Separation Science: Principles and Applications for the Analysis of Bionanoparticles by Asymmetrical Flow Field-Flow Fra
Field-flow fractionation is an analytical technique that allows the separation of particles over a size range, from a few nanometers to several microns in diameter. The separation takes place under mild conditions and is suited for the analysis of neutral
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Introduction Field-flow fractionation (FFF) offers great versatility in the types of sample to be analyzed, and it provides a full sample characterization in a single measurement typically within 30 min (1–3). Various FFF subtechniques are available depending on the field applied to the sample (4–9). They each have specific advantages and drawbacks. For example, sedimentation FFF (SdFFF), in which the field is gravitational, has a much greater resolving power than the other subtechniques, but it is only applicable for particles larger than 100 nm that have a density significantly different from that of the carrier liquid. In flow field-flow fractionation (FlFFF), the field is provided by a crossflow applied perpendicularly to the elution flow. The acronym AF4 (asymmetrical flow field-flow fractionation) refers to FlFFF for which the channel is of trapezoidal shape instead of an elongated hexagon (10). Most commercial FlFFF systems are using AF4, in view of its versatility, relative simplicity, and its wide range in
Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 991, DOI 10.1007/978-1-62703-336-7_30, © Springer Science+Business Media New York 2013
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terms of particle size (11–13). In the following section, we present the principle of the fractionation and review the importance of each parameter as well as describe step-by-step the process to obtain a fractionation of quantum dots (QDs) and optimize the fractionation conditions. Quantum dots are one of the most representative nanoparticles used in the nanosciences. They are nanometer-sized semiconductor crystals, which can be made with core or core-shell architecture, and have been extensively used for imaging applications both in vitro, at the single-cell level (14–16), and in vivo (17–23), mainly in animals bearing tumors. Their main attractiveness relies on the fact that their luminescence emission can be tuned by controlling the size and size distribution of the particles. Because, the emission maxima are directly related to the sizes, it is possible to have a qualitative appreciation of the size distribution of a QD sample by measuring its luminescent emission spectrum. FlFFF can provide quantitative data on the QD’s (and other nanoparticles) size distribution, presence of aggregates, adsorbed proteins (opsonized particles), and nanoparticle decomposition (24). 1.1 Principle of Flow Field-Flow Fractionation
In FFF, the separation of the sample takes place inside a narrow ribbonlike channel clamped between two parallel surfaces through which a field can be applied (Fig. 1). A carrier liquid is pumped through the channel from the inlet (sample injection) to the outlet (detector). A parabolic flow profile (Newtonian flow) is established inside the channel, as in a capillary tube. Flow velocities vary from 0 on the walls to a maximum value in the center of the channel. A field is applied perpendicularly to the flow direction, while the carrier liqu
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