Microthermal Field-Flow Fractionation:
Analysis of Synthetic, Natural, and Biological Macromolecules and Particles

Josef Janča

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April 2008
247 pp.

ISBN: 978-0-9728061-5-2

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ISBN: 978-0-9728061-5-2



Microthermal field-flow fractionation (micro-TFFF) is a new method for the separation, analysis, and characterization of macromolecules and particles. The range of applications includes not only synthetic polymers and polymer-based colloidal particles but also biopolymers and a variety of particles of different origin, natural inorganic as well as biological. There is no doubt that the fundamental principle of micro-TFFF is the same as that of the standard-size TFFF technique invented by J. Calvin Giddings, past Professor at the University of Utah, Salt Lake City, USA. In both, it is thermal diffusion, sometimes called thermophoresis or the Ludwig-Soret effect, that generates the main driving force, which, in combination with secondary effects, imposes one of two principal separation mechanisms: polarization or focusing.


However, the apparent similarity between micro-TFFF and TFFF should not conceal key differences. The miniaturization of the separation channel is not only a mechanical reduction of the channel dimensions, but a rational, theoretically justified choice of the optimal channel size and the accompanying fundamental changes of the sample injection mode, the invention of the hydrodynamic inlet splitting option, and many other modifications of the design and construction details of a compact and versatile micro-TFFF separation unit. The substantial reduction in heating power for a micro-TFFF channel compared with standard-size TFFF channels, represents an economical gain. However, much more important is the fact that the total heat flow produced on the hot side of the channel must be evacuated from the cold wall. Consequently, an accurate temperature of the cold wall is practically impossible to control with the use of a standard-size channel, for which the electric power for heating is on the order of kilowatts times more than for a micro-TFFF channel. The resulting ability of micro-TFFF to control very precisely the temperatures of the hot as well as the cold wall independently opened the opportunity to study the behavior of retained species within a very large range of temperatures. This fact has already contributed to a deeper understanding of the thermal diffusion of macromolecules and particles, and opened the ways for future studies.


During the first 30 or so years of field-flow fractionation (FFF), the research was oriented towards the exploration of various physical fields, thus giving rise to a variety of FFF techniques, such as sedimentation FFF, flow FFF, electric FFF. Nowadays, one of the trends is focused on a deeper elaboration of asymmetrical-flow FFF combined with various detectors, such as automatic pressure transducers, capillary viscometers and multi-angle light-scattering detectors, and also combinations of the results from asymmetrical-flow FFF and size exclusion chromatography. All these methods are in current use in laboratories oriented towards polymer characterization. The second trend is towards the miniaturization of analytical instrumentation with the aim of constructing so-called lab-on-a-chip methods. Micro-TFFF is on this path by exploiting recent inventions in micro-fluidic technologies but also by contributing to the development in this field. One of the great advantages of micro-TFFF, not yet fully explored, is that it is practically unlimited with regard to the range of molar masses or particle sizes of the species that can be fractionated and analyzed. Moreover, the experimental conditions are very versatile because the range of temperatures, pH, etc., is very large and the choice of solvents--often very corrosive ones--also practically without limit due to the materials used for the construction of the micro-TFFF channel. The cleaning of the channel is easy compared with the changing of membranes in asymmetrical-flow FFF. On the other hand, the miniaturization of suitable detectors has not yet followed the rapid growth of micro-TFFF channel technology. Nevertheless, it is hoped that modern detector technologies will lead to miniaturization.


The book is organized into five chapters. Chapter 1 is a historical perspective on the invention and development of FFF in general. The basic principles are briefly described. Chapter 2 is devoted to a detailed description of the theory of FFF starting from the theory of retention, which is related to the formation of various transverse (across-the-channel) concentration distributions of the retained species and their coupling with the flow-velocity profile established inside the channel in the same transverse direction. Zone dispersion is also treated theoretically in detail. This is a very important factor whenever macromolecules or particles are concerned, because of their very low diffusion coefficients. Chapter 3 deals with the theoretical bases that justify the advantages of miniaturization of the TFFF method and thus the invention and development of micro-TFFF. The instrumentation for micro-TFFF is described with regard to optimization of different components within the micro-TFFF setup. Special attention is devoted to practical experimental minimization of the zone broadening caused by the extra-channel elements of micro-TFFF separation system as well as to optimized construction of the channel. Chapter 4 presents a very detailed description of the primary driving forces and separation mechanisms in micro-TFFF and of the secondary effects that can often be exploited to increase the performances of this method. It is shown that the contributions of secondary effects have often been investigated with the use of complementary methods that have no direct relation to micro-TFFF, but their use has been crucial for a complete, rigorous understanding of secondary effects and their use. In Chapter 5, the analytical methodology is analyzed to allow the full and accurate interpretation of experimental data from micro-TFFF. The accuracy, precision, repeatability, and reproducibility of micro-TFFF data are discussed from a theoretical point of view, and the validity of theoretical predictions is justified by the comparative experimental studies carried out in different laboratories. Some typical examples of the methodological applications of micro-TFFF are described at the end of the chapter. The book concludes with tables defining the symbols and abbreviations used throughout.

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