Francesco Dondi

Recent applications in chiral high performance liquid chromatography: A review

The most important and broadly used chiral stationary phases (CSPs) for high performance liquid chromatography (HPLC) are reviewed. After a short description of the state of the art, for each kind of CSP the most important contributions published in the last couple of years are summarized. For the sake of classification, these works have been divided into studies on enantiorecognition mechanisms, new materials, and new applications. Emphasis is given to new, emerging CSPs that seem to possess all requisites to be considered potentially successful chiral separation media in the next future.

Evaluation of the number of components in multi-component liquid chromatograms of plant extracts

Optimization of the chromatographic separation of flavonoid compounds in camomile extracts by the simplex method and the Monte Carlo method is described. Evaluation fo the number of peaks at unit resolution (Rs = 1) is used as the criterion of separation quality. The Davis-Giddings theory is applied in calculating the number of components and the results are validated by numerical simulations. Peak-purity checks for three identified compounds in commercial sample extracts are reported. Capacity factor patterns for 18 flavonoid and phenolic compounds over an extended range of methanol/aqueous buffer (pH 2.8) mobile phases are described.

Characterisation of River Po particles by sedimentation field-flow fractionation coupled to GFAAS and ICP-MS

Abstract A procedure for elemental composition determination of water-borne river particles (Po River) on both size-fractionated and unfractionated submicron particles (0.1–1 μm) by graphite furnace atomic absorption spectroscopy (GFAAS) and inductively coupled plasma-mass spectrometry (ICP-MS) is reported. Sample fractionation was performed using sedimentation field-flow fractionation (SdFFF). The distribution of relative mass vs. particle size was determined using UV detection. Fractions were collected over a narrow size range for scanning electron microscopy. With this combination of techniques the mass, elemental composition, and shape distributions can be obtained across the size spectrum of the sample. The size distributions of the major elements (Al, Fe) were determined by coupling both GFAAS and ICP-MS techniques to the SdFFF. The procedure was validated using a reference clay sample. Satisfactory agreement was found between both the GFAAS and ICP-MS aluminium signal and the UV detector signal. Some discrepancies were observed in the Fe Al ratios when comparing GFAAS and ICP-MS. Thus further investigation is in order to fully assess the role of SdFFF-ICP-MS and SdFFF-GFAAS techniques for elemental characterisation of aquatic colloids. Both GFAAS and ICP-MS signals unambiguously indicate a significantly higher Fe content in the lower size range, which is consistent with previous investigations. Trace element levels in unfractionated Po River particles, determined by both GFAAS and ICP-MS, show good agreement. The high levels of Cu, Pb, Cr and Cd found associated with the colloidal particles underlines the significance of the environmental role played by the suspended matter in rivers in both highly industrialised and intensively cultivated areas.

Separation of particulate environmental samples by SPLITT fractionation using different operating modes

Abstract Procedures for gentle preseparation of particle suspensions prior to sedimentation field flow fractionation (SdFFF) analysis based on the use of Split-flow thin cell (SPLITT) fractionation (SF), were examined for isolating size fractions from both polydisperse standard silica samples and natural river particle samples. Two modes of SPLITT cell operation (conventional SF, full feed depletion SF (FFDSF)) are discussed and their performances compared. Recycling procedures for enhancing the resolution were tested and the degree of the separation attained was checked by optical microscopy and, in the case of the natural river particle samples, by coupling SdFFF with scanning electron microscopy (SEM) of the fractionated samples. The FFDSF has the advantage that no external dilution is introduced during the binary separation, so that the total sample concentration is unaffected and the separation obtained appears to be equally good for the two methods.

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations: A statistical study of complex protein maps

A statistical approach able to extract the information contained in a two‐dimenisional polyacrylamide gel electrophoresis (2‐D PAGE) separation is here reported. The method is based on the quantitative theory of peak overlapping, a procedure previously developed by the authors and here extended to 2‐D separations. The whole map is divided into many strips in order to obtain 1‐D separations on which the statistic procedure is applied: the developed algorithms, on the basis of spot experimental data (intensity and spatial coordinates) permit to estimate the intrinsic number of components and to single out the specific order present in spot positions. The procedure was validated on computer‐simulated maps. Its applicability to real samples was tested on maps obtained from literature sources. The following important information on protein mixtures can be extracted: (i) the number of proteins can be accurately estimated, on the basis of the spatial coordinates and intensities of spots detected in the 2‐D PAGE map; (ii) the model describing distribution of interdistance between adjacent spots can be identified in both the separation dimensions; (iii) the presence of repeated interdistances in spot positions in the maps can be easily singled out: these regularities suggest specific protein modifications.

Continuous split-flow thin cell and gravitational field-flow fractionation of wheat starch particles.

The combined employment of the SPLITT (split-flow thin) cell--a relatively new system for fast, continuous binary separation--and of gravitational field-flow fractionation (GrFFF)--a fractionation technique suitable for micron particle size distribution determination--was investigated for starch separation and characterization. Emphasis is placed on the main advantages of both techniques: operating under gentle earth gravity field, low cost and ease of maintenance. The reproducibility of GrFFF is demonstrated. Both the SPLITT separation and GrFFF fractionation results were checked by optical microscopy. Application examples of typical starch fractionation experiments are reported and discussed.

Solvent selectivity effects in reversed-phase high-performance liquid chromatography of flavonoid compounds

Abstract The selectivity properties of methanol, acetonitrile and tetrahydrofuran as organic modifiers in the reversed-phase high-performance liquid chromatographic separation of flavonoid compounds was studied. Conditions for achieving separations between compound classes are described, in particular the possible separation of glycosides from aglycones in acetonitrile and tetrahydrofuran. Eleven retention contributions as Δlog k ′ are reported, and their dependence on mobile-phase composition is described. Solvent strength values and useful gradient elution conditions are given.

Statistical study of peak overlapping in multicomponent chromatograms: importance of the retention pattern

Abstract A general method is reported for evaluating the statistical degree of overlapping (SDO) in a multicomponent chromatogram, whose retention pattern is known and described by the frequency function of interdistances between subsequent single component peaks (interdistance model, IM). The content of SDO is the expected numbers of singlet, doublet, triplet, etc., peaks, as a function of the number of components, the separation performance and the IM. The proposed method is exploited in practice for three cases of IMs: gamma, normal and uniform, which are known to represent a wide variety of cases. Two different approaches are presented and discussed: analytical expression and simulation computation, according to whether the IM can be analytically integrated or not. The advantages and differences of the two approaches are considered, even in the case of negative interdistances (normal case). A real case of chromatographic separation of a multicomponent mixture, composed of polychlorinated biphenyls, is also exploited. The separation requirements for obtaining a given separation goal are investigated and the relevance of the IM type in determining the overlapping pattern is proved. The basic underlying hypothesis of the present treatment is the stationarity of the component distribution along the chromatogram. Means to check this hypothesis are also reported.

Factors affecting drug adsorption on beta zeolites

The adsorption behaviour of three commonly used drugs, namely ketoprofen, hydrochlorothiazide and atenolol, from diluted aqueous solutions on beta zeolites with different SiO2/Al2O3 ratio (i.e. 25, 38 and 360) was investigated by changing the ionic strength and the pH, before and after thermal treatment of the adsorbents. The selective adsorption of drugs was confirmed by thermogravimetry and X-ray diffraction. The adsorption capacity of beta zeolites was strongly dependent on both the solution pH and the alumina content of the adsorbent. Such a remarkable difference was interpreted as a function of the interactions between drug molecules and zeolite surface functional groups. Atenolol was readily adsorbed on the less hydrophobic zeolite, under pH conditions in which electrostatic interactions were predominant. On the other hand, ketoprofen adsorption was mainly driven by hydrophobic interactions. For undissociated molecules the adsorption capability increased with the increase of hydrophobicity.

Experimental validation of the stochastic theory of size-exclusion chromatography: Retention on single and coupled columns

SummaryThe experimental validation of the recently developed stochastic model of size-exclusion chromatography (SEC), interpreted as a random process in which macromolecules undergo a continuous exchange process between the moving zone between the packing particles and the stagnant zone inside the particles, is attempted on the basis of an extended data set of experimental chromatograms of polystyrene standards, with molar masses ranging from 0.16 to 2200 kDa, on five different columns with widely different pore sizes. Several features of experimental chromatograms of standards eluting within or outside the permeation range, such as band broadening, peak tailing and peak splitting are qualitatively interpreted at the light of the stochastic theory of SEC. Furthermore, some aspects such as the influence of sample polydispersity on peak broadening and of the exclusion effect in the moving zone (hydrodynamic chromatographic effect) on retention are discussed.A new chemometric procedure was developed for estimating theKSEC equilibrium distribution constant without a priori assumptions regarding the extraparticle column volume,V0, and the total volumeVp. This procedure allowed to obtain an unified description of the large retention data set over different columns. All retention data belonging to calibration ranges of the various columns, followed quite similar calibration plots, under the form of KSEC=(1-ρ)m, where ρ is the ratio of macromolecule size to pore size, withm values close to 3 for all columns. A similar approach was able to represent the behaviour of two connected column in series, with consistent values of the pertinent parameters, which validates the stochastic theory of SEC with two pore types of different sizes. Finally, this two-pore model and a mixed SEC-hydrodynamic chromatography model were compared to interpret retention data on single columns around the exclusion limits. The practical application of these approaches in routine analysis, e.g. for monitoring the column degradation during its use, is mentioned.