7,324 research outputs found
METODOLOGIE CROMATOGRAFICHE NELLO STUDIO DEI PROCESSI DI ADSORBIMENTO
No full textIn 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
Introduction to "Numerical determination of the competitive isotherm of enantiomers" by A. Felinger, A. Cavazzini, G. Guiochon [J. Chromatogr. A 986 (2003) 207-225]
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Theoretical and practical aspects in the separation of large biomolecules
No full textForeword to the special issue of Journal of Chromatography A dedicated to: Theoretical and practical aspects in the separation of large biomolecule
Metodo strumentale per l'analisi e la deconvoluzione on‐line di picchi cromatografici multicomponente in condizioni di overloading.
No full textUn sistema cromatografico bidimensionale, non ortogonale, è stato sviluppato per l'analisi on-line e la deconvoluzione di bande cromatografiche multicomponente in condizioni di overloading. Il set-up strumentale prevede l'uso di due pompe binarie, due colonne cromatografiche impaccate con fasi stazionarie simili, interfacciate tramite una valvola a due posizioni per il campionamento on-line, e un detector UV-Vis. Nella prima dimensione i componenti sono iniettati nel sistema in condizioni di overloading; ciò che viene eluito da questa prima colonna viene regolarmente campionato e iniettato nella seconda colonna in condizioni analitiche tramite la valvola multivie.
Questo approccio è stato validato sia in condizioni di eluizione isocratica, sia in gradiente. Esso rende agevolmente possibile, tramite una fase di calibrazione, la conversione on-line dell'intensità dei picchi in overloading da unità di assorbanza ad unità di concentrazione e la loro deconvoluzione nei singoli componenti [1, 2].
L'approccio proposto consente di analizzare in maniera automatica e veloce, sistemi multicomponente in condizioni nonlineari, laddove non sia possibile applicare comuni metodi per il modeling di separazioni cromatografiche (come ad esempio il metodo inverso). L'ottimizzazione di importanti parametri strumentali, come il tempo di campionamento tra la prima e la seconda dimensione in funzione della ritenzione nella seconda dimensione, consente una ricostruzione efficace dei profili di concentrazione dei singoli componenti.
Bibliografia
[1] A. Cavazzini, V. Costa, G. Nadalini, and F. Dondi. Instrumental method for automated on-line fraction analysis and peak deconvolution in multicomponentoverloaded high-performance liquid chromatography. Journal of Chromatography A, 1137 (2006) 36–41.
[2] V. Costa, L. Pasti, N. Marchetti, F. Dondi, A. Cavazzini. Automated instrumental method for on-line fraction analysis and peak deconvolution in gradient multicomponent overloaded high performance liquid chromatography. Journal of Chromatography A, 1217 (2010) 4919–4924
Determination of equilibrium isotherm data: A fundamental piece of information to model preparative chromatography
No full textDeterminazione HPLC iperforina di oli di diversa origine
No full textDeterminazione dell'iperforina in olii di diversa natura (forniti dalla ditta Rimos
Separazione E Purificazione Di Composti Enantiomerici Mediante Hplc Preparativa-Fondo Giovani Ricercatori 2001
No full textMetodologie avanzate di purificazione di enantiomeri. Determinazione delle isoterme di adsorbimento competitivo. Ottimizzazione della separazione in condizioni nonlinear
Sviluppo di un metodo di analisi tramite HPLC del contenuto di luteina e zeaxantina nel latte materno
No full textSviluppo di metodo HPLC per la determinazione di luetina e zeaxantina in campioni di latte in collaborazione con la Ditta Ditta NEOOX Divisione SOOFT Italia S.p.
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