5,268 research outputs found
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
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
The combination acetominophen/tramadol in hematological daily practice
No full textThe combination acetominophen/tramadol implies the use of two molecules which are different in the pharmacological structure but share a biological similarity in kinetics. Their respective actions are directed to different components of the nociceptive afference. Hematological clinical practice daily challenges diseases in with pain may be a very important symptom, ranging from acute leukemia to the common effects of certain chemotherapy regimens. Multiple myeloma gives one of the best example of a disease in which pain can be inflammatory (the compression of a nerve root) or neuropathic (demyelinating process) in origin. The use of acetaminophen/tramadol combination in the common ward or outward activity with myeloma patients indicates how it is possible to reach a good control of chronic pain with a minimum overlapping of the toxicities related to the primary disease as well as to the specific treatment. Furthermore, acetaminophen/tramadol combination is extremely useful also in those clinical situations in which a short term pain control is needed, particularly for the optimal safety profile
Separazioni HPLC preparative su fase chirale. Un'alternativa importante per la separazione di enantiomeri
No full textCromatografia HPLC non-lineare
No full textAl fine di ottenere valori di risoluzione elevati, i cromatografisti analitici scelgono condizioni sperimentali in cui gli effetti dovuti alla non linearità siano il più possibile minimizzati. Una conoscenza accurata di questi stessi fenomeni può portare tuttavia all'ottenimento di condizioni utili per la separazione. La chiave per la comprensione della non linearità e di come essa intervenga nella separazione è rappresentata dalle isoterme di adsorbimento in molte condizioni e specialmente nel caso delle separazioni chirali; la determinazione delle isoterme di adsorbimento rappresenta uno strumento indispensabile per l'interpretazione el'ottimizzazione della separazione
Exploring the potential of zwitterionic teicoplanin-based CSPs by using macroporous superficially porous silica particles 2.7 μm 160Å and 3.4 μm 400Å
No full textDuring the last years, the research in the enantioselective Ultra High Performance Chromatography (eUHPC) is going to push the limits of high efficient and ultrafast analyses. In this work, novel Chiral Stationary Phases (CSPs) were prepared by covalently bonding the teicoplanin selector (TE_A2-2) on Halo 2.7μm 160Å and 3.4μm 400Å Superficially Porous silica Particles (SPP). An innovative bonding protocol allowed to obtain a zwitterionic teicoplanin based CSP, which was used to produce the already known UHPC-FPP-Titan-Tzwitt CSP based on 1.9 μm 120Å Fully Porous monodispersed silica Particles (FPP) and UHPC-SPP-Halo90-Tzwitt CSP 2.0 μm [1-3]. Columns with an internal diameter of 4.6 mm and different lengths (50 and 100 mm) were packed with all CSPs and characterized in terms of permeability, efficiency and thermodynamic under HILIC conditions. The kinetic performance was evaluated through the use of van Deemter curves. The UHPC-SPP-Halo160-Tzwitt 2.7 μm column exhibited extremely high efficiencies on both achiral (>323,000 theoretical plates/meter, N/m; hr: 1.14) and chiral compounds (>240,000 N/m; hr: 1.53) under HILIC conditions, attesting the high potential of this CSP from the kinetic viewpoint. An extreme efficiency was recorded by using the HPC-SPP-Halo400-Tzwitt 3.4 μm column which allowed to achieve an efficiency of 280’000 N/m on naphthalene (hr: 1.05). Furthermore, taking into account the thermodynamic viewpoint, the UHPC-SPP-Halo160-Tzwitt 2.7 μm exhibited the highest resolution power (Rs/tr,2) thanks to its enantioselectivity values because of the higher selector density on the silica matrix. In conclusion, in this study we demonstrate the potential of the use of SPP macroporous silica particles in the UHPLC chiral field opening an interesting scenario in the chiral chromatography area.
[1] O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, J. Chromatogr. A, 1427 (2016) 55–68.
[2] O.H. Ismail, M. Antonelli, A. Ciogli, C. Villani, A. Cavazzini, M. Catani, F. Gasparrini, J. Chromatogr. A, 1520 (2017) 91–102
[3] O.H. Ismail, M. Antonelli, A. Ciogli, M. De Martino, M. Catani, C. Villani, A. Cavazzini, M. Ye, D.S. Bell, F. Gasparrini, J. Chromatogr. A., 1576 (2018), 42-50
Essential Thrombocythemia (ET)
No full textDisease chronic myeloproliferative syndrome
Phenotype / cell stem origin The disease is a chronic myeloproliferative disorder originating from a mutated pluripotent stem cell capable of producing red blood cells, granulocytes and megakaryocytes. In some cases, B-lymphocyte involvement by the clonal proliferation was documented. T-lymphocytes are not involved by the malignant process and nonclonally derived granulocytes may coexist with clonal cells in patients with ET.
Epidemiology ET has an annual incidence of 1.5 to 2.4 patients /100,000. The disease incidence may show a peak around 30 years in females, with a second peak in the elderly with a 1:1 male-to-female ratio. The average age at diagnosis is 50-60 years.
Clinics The disease is diagnosed in the presence of a sustained increase of the platelet count (>600 X 109/L) over at least 1 month without an obvious explanation.
In the majority of patients the disease remains asymptomatic for many years. The disease symptoms are usually related to arterial thrombosis and, less frequently, deep venous thrombosis, which are more frequent in the untreated patient. Death may occur following major ischemic events or leukemic transformation.
Cytology The peripheral blood smear shows thrombocytosis without obvious morphologic abnormalities of the white blood cells and erythrocytes. Megathrombocytes may be seen. The bone marrow is hypercellular with enlarged megakaryocytes, which may tend to aggregate in small clusters. At diagnosis a moderate increase of reticulin fibers may be observed, whereas the presence of marked fibrosis is a diagnostic exclusion criteria.
Treatment Treatment should be considered for patients at risk of thrombosis (age > 60 years, previous ischemic events, platelet > 1500 X 109/L). Low-dose aspirin or other anti-platelet agents are used. Hydroxyurea is effective in reducing the platelet count and the incidence of thrombotic events. Interferon or anagrelide may be used in young patients.
Evolution Leukemic transformation may occur in 3-10% of the cases. Transformation into a stage indistinguishable form idiopathic myelofibrosis was documented in 5% of the cases.
Prognosis The large majority of the patients survive >10 years. No significant difference between life expectancy of ET patients and age-matched subjects was observed in a study.
Cytogenetics
Cytogenetics Morphological Less than 10% of the patients show a clonal chromosome defect at diagnosis. Recurrent abnormalities include total/partial trisomy 1q, trisomy 8 and trisomy 9, del(13q) and del(20q). Rearrangements of Chromosome 17, leading to 17p deletion can be frequently associated with Leukemic transformation.
Cytogenetics Molecular a) Fluorescence in situ hybridization (FISH) and molecular studies :
FISH may be more sensitive than conventional karyotyping for the detection of chromosome deletions
b) Janus Kinase JAK2 mutation :
A valine to phenylalanine substitution at position 617 (JAK2 V617F mutation) is present in 50-75% of the patients leading to constitutive kinase activity. Unlike polycythemia vera, mutated homozygous cells are not found in ET. In 1% of the patients a gain-of-function mutation of the thrombopoietin receptor (MPL) gene can be found, determining activation of the JAK-STAT pathwa
Linear and Nonlinear chromatography: an Introduction to preparative chromatography
No full textStarting on the original model proposed by Giddings and Eyring, the theory of chromatography is revised from a microscopic point of view. The migration of a molecule inside the chromatographic column is described as a sequence of random variables, viz. the times spent in stationary phase (adsorption) and the times spent in mobile phase (desorption). The fundamental parameters used to describe any chromatographic separation are obtained in the framework of the stochastic theory of chromatography. The effects of the surface heterogeneity and mass overloading on the peak shape are discussed. A link between linear and nonlinear chromatography is established
- …
