Direct observation of biomolecule adsorption and spatial distribution of functional groups in chromatographic adsorbent particles

Abstract: Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle.Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles.Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.