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The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions.

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.

Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods. Flow field-flow fractionation (FlFFF) is characterized by the particularly "soft" separation mechanism, which is ideally suited to maintain the native structure of intact proteins. This work describes the original on-line coupling between hollow-fiber FlFFF (HF FlFFF), the microcolumn variant of FlFFF, and electrospray ionization/time-of-flight mass spectrometry (ESI/TOFMS) for the analysis and characterization of intact proteins. The results show that the native (or pseudonative) structure of horse heart myoglobin and horseradish peroxidase is maintained. Sample desalting is also observed for horse heart myoglobin. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI/TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.

Characterization of dispersed samples is an outstanding trend in analytical science. Among flow-assisted separation techniques for dispersed samples, size exclusion chromatography, hydrodynamic chromatography, and field-flow fractionation are the most widely applied. With dispersed analytes separated by these techniques, the UV/vis spectrophotometric detectors work as turbidimeters. To directly convert the analytical signal for quantitative analysis, the extinction properties of the dispersed analyte must be known. A new method is proposed to experimentally obtain-by single-run, flow-assisted separation with UV/vis diode-array detectors-the mass-size (or number-size) distribution function of the analytes when a retention-to-size relationship is either theoretically or empirically available for the chosen separation technique. This approach needs neither standards nor reliance on a method to predict the optical properties of the analytes. Theory and original algorithms are presented. Algorithms are then tested to optimize the numerical routines. Accuracy and robustness of the method are evaluated by simulation, and limitations for the application to experimental data are described. Finally, first application to field-flow fractionation shows validity of the method when applied to a few real cases.

On-line, continuous chemiluminescence detection has been recently applied to gravitational field-flow fractionation as a new method suitable for flow-assisted assays based on the separation between the analyte in solution and the analyte bound to micrometer-sized particles. In this work, flow field-flow fractionation coupled with off-line chemiluminescence detection is exploited for the development of flow-assisted, multianalyte assays. Micrometer-sized, polystyrene beads coated with enzymes suitable for chemiluminescence detection, such as horseradish peroxidase and alkaline phosphatase, are used as model samples. The high size-based selectivity of flow field-flow fractionation and specificity of chemiluminescence detection make possible, in a single run with very short analysis time, the simultaneous, high sensitivity detection of the different enzymes linked to beads of different size.

Steric/hyperlayer flow field-flow fractionation (St/Hyp/FlFFF) is suitable for the separation and characterization of micrometer-sized particles. In this technique, an ultrafiltration membrane is commonly used as the surface of the accumulation wall. St/ Hyp/FlFFF has been recently tested in membraneless mode and an improvement in performance was found. Recovery was also improved and second-order effects were reduced. In the framework of St/Hyp/FlFFF optimization, the effect of sample loading is a problem of a certain importance. For quantitative purposes, the conversion of peaks into mass particle size distributions is of prime importance and, therefore, the conditions in which there is no effect of sample loading on recovery should be investigated. In this paper, systematic work was performed in order to study the effect of sample loading on recovery. We have found the conditions in which recovery is independent of sample loading. For these conditions, the limit of detection for various microme...

Sorting and quantification of deactivated bacteria is an important way of quality control for whole-cell bacterial vaccines. In general, surface features of deactivated bacteria used for whole-cell bacterial vaccines affect the immunoresponse to bacteria-associated antigens. Enumeration of bacteria is also an important process development parameter for these vaccines. Field-flow fractionation (FFF) was previously applied to the separation of bacteria. For the first time, FFF is used for sorting bacteria strains of the same species on the basis of differences in bacterial membrane characteristics. Two FFF techniques, gravitational FFF (GrFFF) and asymmetrical flow FFF (AsFIFFF), are shown to be able to fractionate, distinguish, and quantify different deactivated Escherichia coli strains used for vaccines. E. coli can differ in the presence of fimbriae on the bacterial membrane. Fimbriae affect E. coli pathology and thus the use of E. coli for vaccines. GrFFF and AsFIFFF are able to fractionate fimbriated/ nonfimbriated cells in mixtures of different strains. While GrFFF is characterized by low cost and simplicity, As-FIFFF shows a higher performance in size fractionation with a high-speed separation. Coupled, on-line UV/visible turbidimetry yields the relative numbers of fractionated cells and sample recovery. Scanning electron microscopy and quasi-elastic light scattering are employed as uncorrelated techniques for size and morphology analysis of the E. coli strains.

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.

Interest in low-cost, analytical-scale, highly efficient, and sensitive separation methods for cells and bacteria has recently been increasing. Field-flow fractionation is well suited to the separation of different types of cells, including bacteria. High performance hollow fiber flow field-flow fractionation of such samples is demonstrated here for the first time with potentially disposable channels and high-sensitivity UV/Vis detectors. In this first application, hollow fiber flow field-flow fractionation is used to fractionate bacteria of biotechnological interest such as deactivated Vibrio cholerae, which are employed for whole-bacteria vaccine production. Quite short analysis times, high reproducibility, and low limits of detection are found. Retention of Vibrio cholerae is shown to depend on the mobile phase composition. Two serologically different Vibrio cholerae strains are partly distinguished by their fractogram profiles.

Doxorubicin (DOX) entrapment in porous Fe(III)-trimesate metal organic frameworks (MIL-100(Fe)) nanoparticles was investigated in neutral Tris buffer via UV-vis absorption, circular dichroism (CD), and fluorescence. The binding constants and the absolute spectra of the DOX-MIL-100(Fe) complexes were determined via absorption and fluorescence titrations. A binding model where DOX associates as monomer to the dehydrated Fe3O (OH)(H2O)2 [(C6H3)(CO2)3]2 structural unit in 1:1 stoichiometry, with apparent association constant of (1.1 to 1.8) × 10(4) M(-1), was found to reasonably fit the experimental data. Spectroscopic data indicate that DOX binding occurs via the formation of highly stable coordination bonds between one or both deprotonated hydroxyl groups of the aglycone moiety and coordinatively unsaturated Fe(III) centers. Complete quenching of the DOX fluorescence and remarkable thermal and photochemical stability were observed for DOX incorporated in the MIL-100(Fe) framework.

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.

In previous works, it has been shown that a standard ultraviolet-visible detection system can be used for quantitative analysis of heterogeneous systems (dispersed supermicron particles) in field-flow fractionation (FFF) by single peak area measurements. Such an analysis method was shown to require either experimental measurements (standardless analysis) or an accurate model (absolute analysis) to determine the extinction efficiency of the particulate samples. In this work, an experimental design to assess absolute analysis in FFF through prediction of particles' optical extinction is presented. Prediction derives from the semiempirical approach by van de Hulst and Walstra. Special emphasis is given to the restriction of the experimental domain of instrumental conditions within which absolute analysis is allowed. Validation by statistical analysis and a practical application to real sample recovery studies are also given.

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.

The possibility of an accurate determination of polymer molecular weight and polydispersity by thermal field-flow fractionation (ThFFF) retention measurements is here discussed with reference to the use of only physicochemical data of ordinary and thermal diffusivity, but without need of prior calibration of the ThFFF system. Special emphasis is devoted to the check of linearity conditions of retention data determination, i.e., on the proper sample loading and thermal field strength to be chosen for unbiased polymer specifications determination. Different numerical methods of determining peak profile attributes (non - linear peak fitting procedures by Edgeworth-Cramer series expansions, numerical integration, graphical determination) are compared. The approach is applied to a standard polystyrene sample, with ethylbenzene as polymer solvent and carrier liquid, as extensive physicochemical informations on this polymer-solvent system are available in the literature. In addition, it is shown that th...

Quantitative analysis in field-flow fractionation is becoming a necessary requirement for routine applications, instrumental optimization and scale-up to preparative separations. The use of detection systems which show complex dependence on sample characteristics (i.e. UV spectrometry) has hindered the application of quantitative methods of analysis in field-flow fractionation. A standardless model, shown valid in flow-through, homogeneous systems, is applied here to a heterogeneous system (dispersed supermicron particles) in field-flow fractionation by single peak area measurements. Absolute analysis in the fractionation of spherical silica particles for high-performance liquid chromatography column packing by gravitational field-flow fractionation with UV-Vis detectors is presented. It has been shown that for such samples extinction coefficients are independent of sample concentration and are determined by the size and density of the particles. The accuracy of such an approach to absolute analysis is discussed.

A standardless analysis method with electrothermal atomization/atomic absorption spectroscopy (ETA-AAS) measurements is proposed and applied to the determination of Pb in an urban environment (Bologna, Italy) and compared with results obtained in the same place and time, with the method dictated by Italian law, which is derived by European Community Directives. It was found that the difference previously observed between the two methods is due to the low efficiency of the 0.45-μm-pore-size paper filters, dictated by the official method. The uncovering of the error source validates the proposed method and highlights its accuracy. Moreover, the two methods can be conveniently combined to give two series of data relevant to pollutants associated with low- and high-dimension particles. This capability gives a much more precise picture of the hazard of a pollutant.

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 “Point-Of-Care-Testing” (POCT) system relies on portable and simply operated self-standing analytical devices. To fulfill diagnostic requirements, the POCT system should provide highly sensitive simultaneous detection of several biomarkers of the pathology of interest (multiplexing) in a short assay time. One of the main unsolved issues in POCT devices development is the integration of pre-analytical sample preparation procedures in the miniaturized device. In this work, an integrated POCT system based on gravitational field-flow fractionation (GrFFF) and chemiluminescence (CL) detection is presented for the on-line sample pre-analytical treatment and/or clean-up and analysis of biological fluids. As a proof of principle for the new GrFFF-CL POCT system, the automatic on-line analysis of plasma alkaline phosphatase activity, a biomarker of obstructive liver diseases and bone disorders, starting from whole blood sample was developed. The GrFFF-CL POCT system was able to give quantitative results on blood samples from control and patients with low sample volume (0.5 microL) and reagents consumption, short analysis time (10 minutes), high reproducibility and with a linear range of 50-1400 IU/L.The system can be easily applied to on-line prepare plasma from whole blood for other clinical biomarkers and for other assay formats, based on immunoassay or DNA hybridization.

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.

In this paper the high-temperature thermal field flow fractionation method is exploited for the analysis of polyethylene (PE). The experimental apparatus set-up, obtained by simply modifying a commercial instrument, is presented. The numerical procedure for deriving retention calibration plot versus molecular weight is discussed with reference to the specific polymer-solvent pair, PE-o-dichlorobenzene (ODCB), here employed. Different methods for computing the physicochemical data set of the solvent, necessary for calibration, are compared. The selectivity of the checked PE-ODCB system proves comparable with respect to the values currently found in thermal field-flow fractionation (ThFFF) analysis. Differences are found between PE and polystyrene (PS) analysis in the same solvent. The conditions for high temperature ThFFF operation in PE analysis and their advantages are discussed with respect to the standard SEC technique for PE, PS, and PE-PS copolymer analysis. Molecular weight distributions obtained by ThFFF of two PE commercial samples agree with those obtained by SEC

Gravitational field-flow fractionation (GFFF) is the simplest and cheapest of field-flow fractionation (FFF) techniques, although it is still at an early development stage. The application of GFFF to the determination of particle size distribution (PSD) of silica particles used as chromatographic supports is described. The accuracy of the method is evaluated by comparing PSDs obtained by GFFF with those obtained by laser diffraction, a non-separative technique widely applied to particle characterization. It is ultimately demonstrated that a low-cost GFFF channel can simply replace the column of a standard HPLC system, allowing laboratories that are not specialized in size analysis to perform accurate PSD studies with standard HPLC expertise.

Commercial flow field-flow fractionation (FlFFF) employs macro-scale, flat-type channels. The idea of hollow-fiber (HF) membranes as tubular, micro-column channels for FlFFF (HF FlFFF or, more shortly, HF5) dates back to 1974, with fundamentals on HF5 given in the late 1980s, and outstanding applications reported only over the last 15 years. Compared to flat-channel FlFFF, the key aspect of HF5 lies in the downscaling of the fractionation channel. This implies low-cost, possible disposable usage, and low volume of the channel that allows on-line coupling with highly sensitive detection and characterization techniques. The use of coupled techniques enhances the analysis of macromolecules and micron-sized particles such as intact proteins and whole cells. In this chapter we first report a few basics on HF5 theory and instrumentation. We then focus on technical and methodological developments that have made HF5 reach a performance normally achieved by flat-channel FlFFF. We finally focus on the enhancements obtained by coupling HF5 with powerful methods for detection and characterization of intact proteins and whole cells such as multi-angle light scattering (MALS), time-of-flight (TOF) mass spectrometry (MS), chemiluminescence (CL), and UV/Vis turbidity diode-array detection (DAD).

Whole-cell bioluminescent (BL) bioreporter technology is a useful analytical tool for developing biosensors for environmental toxicology and preclinical studies. However, when applied to real samples, several methodological problems prevent it from being widely used. Here, we propose a methodological approach for improving its analytical performance with complex matrix. We developed bioluminescent Escherichia coli and Saccharomyces cerevisiae bioreporters for copper ion detection. In the same cell, we introduced two firefly luciferases requiring the same luciferin substrate emitting at different wavelengths. The expression of one was copper ion specific. The other, constitutively expressed, was used as a cell viability internal control. Engineered BL cells were characterized using the noninvasive gravitational field-flow fractionation (GrFFF) technique. Homogeneous cell population was isolated. Cells were then immobilized in a polymeric matrix improving cell responsiveness. The bioassay was performed in 384-well black polystyrene microtiter plates directly on the sample. After 2 h of incubation at 37 °C and the addition of the luciferin, we measured the emitted light. These dual-color bioreporters showed more robustness and a wider dynamic range than bioassays based on the same strains with a single reporter gene and that uses a separate cell strain as BL control. The internal correction allowed to accurately evaluate the copper content even in simulated toxic samples, where reduced cell viability was observed. Homogenous cells isolated by GrFFF showed improvement in method reproducibility, particularly for yeast cells. The applicability of these bioreporters to real samples was demonstrated in tap water and wastewater treatment plant effluent samples spiked with copper and other metal ions.

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.

A new system design and setup are proposed for the combined use of asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) within the same instrumentation. To this purpose, three innovations are presented: (a) a new flow control scheme where focusing flow rates are measured in real time allowing to adjust the flow rate ratio as desired; (b) a new HF5 channel design consisting of two sets of ferrule, gasket and cap nut used to mount the fiber inside a tube. This design provides a mechanism for effective and straightforward sealing of the fiber; (c) a new AF4 channel design with only two fluid connections on the upper plate. Only one pump is needed to deliver the necessary flow rates. In the focusing/relaxation step the two parts of the focusing flow and a bypass flow flushing the detectors are created with two splits of the flow from the pump. In the elution mode the cross-flow is measured and controlled with a flow controller device. This leads to reduced pressure pulsations in the channel and improves signal to noise ratio in the detectors. Experimental results of the separation of bovine serum albumin (BSA) and of a mix of four proteins demonstrate a significant improvement in the HF5 separation performance, in terms of efficiency, resolution, and run-to-run reproducibility compared to what has been reported in the literature. Separation performance in HF5 mode is shown to be comparable to the performance in AF4 mode using a channel with two connections in the upper plate. (C) 2010 Elsevier B.V. All rights reserved.

Flow field-flow fractionation (F4) is the gentlest flow-assisted separation technique for analysis of macromolecules. The use of an empty channel as separation device and of a second mobile phase flow as perpendicular field enable F4 to separate analytes under native conditions without any modification of their original structure. Because of this unique peculiarity, F4 has been shown to be ideal for "gentle" separation of biological samples, for example intact proteins and protein complexes, since its early development. Today's F4 is an appealing technique which complements most established separation techniques, for example liquid chromatography and electrophoresis. The number of applications that show the unique advantages of F4 for analysis of protein samples is constantly increasing. In particular, F4 is finding increasing application on very high-molecular-weight species such as protein oligomers, aggregates, and complexes. This review critically discusses recent literature on the application of F4 to proteins. Either stand-alone or coupled with other characterization techniques, F4 is particularly promising for quality control of protein therapeutics, characterization of amyloid proteins, lipoprotein profiling, and as a pre-MS separation step in proteomics. © 2010 Springer-Verlag

Chemical modifications of nanoparticles (NPs) are often necessary to improve their features as spectroscopic tracers or chemical sensors, or to increase water solubility and biocompatibility of NPs for applications in nano-biotechnology. The description of newly designed functional NPs is rapidly expanding. However, the full exploitation of technologies based on functional NPs requires accurate, precise, and rugged methods for their analysis and characterization. When quality control protocols for industrial NP production are required, these methods must be applied on a routine basis. Since many properties of functional NPs are size-dependent, particle size distribution analysis provides fundamental information. The actual presence and distribution of functional groups in the NPs as well as their chemical features in the nanodispersed state are also fundamental aspects to be additionally analyzed. However, all these tasks cannot be afforded by a single method. Separation methods are necessary to isolate the newly synthesized NPs from the reagents in solution, and then coupled methods can characterize the isolated NPs. Flow field-flow fractionation (F4) is increasingly used as a mature separation method to size-sort and isolate NPs for their further analysis or size characterization by multiangle light scattering (MALS) detection. In this work, firstly the application of F4-MALS to different types of functional NPs is concisely overviewed. We then illustrate our recent applications of F4-MALS coupled with spectroscopic methods for the analysis and characterization of functional NPs. We finally provide an outlook of what we believe are the trends to make F4 soon become the required method in routine-based analytical platforms for quality control protocols of industrial-scale, functional NP production. © 2010 Springer-Verlag

Mass spectrometry (MS) provides analyte identification over a wide molar-mass range. However, particularly in the case of complex matrices, this ability is often enhanced by the use of pre-MS separation steps. A separation, prototype technique for the “gentle” fractionation of large/ultralarge analytes, from proteins to whole cells, is here described to reduce complexity and maintain native characteristics of the sample before MS analysis. It is based on flow field-flow fractionation, and it employs a micro-volume fractionation channel made of a ca. 20 cm hollow-fiber membrane of sub-millimeter section. The key advantages of this technique lie in the low volume and low-cost of the channel, which makes it suitable to a disposable usage. Fractionation performance and instrumental simplicity make it an interesting methodology for in-batch or on-line pre-MS treatment of such samples.

The demand for direct, robust and quantitative determination of analytes even in complex matrices of different origin produced an increase interest in flow-assisted techniques for automatization of chemical analysis with high throughput and reproducibility. With this aim, thanks to simple instrumentation, high sensitivity and low background signal, chemiluminescence, bioluminescence and electrochemiluminescence represent suitable detection techniques to which flow-analysis can be coupled. In this Chapter the fundamental, instrumental setup and recent application are reviewed for flow-injection analysis and its variants, and for field-flow fractionation, a flow-assisted technique suitable to separate analytes with high molar mass, for the development of assays with reliability, rapidity and robustness. A discussion on the miniaturization of these approaches to add features such as low reagent consumption and reduced analysis time is also presented.

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.

Synthesis and applications of new functional nanoparticles are topics of increasing interest in many fields of nanotechnology. Chemical modifications of inorganic nanoparticles are often necessary to improve their features as spectroscopic tracers or chemical sensors, and to increase water Solubility and biocompatibility for applications in nano-biotechnology. Analysis and characterization of structured nanoparticles are then key steps for their synthesis optimization and final quality control. Many properties of structured nanoparticles are size-dependent. Particle size distribution analysis then provides fundamental analytical information. Asymmetrical flow field-flow fractionation (AF4) with multi-angle light scattering (MALS) detection is able to size-separate and to characterize nanosized analytes in dispersion. In this work we focus on the central role of AF4-MALS to analyze and characterize different types of structured nanoparticles that are finding increasing applications in nano-biotechnology and nanomedicine: polymer-coated gold nanoparticles, fluorescent silica nanoparticles, and quantum dots. AF4 not only size-fractionated these nanoparticles and measured their hydrodynamic radius (r(h)) distribution but it also separated them from the unbound, relatively low-M(r) components of the nanoparticle structures which were still present in the sample Solution. On-line MALS detection on real-time gave the gyration radius (r(g)) distribution of the fractionated nanoparticles. Additional information on nanoparticle morphology was then obtained from the r(h)/r(g) index. Stability of the nanoparticle dispersions was finally investigated. Aggregation of the fluorescent silica nanoparticles was found to depend on the concentration at which they were dispersed. Partial release of the polymeric coating from water-soluble QDs was found when shear stress was induced by increasing flowrates during fractionation. (C) 2009 Elsevier B.V. All rights reserved.

Field-flow fractionation (FFF) is a mature technique in bioanalysis, and the number of applications to proteins and protein complexes, viruses, derivatized nano- and micronsized beads, sub-cellular units, and whole cell separation is constantly increasing. This can be ascribed to the non-invasivity of FFF when directly applied to biosamples. FFF 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 field. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media and without using degrading mobile phases. The fractionation device can be also easily sterilized, and analytes can be maintained under a bio-friendly environment. This allows to maintain native conditions of the sample in solution. In this review, FFF principles are briefly described, and some pioneering developments and applications in the bioanalytical field are tabled before detailed report of most recent FFF applications obtained also with the hyphenation of FFF with highly specific, sensitive characterization methods. Special focus is finally given to the emerging use of FFF as a pre-analytical step for mass-based identification and characterization of proteins and protein complexes in proteomics.

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.

Flow field-flow fractionation (FlFFF) has been utilized for size-based separation of rat liver mitochondria. Collected fractions of mitochondria of various sizes were examined by confocal microscopy, and mitochondria of each fraction were lysed and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the comparison of protein patterns in differently sized mitochondria by densitometric measurements, and for protein characterization of some gel spots with nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). FlFFF fractions of the mitochondria were also tryptically digested for shotgun proteomic characterization of mitochondrial proteins/peptides by nLC-ESI-MS-MS. Peak area (integrated ion counts) of some peptides extracted from LC-MS chromatograms were examined at different fractions for the quantitative comparison. Among 130 proteins, 105 unique proteins were found to be mitochodrial from the off-line combination of FlFFF and nLC-ESI-MS-MS analysis. It also showed that 23 proteins were found in all fractions but some proteins were found exclusively in certain fractions. Among 25 proteins listed from other subcellular species, seven proteins were known to exist in mitochondria as well as in other subcellular locations, which may support the possible translocation or multiple localizations of proteins among organelles. This study demonstrated effective use of FlFFF for the isolation and/or enrichment of intact mitochondria isolated from cells, as well as its potential use for the fractionation of other subcellular components in the framework of subcellular functional proteomics.

The determination of the particle size distribution and mass ratio of two types (small and large) of starch granules is of great significance in the food industry, since the size of the granules influences the gelatinization, digestibility and thermomechanical behavior of starch. In order to achieve that, the ideal experimental conditions for sample analysis should be determined. The influence of the various surfactants (FL-70, a mixture of anionic and nonionic surfactants; Triton X-100, a nonionic surfactant; and SDS, an ionic surfactant) used during the analysis on these results was investigated, as the chemical structure of the suspending medium affects the interaction forces between the starch granules themselves, as well as the starch granules and the accumulation wall. Two different mathematical methodologies were applied, which were found to lead to approximately similar conclusions concerning the mass ratio of starch granules. The kind of detergent used was found to have a strong influence on the results of the experimental analysis. The total experimental procedure was performed by sedimentation field-flow fractionation (SdFFF). The results were compared with those obtained by scanning electron microscopy (SEM) or with those found in the literature.

Up to 80 million cases of food-borne illness occur yearly in the US. Approximately 30% of these cases are caused by bacteria and their related toxic products (1)(2). The bacteria species that are most frequently responsible for food contaminations are Salmonella typhimurium (meat, milk, and eggs), Escherichia coli O157:H7 (meat), Staphylococcus aureus (milk, cream, and meat), Clostridium perfringens (sausages, preserved food), Campylobacter jejuni (poultry, eggs), Vibrio parahaemolyticus (shellfish), Yersinia enterocolitica (meat, milk), and Listeria monocytogenes (dairy products) (3). Conventional microbiological methods for the identification of pathogenic bacteria are labor-intensive (several enrichment steps) and time-consuming (2 to 3 days to obtain results) (4). Currently, new rapid procedures based on immunological, DNA hybridization, or biosensing methods have been proposed (5)(6). However, many of these rapid tests are expensive, laborious, and all of them are based on cultural enrichment to enhance sensitivity and selectivity before analysis. Simpler, faster and more sensitive diagnostic techniques are required to improve food safety (food production, processing, storage and distribution) and to screen for potential bacterial infections in humans. Several immuno-based detection methods for infectious agents have been developed recently (7)(8). Such methods are of great diagnostic importance because they offer unique detection specificity and sensitivity, with relatively simple and low-cost assay formats. Most commercially available immunological methods for the detection of bacteria are based on conventional sandwich immunoassays, which involve the formation of an immunocomplex with an immobilized antibody, the target bacteria, a second labeled antibody, and the separation of free and bound antibody fractions by washing steps. Unfortunately, these separation steps reduce the assay production, so alternative approaches are welcome. In general, flow-assisted immunoassays offer advantages over conventional microtiter solid-phase formats, mainly related to easier automation and faster kinetics of the immunological reaction (9)(10), which takes place …

Determination of total cholesterol (TC), HDL and LDL cholesterol, and triglycerides (TG) is used to assess lipoprotein abnormalities and coronary artery disease (CAD) risk. Furthermore, studies have demonstrated that small, dense LDL particles penetrate more easily into the arterial intima (1), exhibit increasing binding to arterial wall proteoglycans(2), and are more prone to oxidative stress(3). Small, dense LDL particles also have a prolonged plasma half-life because of their lower binding affinity for the LDL receptor(4). The presence of small, dense LDL particles in plasma is, therefore, considered to be proatherogenic. Analytical methods for LDL size characterization are therefore expected to improve classification, diagnosis, and therapy of dyslipidemic patients. However, simple methods for routine LDL size measurement are not yet available. LDL size is mainly measured by nondenaturing PAGE (5), which is time-consuming and may be unsuitable for large numbers of samples. Easier methods for measuring LDL size have been proposed, including high-performance gel-filtration chromatography (HPGC)(6). Both PAGE and HPGC can give accurate size estimations only if appropriate standards are available. Moreover, recent studies have shown that LDL particles may be discoidal, with diameter and height that are not significantly correlated(7). PAGE and HPGC cannot give information on particle conformation. Flow field-flow fractionation (FlFFF) is a separation technique in which macromolecules and particles are separated by the combined actions of an axial flow and a perpendicular cross-flow (8). No stationary phase is present, and the separation mechanism is sufficiently gentle not to alter the native structure of proteins and protein complexes(9). Proteins are fractionated according to their difference in diffusion, with retention time that is inversely proportional to the analyte diffusion coefficient. In principle, with the use of FlFFF it is possible to obtain the hydrodynamic radius (Rh) of proteins from their retention time …

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution.

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