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The rapid development of nanoproducts in pharmaceutical field highlights the need for robust analytical methods to ensure their quality and stability. Nanoparticles of different nature and composition (NPs) are employed for many purposes, such as the improvement of drug solubility/bioavailability and the controlled delivery of drugs. Among NPs features, particle size distribution (PSD) plays a fundamental role in determining NPs properties. Nevertheless the high development of different NPs, authorities such as the FDA and the European Union highlight the lack of robust characterization methods and quality control for nanomaterials. Among the techniques for the size-characterization of particles, Field-Flow Fractionation (FFF) represents a competitive choice: due to the absence of a stationary phase, the separation mechanism is gentle with total maintenance of the native properties of the analytes. In this paper the microcolumn variant of FFF, the Hollow-Fiber Flow FFF (HF5), is coupled on-line with Multi-Angle Light Scattering (MALS) for the development of methods for the characterization of NPs as quality control in new pharmaceutical field. The HF5-MALS was applied to the size c haracterization in different preservation conditions of silver polyvinylpyrrolidone-stabilized NPs (AgPVP) used for their antimicrobial activity. The ratio of gyration and hydrodynamic radii of AgPVP was evaluated for fresh and aged NPs, suggesting in this case aggregation rearrangement. The influence of different PVP coating and dilution factor was also studied. Finally the metal ion release was determined in relationship to these shape modifications. The HF5-MALS method is robust and reproducible and it can be considered as an important tool for the development of analytical platform for quality control of NPs.

Due to the increased use of silver nanoparticles in industrial scale manufacturing, consumer products and nanomedicine reliable measurements of properties such as the size, shape and distribution of these nano particles in aqueous medium is critical. These properties indeed affect both functional properties and biological impacts especially in quantifying associated risks and identifying suitable risk-mediation strategies. The feasibility of on-line coupling of a fractionation technique such as hollow-fiber flow field flow fractionation (HF5) with a light scattering technique such as MALS (multi-angle light scattering) is investigated here for this purpose. Data obtained from such a fractionation technique and its combination thereof with MALS have been compared with those from more conventional but often complementary techniques e.g. transmission electron microscopy, dynamic light scattering, atomic absorption spectroscopy, and X-ray fluorescence. The combination of fractionation and multi angle light scattering techniques have been found to offer an ideal, hyphenated methodology for a simultaneous size-separation and characterization of silver nanoparticles. The hydrodynamic radii determined by fractionation techniques can be conveniently correlated to the mean average diameters determined by multi angle light scattering and reliable information on particle morphology in aqueous dispersion has been obtained. The ability to separate silver (Ag(+)) ions from silver nanoparticles (AgNPs) via membrane filtration during size analysis is an added advantage in obtaining quantitative insights to its risk potential. Most importantly, the methodology developed in this article can potentially be extended to similar characterization of metal-based nanoparticles when studying their functional effectiveness and hazard potential.

Monoclonal antibodies (mAbs) are promising reagents both for the manufacture of drug substances and for their employment as a drug themselves, but to be approved for utilization, according to FDA recommendations and WHO guidelines, they have to undergo verifications regarding their purity, stability and percentage of aggregates. Moreover, stability tests of lots have to be performed in order to verify molecular size distribution over time and lot-to-lot consistency. Recent works in literature have highlighted the need for suitable, sensitive and reliable complementary analytical techniques for the characterization of mAbs and quantification of aggregates. Size-exclusion chromatography (SEC) is the reference technique in the biopharmaceutical industry for its robustness, high performance and simple use; however it presents some limitations especially toward the separation and detection of aggregates with high molecular weight. On the other hand, flow field-flow fractionation (F4) in its miniaturized version (hollow fiber flow field-flow fractionation, HF5) shows comparable performances with interesting additional advantages: a broad size range, gentle separation mechanism with low dilution factor and higher sensitivity. To propose HF5 as a complementary technique for evaluating aggregates' content in mAbs samples, a comparative study of both SEC and HF5 performances has been made. In this work, SEC and HF5 were coupled with UV and multi-angle light scattering detection and employed first in separating standard samples of proteins mixture used as a sample model. Then, a screening of mobile phases and an evaluation of separation performances was performed on a therapeutic mAbs formulation, demonstrating the complementarities between SEC and HF5 and their possible use as a separative platform approach for the characterization and quality control of protein drugs. © 2014 Elsevier B.V.

Reinjection of one ore more collected fractions of eluted samples is recognized as a useful procedure in analytical separation techniques, among which field-flow fractionation (FFF), to improve the actual separation of complex samples. Hollow-fiber flow FFF (HF5) is a micro-channel subset of flow FFF (F4), which has recently reached a performance comparable to that of standard, flat-channel F4. To further improve HF5 of complex protein samples, we present a new device and method for in-line, reinjection HF5 that we call tandem H F5 (HF5/HF5). HF5 is ideally suited for tandem operation because (1) small channel volume and low operation flow rates allow reducing dilution and volume of the collected fractions, and (2) the relaxation/focusing step that takes place between the 1st and 2nd run (refocusing) allows reestablishing the volume and concentration of the sample plug before the 2nd elution. HF5/HF5 proves particularly effective in the case of oligomeric proteins since it allows collecting and reinjecting the bands that correspond to each separated oligomeric form. This provides information on the dynamic equilibria between the different oligomers. For HF5/HF5 operations, a modified, prototype HF5 instrumentation is presented which includes a "trap" constituted of a four-port, two-way valve positioned downstream the UV detector and a collection loop. The effect of refocusing conditions on HF5/HF5 performance is investigated by varying refocusing time. With a complex protein samples such as blood serum, HF5/HF5 can improve detectability of the low abundance components since overloading effects due to high-abundance components are reduced. This is shown for serum lipoproteins: while after the 1st run high density lipoproteins (HDLs) are not separated from high-abundance serum proteins, after the 2nd run it is shown possible to separate the HDL subclasses. (C) 2011 Elsevier B.V. All rights reserved.

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.

Alterations of lipoproteins (LPs) and related lipid levels in blood serum are correlated to the risk of coronary artery disease (CAD). Fast, possibly automated methods to obtain complete, multi-parametric LP profiles are therefore welcome to be developed for routine, clinical analysis practice. In this work, asymmetrical flow field-flow fractionation (AF4) with on-line, dual post-fractionation reaction detection (PFRD) is applied to develop a method for single-run, simultaneous quantification of cholesterol (CHOL) and triglycerides (TGs) in each fractionated LP class. The enzymatic reagents used for the post-fractionation reaction are available as commercial kits for certified, standard clinical protocols for the analysis of CHOL and TGs in serum. Using CHOL and glycerol as reference standards, a new procedure is applied to optimize the experimental conditions for PFRD-based, quantitative analysis. Upon optimized PFRD and AF4 conditions, results obtained for the determination of total CHOL (TC), TGs, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) in a set of serum samples from healthy donors are found in agreement with the values provided by a clinical laboratory. The intra-day and inter-day precisions of the method were found always lower than 10% (CV). When the method was applied to serum samples from patients affected by sepsis, differences in CHOL and TG profiles between patients and healthy donors were observed.

Proteome analysis requires a comprehensive approach including high-performance separation methods, mass spectrometric analysis, and bioinformatics. While recent advances in mass spectrometry (MS) have led to remarkable improvements in the ability to characterize complex mixtures of biomolecules in proteomics, a proper pre-MS separation step of proteins/peptides is still required. The need of high-performance separation and/or isolation/purification techniques of proteins is increasing, due to the importance of proteins expressed at extremely low levels in proteome samples. In this review, flow field-flow fractionation (F4) is introduced as a complementary pre-analytical separation method for protein separation/isolation, which can be effectively utilized for proteomic research. F4 is a set of elution-based techniques that are capable of separating macromolecules by differences in diffusion coefficient and, therefore, in hydrodynamic size. F4 provides protein separation without surface interaction of the analyte with packing or gel media. Separation is carried out in an open channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly-applied, secondary flow of the fluid. Therefore, biological analytes such as proteins can be kept under a bio-friendly environment without losing their original structural configuration. Moreover, proteins fractionated on a size/shape basis can be readily collected for further characterization or proteomic analysis by MS using, for instance, either on-line or off-line methods based on electrospray ionization (ESI) or matrix-assisted laser desorption-ionization (MALDI). This review focuses on the advantages of F4 compared to most-assessed separation/isolation techniques for proteomics, and on selected applications based on size-dependent proteome separation. New method developments based on the hyphenation of F4 with on-line or off-line MS, and with other separation methods such as capillary isoelectric focusing (CIEF) are also described.

Hollow-fiber flow field-flow fractionation is here applied to untreated, whole human blood serum. Matrix-assisted, laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) of serum fractions shows mass signals in the M r range where low-abundance, serum protein components are known to be present, though a membrane of nominal 30 000 Da cutoff was employed for the fractionation device. Using diluted sera spiked with low amounts (0.06–0.1%, w/w) of an artificial mixture constituted the human adrenocorticotropic hormone fragments 18–39 ( M r = 2465.7) and 7–38 ( M r = 3659.2), and of bovine insulin ( M r = 5734), horse cytochrome c ( M r = 12 384) and chicken lysozyme ( M r = 14 388), a hybrid fractionation/microfiltration mechanism shows to govern the separation of the low- M r components.

Films based on TiO2 nanoparticles (NPs) have been successfully used as sensing elements in chemical sensors. TiO2 colloidal suspensions can be obtained by spontaneous hydrolysis in acidic solutions of Ti(IV) compounds. The obtained TiO2 NPs can be employed to build up nanostructured films. With the purpose of preparing TiO2-based nanostructured, imprinted materials as sensing elements for piezoelectric sensors, we obtained TiO2 NP dispersions by hydrolyzing potassium titanyl oxalate in the presence of a target analyte (tyrosine). Since morphological properties of the synthesized NPs are known to influence the nanostructured film characteristics, an analytical strategy to characterize such colloidal systems can combine a size-based separation method with spectroscopic analysis to correlate the particle size distribution (PSD) with the particle-target interaction properties able to determine the sensing efficiency. In this work, we present the characterization of colloidal tyrosine-TiO2 NP systems by flow field-flow fractionation (FlFFF) with online, UV/Vis absorption detection and offline fluorescence analysis. FlFFF eliminates the possible contribution of free tyrosine to the absorption and fluorescence properties of the NPs. FlFFF also fractionates NPs on a size basis. Particle size distribution (PSD) profiles of the fractionated NPs are then obtained by conversion of the multi-wavelength UV/Vis fractograms. Size of the fractionated NPs is finally related to fluorescence properties of the collected NPs fractions. Good correlation between the fluorescence intensity, which is proportional to the tyrosine uptake, and the FlFFF-based, NP mass-size frequency distribution finally confirms the existence of tyrosine-TiO2 NP interaction.

Water-soluble, functionalised multi-walled carbon nanotubes (MWNT) have been separated and purified from amorphous material through direct flow field-flow fractionation. MWNT subpopulations of relatively homogeneous, different length were obtained by collecting fractions of the raw, highly polydispersed (200–5000 nm) functionalised MWNT sample.

Performance of gravitational field-flow fractionation (GFFF) is improved here with respect to the ability to fractionate and distinguish different varieties of wine-making yeast from Saccharomyces cerevisiae. A new GFFF channel with non-polar walls has been employed to enhance fractionation selectivity and reproducibility. Since GFFF retention depends from first principles on particle size, Coulter counter measurements were performed in order to compare size distribution profiles with GFFF profiles. From such a comparison, GFFF was shown to be able to reveal differences in yeast cells other than size. This could make use of GFFF for screening different varieties of wine-making yeast towards future quality assessment procedures based on a possible correlation between yeast cell morphology indexes and quality indexes.

Gravitational field-flow fractionation (GrFFF) is applied to the fractionation of active dry wine yeast. An experimental approach to the analysis of the effects that field variation by changing mobile phase composition and flow-rate have on the fractionation process of standard particles (polystyrene) was first developed to further obtain effective fractionation of wine yeast by GrFFF. Scanning electron microscopy and Coulter counter particle size measurements were used to monitor the fractionation extent and capabilities of GrFFF to describe the distribution of yeast cells populations.

In previous papers it has been demonstrated that by using a special atomiser and power supply it was possible with elements of high volatility like Hg, Cd, Pb and Ag to obtain absorbance vs. time curves of a form consistent with the assumption that all atoms present in a condensed state at the beginning of the atomisation step are simultaneously present in the vapour phase inside the atomiser. In fact, these absorbance vs. time curves closely fit a model, which assumes a very rapid atomisation and homogenisation of the atoms at the centre of the atomiser followed by diffusion out of the graphite tube. The absorbance in these curves is, theoretically, an asymptotic maximum, which is related, if the experimental conditions are well chosen, to the total number of atoms injected, through the Lambert–Beer law. In this case the absorbance data can be used, if the proportionality constant is known, for standardless analysis. The increase of the atomisation temperature, as a consequence of a new design of the atomisation system, has made it possible to extend the method to elements of medium volatility like Co, Cr, Cu, Fe, Mn and Ni. The elements cited are those so far analysed but it is easy to predict that the method can be applied to the majority of the elements, thus greatly increasing its usefulness in a large number of real samples.

The absorbance vs. time curves that can be obtained in electrothermal atomic absorption spectroscopy with special atomizers and high heating rates can show a very steep increase of the signal from the baseline at the beginning of the atomization step, followed by a more or less extended flat region and by an exponential decay. This type of curve can be mathematically described by simple equations if rather drastic assumptions are made, i.e. if the atom supply is a delta function, if the absorbing species are homogeneously distributed in a plane at the center of a container of constant cross-section, and if the atom removal occurs only by diffusion. In this paper, some experimental curves are presented which are in satisfactory agreement with the theoretical model, thus supporting the claim that, with our atomization system and power supply, the existence of the flat region is indeed justified. With a new atomizer, specifically designed, it is possible to measure the diffusion coefficient of atoms in a gas at high temperature. However, for accurate measurements, the model must be refined and better measurements of the temperature of the atomizer must be obtained.

The fundamental parameter (cm 2 /atom) relating the absorbance and the number of atoms, called “atomic absorptivity” (C.Th.J. Alkemade et al. Metal Vapours in Flames, Pergamon, New York, 1982) and herewith referred to as the “spectroscopic constant” k , can be either theoretically calculated or derived from experimental calibration curves. The comparison between these two values is useful because the theory can be refined and the operating conditions chosen with more confidence. One of the most important parameters needed is the variation of k with temperature, since a good control of the atomizer temperature is not easy due to difficulties in calibrating the sensor. In fact, if k is constant, there is no need to control the temperature, while if its variation is known, one can calculate the error caused by such inaccuracy. In this paper, some experimental and theoretical data for Hg, in the temperature interval 300–2400 K, are discussed.

Effects of mobile phase composition can play an effective role in modulating the retention of particles in gravitational field-flow fractionation (GFFF), the simplest and cheapest among field-flow fractionation (FFF) techniques. In the framework of an optimized procedure for the GFFF characterization of particulate systems, an experimental approach to the effects of the mobile phase composition on the retention of silica particles retention is presented. The role of the ionic strength and the presence of surfactant are emphasized, with special regards to the shape of the particles. Moreover, the first experimental evidence of potential-barrier GFFF is reported.

The analysis of Pb associated with air aerosols by its accumulation through electrostatic precipitation directly in a graphite tube, which is subsequently used as an atomizer in electro thermal atomization-atomic absorption spectroscopy (ETA-AAS), has already been tested and found very convenient for its simplicity and speed. However, its use as a practical and useful method of analysis was hampered by the necessity of a laborious and difficult calibration with high risks of systematic errors. These difficulties can be bypassed by resorting to an absolute or standardless method of analysis. Standardless methods of analysis have been proposed for ETA-AAS measurements and recently applied to Cd and Hg. With the use of a new and more powerful power supply this method has been extended to Pb so that a very easy, fast, and almost on line way of measuring the level of Pb in air aerosols is possible. The system presented in this paper is manually operated but its automation, in order to obtain an automatic control station, is only a matter of convenience.

A new power supply for electrothermal atomization — atomic absorption spectrometry (ETA-AAS) with associated equipment for temperature control is presented. The system is composed of two high current commercial batteries and a MOSFET switch which controls the temperature of the atomizer by switching the current in an on-off way through a feedback loop connected to a personal computer and suitably programmed peripherals. The delivered power can be changed from 0 to 100% in 1% steps which gives the possibility of roughly controlling both the temperature at low power when the temperature sensor is not working and the heating rate. With this system, curves for which the simultaneous presence of all atoms injected in the atomizer is claimed, have been extended to Pb, Ag, Tl and Zn. With a more conventional power supply previously used, such a claim could be made only for Cd and Hg.

The apparatus presented in this paper is able to simultaneously confine all the atoms injected into the atomizer within the optical beam. As a consequence, during the time interval fulfilling this condition, the absorbance is constant. Experimental absorbance vs time curves, with Cd as analyte, show a constant value for more than 0.2 s. The curve shapes are in agreement with those theoretically derived for pure diffusion. This finding should lead to a method for absolute analysis that is more accurate than the methods used in ETA-AAS up to now. In fact, with this approach, one has to measure a constant signal while in the case of area measurements one has to integrate a signal that exponentially decays toward the baseline value. Data presented in this paper refer to the most volatile elements (Cd, Hg and Pb) in simple matrices. The possibility of extending the presented method to complex matrices and less volatile elements is dependent on the availability of both a more powerful power supply system and well-suited atomizer design.

The retention behaviour of selected flavonoid compounds was analysed and correlated with structural properties, as elucidated by X-ray crystallographic and 13 C NMR spectroscopic studies. A detailed analysis of group retention contribution (Δlog k′ ) makes it possible to establish how the introduction of an OH group in the molecule affects the overall solute polarity and, therefore, its retention, in relation to the position of the OH group and the molecular environment. X-ray structural information and 13 C NMR chemical shifts show the formation of strong intramolecular hydrogen bonding (HB) in 5-hydroxy-flavones and a weaker intramolecular HB in 3-hydroxy-substituted compounds. Δlog k′ values may be explained in terms of these intramolecular HB properties; moreover a quantitative relationship Δlog k′ values and chemical shifts was found.

The development of new “pre-MS” methods for protein isolation/separation from complicated biological samples is a fundamental requirement for successful, MS-based approaches to proteomics. Flow field-flow fractionation (F4) has been applied as pre-MS step for proteomics. Hollow-fiber F4 (HF5) is the innovative, microcolumn version of F4. HF5 employs a piece of hollow fiber as separation channel and current HF5 shows performance comparable to commercial F4. The reduced channel volume and flowrates, the low dilution of fractionated analytes, and the potentially disposable usage to exclude contaminations or carryover then make HF5 ideally suited as online or offline pre-MS separation step for the characterization of complex protein samples in native form. We have developed different applications based on HF5 in combination with MS or LC-MS for the fractionation, purification and characterization of native proteins, and for proteomic analysis of complex protein samples.

The reversed-phase high-performance liquid (HPLC) and thin-layer chromatographic (TLC) behaviour of flavonoids with methanol, tetrahydrofuran and acetonitrile as organic modifiers was compared. Twenty-six different correlation cases, together with their statistical parameters, are presented and discussed. Both TLC and HPTLC plates were considered. The dependence of TLC incremental Rm, values from different group substitutions on solvent type, solvent composition and type of plate is considered. A method for establishing HPLC gradient elution conditions by using TLC data is discussed.

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.

Optimization of the linear gradient elution of a crude extract of Swertia herb (Gentianaceae) was performed by means of sequential methods (Fibonacci and simplex) using a reversed-phase partition system and three different organic modifiers. The optimum gradient elution separations obtained with methanol, acetonitrile and tetrahydrofuran as different organic modifiers are discussed and compared by using the linear solvent strength theory. The Poisson character of the optimized separations was checked and the Davis-Giddings statistical overlap theory was applied in order to calculate the number of components. The value obtained is independent of both the organic modifier and column type. The quality of the best separation obtained was close to the absolute optimum expected from the Davis-Giddings theory. The usefulness of dealing with two or more optimized gradient separations is discussed. Solvent strengths are reported for selected compounds (amarogentin, amaroswerin and gentiopicroside).