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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.

Very monodisperse water-soluble silica core-surfactant shell nanoparticles (SCSS NPs) doped with a rhodamine B derivative were prepared using micelles of F127 as nanoreactors for the hydrolysis and condensation of the silica precursor tetraethoxysilane (TEOS). The functionalization of the rhodamines with a triethoxysilane group allowed the covalent binding of the fluorophores to the silica core: no leaking of the dye was observed when the NPs were purified either by ultrafiltration (UF) or dialysis. The diameter of the core (d(c) = 10 ± 1 nm) was determined by TEM and subtracted from the hydrodynamic diameter, measured by DLS, (d(H) = 24 nm, PdI = 0.1) to calculate the shell thickness (∼7 nm). The presence of a single population of NPs with a radius compatible with the one measured by DLS after UF was confirmed by AF4-MALS-RI measurements. The concentration of the NPs was measured by MALS-RI. This allowed us to determine the average number of rhodamine molecules per NP (10). The ability of the NPs to host hydrophobic species as cyanines in the SS was confirmed by fluorescence anisotropy measurements. Steady-state and time-resolved fluorescence measurements allowed us to observe the occurrence of a very efficient Förster resonance energy transfer process from the covalently linked rhodamines to the hosted cyanines. In particular, the analysis of the TCSPC data and steady-state measurements revealed that the adsorption of a single cyanine molecule causes an almost complete quenching of the fluorescence of the NP. Thanks to these observations, it was possible to easily determine the concentration of the NPs by fluorescence titration experiments. Results are in good agreement with the concentration values obtained by MALS-RI. Finally, the hosted cyanine molecule could be extracted with (±)-2-octanol, demonstrating the reversibility of the adsorption process.

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.

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).

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.

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.

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 …

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.

Oligothiophenes (TFs) with blue, green and orange emission have been used for the first time as doping fluorophores of silica nanoparticles (SiO(2)NPs). High purification of the new nanoparticles (TFsSiO(2)NPs) from free molecular fluorophores was achieved by means of asymmetrical flow field-flow fractionation on-line combined with multi-angle light scattering and fluorescent detection (AF4-MALS-FD). The synthesis, structural, compositional and optical characterizations of the new TFsSiO(2)NPs are reported. We show that the tailored co-assembly of TFs in bi- and tricomponent TFsSiO(2)NPs allows for the fine-tuning of the emission of the nanoparticles from blue to white by means of FRET processes between adjacent TFs. These unique optical signatures make TFsSiO(2)NPs potentially effective tools for fluorescent sensing and labeling.