Flow field-flow fractionation: Critical overview.

This overview regards some critical issues in performing flow field-flow fractionation (flow FFF, FlFFF, AF4, HF-FlFFF, HF5). It includes the channel thickness, void time, channel-flow parabolic profile, channel-flow velocity gradient, uniformity of the cross-flow, sample injection time, relaxation/focusing time, width of sample starting zone, retention level, theoretical and experimental zone broadening, hydrodynamic threshold immobilisation/re-mobilisation, sample loss and adsorption, membrane fouling, sample mass overloading, problems with symmetrical channels, non-spherical sample particles, and method development. Good method development practice (GMDP) and good fractogram practice (GFP) is suggested

Comments on the separation efficiency of asymmetrical flow field-flow fractionation in channels of constant channel and crossflow velocities leading to constant separation efficiency.

It is shown theoretically that a claim in the literature about the overall separation efficiency of asymmetrical flow FFF channels being improved by geometries that permit a uniform channel flow velocity throughout the channel length is untrue

Asymmetrical Flow Field-Flow Fractionation as a Method to Study the Behavior of Humic Acids in Solution

Asymmetrical flow field-flow fractionation (AsFlFFF) is used to measure the size distributions of humic acids in solution. Shifts in the size distribution are used to study the behavior of these amphiphilic macromolecules in solution with changes in pH, ionic strength, and humic acid concentration. Humic acid concentrations are increased by on-channel focusing of the injected samples. As the concentration is increased, the humic acids aggregate, but the effect varies with solution conditions. As the concentration of calcium chloride is increased, size distributions broaden and become multimodal. The decrease in size is consistent with reports that divalent cations induce conformational changes by the formation of intramolecular bridges between carboxylate moieties; multimodality indicates intermolecular interactions as well. Lowering the pH also promotes intermolecular interactions, with severe aggregation occurring below pH 4. The work demonstrates the utility of AsFlFFF in studies of the hydrodynamic behavior of amphiphilic macromolecules. © 1997 John Wiley & Sons, Inc. J Micro Sep9: 535–543, 199

Mass overloading in the flow field-flow fractionation channel studied by the behaviour of the ultra-large wheat protein glutein.

Flow field-flow fractionation (FFF) has previously been used in successful fractionation and characterisation of the ultra-large wheat protein glutenin. The many parameters, which may influence the retention behaviour, especially when analysing extremely high-molecular-mass samples such as glutenin, are here reported. Size determination from the sample retention time, using FFF theory, will as a result have a very low accuracy. The need for direct molecular mass determination, such as by light scattering, in combination with FFF, in order to do accurate size measurements of glutenin is pointed out as well as the importance to minimise the overloading

Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field-flow fractionation.

The evaluation of the translation capacity of cells that produce recombinant proteins can be made by monitoring their ribosomal composition. In a previous use of asymmetrical flow field–flow fractionation (AsFlFFF) for this purpose the overall analysis time was more than 1 h and 40 min, based on a standard protocol for cell harvest, washing, cell disruption, and the final 8-min AsFlFFF determination of ribosome and subunits. In the present work the overall analysis time was reduced to 16 min. The washing step was deleted and a time-consuming freeze-thaw cycle. Cell disruption was obtained by a time-minimized lysozyme and detergent treatment for 1.5 min, respectively. The ribosomal material was finally fractionated and quantified in only 6 min, without previous centrifugation, using AsFlFFF. The great time reduction will enable the future use of AsFlFFF at-line to a growing cell cultivation, continuously monitoring the change in ribosomal composition or in other applications requiring high sample throughput. To demonstrate the high efficiency of the method the ribosome and tRNA composition in an Escherichia coli cultivation was monitored every half an hour, giving 18 measurements across the complete growth curve, a frequency of data enough to make decisions about induction or termination of the cultivation

Size separation of supermicrometer particles in asymmetrical flow field-flow fractionation. Flow conditions for rapid elution

The performance of lift-hyperlayer asymmetrical flow field-flow fractionation using rapid elution conditions was tested through the separation of standard polystyrene latex particles of diameters from 2 to 20 mum. Optimization of flowrates was studied not only in order to obtain efficient and rapid separation; but also to work under conditions of various shape and steepness of the axial flow velocity gradient. Using extreme flow conditions, the five widely spaced particle sizes, 20.5-, 15.0-, 9.7-, 5.0-, and 2.0-mum diameter, could be resolved in 6 min, whereas for the narrower size range of 20.5-5.0 mum, 1 min was enough. The size selectivity in the size range 9.7-2.0 mum was studied as a function of flowrates and particle size and was found to be constant. A particle trapping device made it possible to separate particles of sizes >10 mum; which has previously proven to be difficult in asymmetrical channels

Field-Flow Fractionation

Not available

Flow FFF–basics and key applications

The 1990s and 2000s have seen a rapidly growing use of flow field-flow fractionation (flow FFF, FlFFF). As of today hundreds of publications in many different application areas are presented each year in which flow FFF has been used or is referred to. In this chapter a brief historical overview of flow FFF is given. Channel designs and basic principles are discussed as well as approaches to development of rapid high resolution separations. Finally, an overview of key applications is included with pioneering and ground-breaking papers from literature