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Francesco Dondi

Publications and source records attributed to Francesco Dondi.

At least 19 recordsLinked to original sources

Chromatography as Lévy stochastic process.

The Stochastic Theory of Chromatography has been revised in light of some of the most relevant Lévy's findings in Theory of Probability, including the so-called Lévy's distance, the characteristic function and the theory of infinitesimally divisible distributions. These concepts represent the key to exploit and understand, at a molecular basis, phenomena typical of chromatographic separations under linear conditions, such as peak tailing and splitting. In particular, Lévy's distance has been used to quantify the degree of convergence of real peaks towards an ideal Gaussian shape; the characteristic function properties, introduced by Lévy to deal with the problem of the addition of independent random variables, have been employed to solve a wide variety of chromatographic models (including adsorption on heterogeneous surfaces) and to interpret mobile phase dispersion from a probabilistic point of view. Finally, Lévy's studies concerning infinitesimally divisible distributions have allowed to introduce in the stochastic description of chromatography, effects associated to dispersion in mobile phase. It has been demonstrated that, according to Lévy's canonical representation of stochastic processes, the basis of chromatography is a mobile phase Poisson Process. Represented as a Lévy's process, the microscopic-probabilistic model of chromatography permits the establishment of a connection between single-molecule properties and their statistical fluctuations and shapes of real chromatographic peaks allowing, at the same time, for the constitution of a link between different branches of physical sciences.

Chromatography↗

High-performance liquid chromatographic separation of dihydropyrimidine racemates on polysaccharide-derived chiral stationary phases.

The chromatographic behavior of a set of racemic dihydropyrimidines (DHPMs) has been evaluated on two polysaccharide-derived chiral stationary phases under normal phase conditions. One of these is coated, the other chemically immobilized. The outstanding solvent compatibility of the immobilized chiral stationary phase (CSP) permits the use of solvents such as ethyl acetate (EtOAc) that are unsuitable for coated supports, for which traditional 2-propanol:hexanes mixtures have been employed. Drastic changes in the chromatographic retention and resolution of DHPMs and, in general, in the separation performances have been observed for the two systems. From a practical point of view, EtOAc has been proven to be a better choice for the separation of this important class of compounds. By comparing molecules different in specific positions of their scaffolds, hypotheses concerning the role of individual chemical groups on retention and selectivity have been done. These effects have been quantified, in some cases, in terms of standard Gibbs energy variations. Even if no chromatographic measurements have been made under nonlinear conditions, clear indications of the potential use of immobilized chiral adsorptive media operated with EtOAc:hexanes mixtures for preparative separations of DHPMs have been evidenced.

Amylose↗

Identification and quantification of homologous series of compound in complex mixtures: autocovariance study of GC/MS chromatograms.

The paper describes a method for determining homologous classes of compounds in a multicomponent complex chromatogram obtained under programming elution conditions. The method is based on the computation of the autocovariance function of the experimental chromatogram (EACVF). The EACVF plot, if properly interpreted, can be regarded as a "class chromatogram" i.e., a virtual chromatogram formed by peaks whose positions and heights allow identification and quantification of the different homologous series, even if they are embedded in a random complex chromatogram. Theoretical models were developed to describe complex chromatograms displaying random retention pattern, ordered sequences or a combination of them. On the basis of theoretical autocovariance function, the properties of the chromatogram can be experimentally evaluated, under well-defined conditions: in particular, the two components of the chromatogram, ordered and random, can be identified. Moreover, the total number of single components (SCs) and the separated number of the SCs belonging to the random and ordered components can be determined, when the two components display the same concentration. If the mixture contains several homologous series with common frequency and different phase values, the number and identity of the different homologous series as well as the number of SCs belonging to each of them can be evaluated. Moreover, the power of the EACVF method can be magnified by applying it to the single ion monitoring (SIM) signals to selectively detect specific compound classes in order to identify the different homologous series. By this way, a full "decoding" of the complex multicomponent chromatogram is achieved. The method was validated on synthetic mixtures containing known amount of SCs belonging to homologous series of hydrocarbon, alcohols, ketones, and aromatic compounds in addition to other not structurally related SCs. The method was applied to both the total ion monitoring (TIC) and the SIM signals, to describe step by step the essence of the procedure. Moreover, the systematic use of both SIM and TIC can simplify the decoding procedure of complex chromatograms by singling out only specific compound classes or by confirming the identification of the different homologous series. The method was further applied to a sample containing unknown number of compounds and homologous series (a petroleum benzin, bp 140-160 degrees C): the results obtained were meaningful in terms of both the identified number of components and identified homologous series.

Journal Article↗

Decoding 2D-PAGE complex maps: relevance to proteomics.

This review describes two mathematical approaches useful for decoding the complex signal of 2D-PAGE maps of protein mixtures. These methods are helpful for interpreting the large amount of data of each 2D-PAGE map by extracting all the analytical information hidden therein by spot overlapping. Here the basic theory and application to 2D-PAGE maps are reviewed: the means for extracting information from the experimental data and their relevance to proteomics are discussed. One method is based on the quantitative theory of statistical model of peak overlapping (SMO) using the spot experimental data (intensity and spatial coordinates). The second method is based on the study of the 2D-autocovariance function (2D-ACVF) computed on the experimental digitised map. They are two independent methods that are able to extract equal and complementary information from the 2D-PAGE map. Both methods permit to obtain fundamental information on the sample complexity and the separation performance and to single out ordered patterns present in spot positions: the availability of two independent procedures to compute the same separation parameters is a powerful tool to estimate the reliability of the obtained results. The SMO procedure is an unique tool to quantitatively estimate the degree of spot overlapping present in the map, while the 2D-ACVF method is particularly powerful in simply singling out the presence of order in the spot position from the complexity of the whole 2D map, i.e., spot trains. The procedures were validated by extensive numerical computation on computer-generated maps describing experimental 2D-PAGE gels of protein mixtures. Their applicability to real samples was tested on reference maps obtained from literature sources. The review describes the most relevant information for proteomics: sample complexity, separation performance, overlapping extent, identification of spot trains related to post-translational modifications (PTMs).

Electrophoresis, Gel, Two-Dimensional↗

Determination of calibration function in thermal field flow fractionation under thermal field programming.

A new procedure for determining the calibration function able to relate retention and operative parameters to molecular weight of the species in thermal field flow (ThFFF) under thermal field programming (TFP) conditions is presented. The procedure involves determining the average values of retention parameters under TFP and determining a numerical function related to the temperature variations that occur during TFP. The calibration parameters are obtained by a procedure fitting the retention and operative parameters that hold true at the beginning of the TFP. The procedure is closely related to the one previously developed to calibrate the retention time axis under TFP ThFFF and, together, they constitute a full calibration procedure. Experimental validation was performed with reference to polystyrene (PS)-decalin and PS-THF systems. The calibration functions here obtained were compared to those derived by the classical procedure at constant thermal field ThFFF to obtain the calibration function at variable cold wall temperatures. Excellent agreement was found in all cases proving "universality" of the ThFFF calibration concept, i.e. it is independent of the particular system on which it was determined and can thus be extended to ThFFF operating under TFP. The new procedure is simpler than the classical one since it requires less precision in setting the instrumentation and can be obtained with fewer experiments. The potential applications for the method are discussed.

Journal Article↗

Modeling of overloaded gradient elution of nociceptin/orphanin FQ in reversed-phase liquid chromatography.

The Reversed-phase (RP) gradient elution chromatography of nociceptin/orphanin FQ (N/OFQ), a neuropeptide with many biological effects, has been modeled under linear and non-linear conditions. In order to do this, the chromatographic behavior has been studied under both linear and nonliner conditions under isocratic mode at different mobile phase compositions--ranging from 16 to 19% (v/v) acetonitrile (ACN) in aqueous trifluoracetic acid (TFA) 0.1% (v/v)-on a C-8 column. Although the range of mobile phase compositions investigated was quite narrow, the retention factor of this relatively small polypeptide (N/OFQ is a heptadecapeptide) has been found to change by more than 400%. In these conditions, gradient operation resulted thus to be the optimum approach for non-linear elution. As the available amount of N/OFQ was extremely reduced (only a few milligrams), the adsorption isotherms of the peptide, at the different mobile phase compositions examined, have been measured through the so-called inverse method (IM) on a 5 cm long column. The adsorption data at different mobile phase compositions have been fitted to several models of adsorption. The dependence of the isotherm parameters on the mobile phase composition was modeled by using the linear solvent strength (LSS) model and a generalized Langmuir isotherm that includes the mobile phase composition dependence. The overloaded gradient separation of N/OFQ has been modeled by numerically solving the equilibrium-dispersive (ED) model of chromatography under a selected gradient elution mode, on the basis of the previously determined generalized Langmuir isotherm. The agreement between theoretical calculations and experimental overloaded band profiles appeared reasonably accurate.

Chromatography, Liquid↗

Adsorption equilibria of benzodiazepines on a hybrid polymeric chiral stationary phase.

The chromatographic behavior of a series of racemic benzodiazepines was evaluated under linear and nonlinear conditions on a new hybrid polymeric (DACH-ACR) chiral stationary phase (CSP). Differently substituted benzodiazepines were employed as probes to make hypotheses concerning possible molecular interaction mechanisms originating between target compounds and active sites on the CSP. Hydrogen bonds were found to be pivotal for chromatographic retention and chiral selectivity. The competitive effect from a mobile-phase (MP) modifier able to interact with the CSP through H-bonds was investigated. The performance of the polymeric DACH-ACR CSP for preparative purposes was also evaluated. The competitive adsorption isotherms of two benzodiazepines, lorazepam and temazepam, were measured at different MP compositions through the so-called inverse method. The adsorption data were fitted with a competitive bi-Langmuir adsorption isotherm. Enantiomeric separations under nonlinear conditions were modeled by using the equilibrium dispersive (ED) model of chromatography. Theoretical overloaded band profiles (obtained by solving the system of partial differential equations described by the ED model) matched, in a significantly accurate way, the profiles experimentally measured.

Adsorption↗

Stochastic theory of size exclusion chromatography: peak shape analysis on single columns.

The stochastic theory of size exclusion chromatography (SEC) was applied to analyze the peak shape of chromatograms obtained with a wide range of polystyrene standards on various columns. The columns were packed with stationary phases of different pore sizes. The stochastic-dispersive model of SEC results in a peak shape model that fits well the symmetrical and asymmetrical peaks observed in SEC. From the peak shape parameters obtained after nonlinear parameter estimation, information can be gained regarding the fundamental characteristics of the size exclusion process. When a series of polymer standards are analyzed on one column-in a manner similar to other methods of inverse chromatography-the stationary phase can be characterized. The dependence of the ingress and the egress processes on the relative size of the macromolecule to the pore size was determined. We found that for small molecules the selectivity in SEC arises from the ingress process, while when the size of the macromolecule is comparable to that of the pore-i.e., close to the exclusion limit-the egress process will also strongly affect the selectivity.

Journal Article↗

Single-molecule observation and chromatography unified by Lévy process representation.

A renewed stochastic model of chromatography is proposed able to establish a conceptual link between the single-molecule dynamics observations in a given chromatographic system and chromatographic experiment results in the same media. The chromatographic peak is expressed through its Fourier transform as a function of the experimental sorption time distribution. The pertinent numerical procedure necessary for obtaining the chromatographic peak is described, and the numerical programming code is given. Two types of cases were considered, the first one where the sole discrete sorption time distribution is available, and the second one where the adsorption mechanism is made of a mixture of continuous and discrete sorption time distributions. The method is applied to experimental data found in the literature by determining typical chromatographic peak shapes on the basis of the experimental interface adsorption data. This renewed stochastic approach is based on the so-called Levy canonical description of stochastic processes and appears to be the most general basis for handling separation processes from a stochastic point of view.

Journal Article↗

Decoding two-dimensional polyacrylamide gel electrophoresis complex maps by autocovariance function: a simplified approach useful for proteomics.

This paper describes a mathematical approach applied for decoding the complex signal of two-dimensional polyacrylamide gel electrophoresis maps of protein mixtures. The method is helpful in extracting analytical information since separation of all the proteins present in the sample is still far from being achieved and co-migrating proteins are generally present in the same spot. The simplified method described is based on the study of the 2-D autocovariance function (2D-ACVF) computed on an experimental digitized map. The first part of the 2D-ACVF allows for the estimation of the number of proteins present in the sample (2D-ACVF computed at the origin) and of the separation performance (mean spot size). Moreover, the 2D-ACVF plot is a powerful tool in identifying order in the spot position, and singling it out from the complex separation pattern. This method was validated on synthetic maps obtained by computer simulation to describe 2-D PAGE real maps and reference maps retrieved from the SWISS-2DPAGE database. The results obtained are discussed by focusing on specific information relevant in proteomics: sample complexity, separation performance, and identification of spot trains related to post-translational modifications.

Analysis of Variance↗

Spot overlapping in two-dimensional maps: a serious problem ignored for much too long.

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.

Adrenal Gland Neoplasms↗

Validation of a human immunoturbidimetric assay to measure canine albumin in urine and cerebrospinal fluid.

The aim of this study was to validate an automated immunoturbidimetric assay used to quantify human albumin in urine and to accurately measure canine albumin concentrations in both urine and cerebrospinal fluid. The partial homology existing between human and canine albumin limited the accuracy of the human assays in measuring canine albumin without method modifications. Thus, the assay was modified by calibrating the analyzer with calibrators made in the laboratory containing known concentrations of canine albumin. To prepare the set of calibrators, the albumin concentration of pooled sera of healthy dogs was assessed in 5 replicates using the BromocresolGreen assay. Pooled samples were aliquoted and serially diluted to obtain the expected concentrations of albumin (0.5, 1, 5, 13, and 30 mg/dl) for establishing the canine calibration curve. Thereafter, the performance was assessed by analyzing canine urine and CSF The modified assay accurately quantified canine albumin in both specimens, as indicated by the following. Intra- and interassay variability was 0.92% and 2.74%, respectively; recovery was 99.66% and 99.07% in urine and 105.02% in CSF No interference was detected when hemolysate and glucose were added to urine. The test was linear within the verified range (0-225 mg/dl). These results demonstrate that the modified human albumin immunoturbidimetric assay can be a useful tool in the veterinary diagnostic laboratory. It is accurate and tends itself to automatization on chemistry analyzers.

Albumins↗

Correspondence between chromatography, single-molecule dynamics, and equilibrium: a stochastic approach.

We report a detailed study concerning the correspondence between separations by chromatography, dynamic quantities coming from single-molecule measurements at the interfaces, and phase partition equilibrium by using the unifying approach of the stochastic description. The fundamental hypotheses allowing establishing the proper links between the three experimental techniques are discussed, and the full correspondence between the different quantities is determined from basic principles. The expressions of the errors on the retention factor which are intrinsically linked to the separation process, and which arise from peak splitting, peak tailing, stochastic bias, injection step, and number of the analyte molecules, are derived under general conditions and discussed in detail. Reference is made to the growing area of microsystems or nanosystems and chip technology, with numerical examples. How to determine the impact of single-molecule dynamics observations on the chromatographic peak shape of the experimentally observed sorption time distribution and, in general, of the behavior of the species at the stationary phase (surfaces, interfaces) is pointed out.

Journal Article↗

Programmed field decay thermal field flow fractionation of polymers: a calibration method.

The universal calibration procedure typical of thermal field flow fractionation (ThFFF) under constant thermal field operation was extended to thermal field programming (TFP) operation. The method requires knowledge of the following: (a) the programming function, which only depends on the thermal field decay function, (b) the physicochemical properties of the solvent, and (c) the calibration plot under varying channel cold wall temperatures (T(c)). Two field flow fractionation field programming conditions, with either a constant or a variable in time carrier flow velocity, are exploited. The method is based on determination, for each retention time position, of the average lambda retention value typical of TFP ThFFF. This parameter is then used to obtain the calibration plot (i.e., the molecular weight of the species as a function of the retention time position) by using the programming function and the calibration plot under varying T(c) values. The procedure approximation errors are also derived as a function of the programming type and solute-solvent system. To properly test the procedure, the calibration plot for the system constituted by polystyrene (PS) in cis-trans Decalin was determined, under varying conditions T(c) and thermal gradients, by using a set of monodisperse PS standards of different molecular weights (M). The procedure was first validated by simulation under two typical cases of TFP ThFFF operation. The approximation errors were found acceptable (in the worse cases, the accuracy in M prediction was 3%) and are in agreement with the theory. The procedure was then experimentally validated under varying programming decay function conditions. The reproducibility and accuracy of the M determination are both better than 2%.

Journal Article↗

Equivalence of the microscopic and macroscopic models of chromatography: stochastic-dispersive versus lumped kinetic model.

The microscopic model of chromatography is a stochastic model that consists of two fundamental processes: (i) the random migration of the molecules in the mobile phase, and (ii) the random adsorption-desorption of molecules on the stationary phase contained in a chromatographic column. The diffusion and drift of the molecules in the mobile phase is described with a simple one-dimensional random walk. The adsorption-desorption process is modeled by a Poisson process that assumes exponential sojourn times of the molecules in both the mobile and the stationary phases. The microscopic, or molecular model of chromatography studied here turns out to be identical to the macroscopic lumped kinetic model of chromatography, whose solution is well known in chromatography. A complete equivalence of the two models is established via the identical expressions they provide for the band profiles.

Chromatography↗

Decoding two-dimensional complex multicomponent separations by autocovariance function.

A new method for decoding two-dimensional (2D) multicomponent separations based on the use of the 2D Autocovariance function (2D-ACVF) has been developed. Theoretical models of single component (SC) spot distributions in 2D separations, both random and structured, are developed as the basis for a nonlinear estimation of both sample and separation system parameters from experimental 2D separations. The number of SCs, the average spot size, the spot capacity, and the saturation factor can be evaluated in the case of random SC spot patterns. The procedure was validated by extensive numerical simulation under conditions close to those usually found in GC x GC or 2D-polyacrylamid gel electrophoresis of proteins. The worse precision degree was no greater than 10% in the case of maximum spot density. This imprecision was fully accounted for, and it seems acceptable owing to the intrinsic statistical character of the estimation method. Structured multicomponent 2D separations, where SCs are linked by linear relationships, give rise to specific structured patterns in 2D-ACVF plots from which the parameters (phase and frequency) of the structured SC sequences can be evaluated: the study of 2D-ACVF makes it possible to decode multicomponent 2D separation, that is, to determine the number, relative abundance, and structural similarities of the single components. Pertinent expressions of the theoretical 2D-ACVF were derived for simple cases, and a procedure for decoding cases of structured 2D separations was developed and applied. It was shown that 2D separations containing both random and structured patterns of SC spots give rise to 2D-EACVF, which is the superimposition of the two component parts. This feature allows one, in principle, to decode the two components. The relevance of these results for Giddings sample dimensionality and separation dimensionality and their effective experimental evaluation is discussed.

Journal Article↗

Study of mechanisms of chiral discrimination of amino acids and their derivatives on a teicoplanin-based chiral stationary phase.

The behavior of a series of amino acids and some of their methyl ester hydrochloride, N-acetyl and N-tert-butyloxycarbonyl derivatives has been investigated on a teicoplanin-based chiral stationary phase by changing the chromatographic conditions, namely, the type and amount of mobile phase organic modifier and the ionic strength of the solutions. By using species with significantly different characteristics and chemical reactivity, some general conclusions regarding the chiral recognition process on this kind of stationary phase have been formulated. The importance of the carboxylic moiety for the formation of the complex between enantiomers and the aglycone basket of teicoplanin has been demonstrated via chromatography. Additionally, the increased possibility to make an hydrogen bond between the amidic hydrogen of the acetylated compounds and an amidic group on the stationary phase has been proposed to be pivotal for the stability of the complex aglycone D-enantiomer. Phenomena leading to the exclusion from the chiral stationary phase of one or both enantiomers have been rationalized by considering the ionic interactions between stationary phase, molecules to be separated and the surrounding medium and/or steric hindrance effects. The understanding of some of the observed phenomena may be important for optimizing the performance of the separation on aglycone-based media.

Acetylation↗