Flow field-flow fractionation with multiangle light scattering detection for the analysis and characterization of functional nanoparticles

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. \ua9 2010 Springer-Verla

Flow field-flow fractionation: recent trends in protein analysis

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. \ua9 2010 Springer-Verla

Hollow-fiber flow field-flow fractionation

none5noneA. Zattoni; D.C. Rambaldi; S. Casolari; B. Roda; P. ReschiglianA. Zattoni; D.C. Rambaldi; S. Casolari; B. Roda; P. Reschiglia

Tandem hollow-fiber flow field-flow fractionation

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