METODOLOGIE CROMATOGRAFICHE NELLO STUDIO DEI PROCESSI DI ADSORBIMENTO

In analytical chromatography, the sample we analyze is usually rather dilute and allows the development of a rather straightforward method. Due to the minute concentrations we deal with in analytical chromatography, we face a linear behavior. The retention time of the analytes and the selectivity of a given separation can be forecast by simple rules that tremendously help us to develop efficient and fast separations. However, when we increase the sample size and a finite amount of sample is introduced in a chromatographic column, we leave the shelter of linear chromatography and have to cope with more complex peak shapes and phenomena. When the amount of the sample is comparable to the adsorption capacity of the zone of the column the migrating molecules occupy, the analyte molecules compete for adsorption on the surface of the stationary phase. The molecules disturb the adsorption of other molecules, and that phenomenon is normally taken into account by nonlinear adsorption isotherms. The nonlinear adsorption isotherm arises from the fact that the equilibrium concentrations of the solute molecules in the stationary and the mobile phases are not directly proportional. The stationary phase has a finite adsorption capacity; lateral interactions may arise between molecules in the adsorbed layer, and those lead to nonlinear isotherms. If we work in the concentration range where the isotherms are nonlinear, we arrive to the field of nonlinear chromatography where thermodynamics controls the peak shapes. The retention time, selectivity, plate number, peak width, and peak shape are no longer constant but depend on the sample size and several other factors. In addition to be a fundamental piece of information to modeling and optimizing preparative separations, adsorption isotherm determination is the key to study analytestationary phase interactions. Besides they allow for the characterization in terms of adsorption energy distribution of the surface heterogeneity. If biomolecules (peptides, proteins, etc.) or molecules with biological activity (such as drugs, etc.) were chemically bound to the stationary phase one speaks in terms of bioaffinity chromatography. In these cases, adsorption isotherm measurements are a powerful tool to study molecular recognition processes between the adsorbed biomolecules and any analyte injected into the chromatographic column. During this PhD thesis, different aspects of fundamentals of adsorption processes at the liquid-solid interface have been considered. In parallel, we focused on the setup of instrumental techniques for the automatic determination of adsorption isotherms. For that which concerns the study of fundamentals of adsorption, stationary phases traditionally employed in liquid chromatography (C18) have been characterized by investigating the adsorption from binary mixtures of compounds with different chemico-physical properties. This kind of study was essentially realized by measuring excess isotherms. Through them, the preferential adsorption of one component with respect to the other constituting the mixture can be determined. These studies are important to understand the role of so-called organic modifiers in the chromatographic separation process. In fact, in reversed phase chromatography the organic modifier is usually considered as a mean to increase mobile phase analyte solubility, while its active role in the adsorption process is often underestimated. We then focused on bioaffiniy recognition studies by applying nonlinear concepts to the characterization of adsorption of peptides and dipetides on a macrocyclic antibiotic (Teicoplanin) chemically bounded to silica gel. It was demonstrated that nonlinear chromatography can be considered a valid alternative to other techniques in use for the determination of binding constants (such as ELISA, Surface Plasmon Resonance, etc.). The fundamental advantage of nonlinear studies is that they permit to distinguish between selective and non-selective interactions in the molecular recognition process, which is not achievable by other investigation techniques. Adsorption equilibria of insulin (a relatively small protein, about 5800 Da) in different experimental conditions were studied with the purpose of understanding the mechanisms responsible for the chromatographic behavior of this protein. In fact, insulin retention is characterized by a very large sensitivity to the mobile phase composition. Nonlinear adsorption data were coupled with circular dichroism and mass spectrometry measurements. Our purpose was to understand if tertiary structure modifications or agglomerate formation (dimers, trimers, etc.) could be at the origin of the observed phenomena. Besides thermodynamic aspects, kinetic phenomena are fundamental to characterize the chromatographic process. In chromatography, in particular, we refer to mass transfer kinetics, that is the ensemble of the processes involved in the transfer of solute molecules from the mobile to the stationary phase. In chromatography, these include axial dispersion (molecular and eddy diffusion), external and internal mass transfer (pore diffusion and surface diffusion), and adsorption-desorption kinetics. In this work, mass transfer phenomena on a new kind of monolithic columns, obtained through gamma-ray polymerization, were studied. The combined use of Van Deemter and kinetic plots allowed to correlate column efficiency to the length of cross-linkers used in polymerization. In addition to these fundamental studies, the other part of this work was about the set-up of instrumentation for different kinds of purposes. A pseudo-bidimensional system that allows for the deconvolution and online sampling of chromatographic peaks measured under nonlinear and gradient conditions was developed. The importance of this system is that it permits to achieve the information necessary for the determination of adsorption isotherms in an optimized and automatic manner by exploiting numerical procedures, known in literature as inverse methods. A second application was in the field of pharmaceutics. By using a system which combines size exclusion and polar reversed-phase columns, a method for the simultaneous purification and determination of iomeprol, a radiopharmaceutical analyte, in human plasma was developed and validated.