisplacement behavior in Q Sepharose HP. To this end, displacement chromatographyof two intrinsic fluorescent proteins, enhanced green fluorescent protein (eGFP) andred fluorescent protein (RFP), were successfully developed using sodium saccharin(NaSac) as a displacer. The results indicated that both the proteins could be effectivelydisplaced in the single-component experiments by50mmol/L NaSac at120and140mmol/L NaCl whereas a fully developed displacement train with eGFP and RFP wasonly observed at120mmol/L NaCl in binary component displacement. At140mmol/L NaCl, there was a serious overlapping of the zones of the two proteins,indicating the importance of induced-salt effect on the formation of an isotachicdisplacement train.CLSM provided particle-scale evidence that induced-salt effect occurredlikewise in the interior of an adsorbent and was synchronous to the introduction of thedisplacer. CLSM results at140mmol/L NaCl also demonstrated that both the proteinshad the same fading rate at50mmol/L NaSac in the initial stage, suggesting the samedisplacement ability of NaSac to both the proteins. In the final stage, the fading rate ofRFP in the adsorbent became slow, particularly at lower displacer concentrations. Inthe binary component displacement, the two proteins exhibited distinct fading rates ascompared to the single component displacement. It suggested that the co-adsorbedproteins had significant influence on the formation of an isotachic train and thedisplacement chromatography of the proteins.