FOCUS
Third International Symposium on
Michel Martin Ecole Supérieure de Physique et Chimie Industrielles Laboratoire de Physique et Mécanique des Milieux Hétérogènes (URA CNRS 857) 10, rue Vauquelin 75231 Paris Cedex 05 France
F F F '92, the Third I n t e r n a t i o n a l Symposium on Field-Flow Fractionation, convened Oct. 5 - 7 , 1992, in Park City, UT. Previous symposia were held in Park City (June 1989) and in Salt Lake City ( F e b r u a r y 1991). The third meeting, sponsored by the University of Utah's FieldFlow Fractionation Research Center (FFFRC), was chaired by J. C. Giddings, director of the center. About 75 academic and industrial scientists from 12 countries (Australia, Canada, France, Germany, Israel, Italy, J a p a n , Sweden, Switzerland, The Netherlands, the United States, and Venezuela) gathered in a stimulating environment that promoted lively 0003 - 2700/93/0365 -315A/$04.00/0 © 1993 American Chemical Society
discussions among the participants. In three busy days, 30 lectures and 42 posters were presented, and part i c i p a n t s came away with a good overview of the state of FFF. It is noteworthy that the number of presentations devoted to F F F applications has increased steadily since the first symposium; at F F F '92, twot h i r d s of t h e lectures and t h r e e fourths of the posters were devoted to applications.
Environmental analyses The first lecture session was devoted to e n v i r o n m e n t a l a n a l y s e s . Ron Beckett (Monash University, Australia) discussed the physical characterization and quantitation of bacteria and algae in river water by sedimentation F F F . This technique is well suited for the separation of sub- and supermicrometer particles of various types, including cellular bioorganisms. Elution fractions from the FFF unit were collected and analyzed by epifluorescence microscopy to determine the cell volumes that, in combi-
nation with FFF retention times, allowed cell density to be calculated. The cell biomass, an important measure of microbial activity and production in aquatic systems, was then directly calculated by using literature values (for the average density of dried bacteria mass) and a calibration factor (relating detector signal and cell concentration). Howard Taylor (U.S. Geological Survey) reported on the combination of sedimentation F F F with inductively coupled plasma MS. The high sensitivity of the detection system is particularly well suited for the relatively small sample sizes used in FFF. The m u l t i e l e m e n t m e a s u r e m e n t capability allows the use of data from fractograms to reconstruct the average molecular composition of clay samples. David Chittleborough (University of Adelaide, Australia) reported on field studies of pollutant transport t h r o u g h soils by mobile colloids present in groundwater. Sedimentation FFF was used to size and sepa-
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FOCUS rate colloids in the 0.08- l-μπι range; flow FFF was used to determine the molecular weight distributions of humic substances down to 300 Da and to fractionate colloids and macromolecules in the 1-100-nm range. Richard Keil (University of Wash ington) used split-flow thin (SPLITT) cells to separate m a r i n e sediments into size classes ranging from < 1 μπι to > 260 μπι, using the natural gravitational field. SPLITT cells are similar to FFF channels ex cept that a flow splitter placed at the inlet allows independent and contin uous introduction of sample and car rier liquid s t r e a m s . Another flow splitter at the outlet separates the ef fluent into two streams. The proper adjustment of the inlet and outlet flow ratios allows selection of the size cutoff for particles eluting into the u p p e r a n d lower o u t l e t flow s t r e a m s . SPLITT cells have been used to physically isolate and con centrate selected sedimentary phases from composite samples, allowing evaluation of individual components of complex sedimentary mixtures.
Pharmaceutical applications Another emerging field of applica tions presented at the symposium concerns the analysis of pharmaceu tical products. Continuous SPLITT fractionation has also proved useful for r a p i d m e a s u r e m e n t s of d r u g b u r s t s from liposomes (because of sudden, large dilutions), as shown by Shulamit Levin (Hebrew University, Israel). The SPLITT cell is then used as a membraneless dialysis cell for preliminary, in vitro screening of drug-carrying liposomes. The fastdiffusing released drug emerges in the two outlet streams, whereas the colloids emerge at only one outlet. Jianmin Li (University of Utah) showed that the combination of sedi mentation FFF and photon correla tion spectroscopy provides informa tion not only on size distribution but also on droplet density of pharmaceutically important emulsions. This capability allowed speculation about possible emulsion s t r u c t u r e s t h a t were later confirmed by freeze frac ture electron microscopy. Becky Arlauskas (Alliance Phar maceutical) displayed fractograms of perfluorocarbon dispersions (devel oped as potential substitutes for red blood cells) in which the vesicle peak is clearly separated from the emul sion peak.
Particle characterization Several presentations were devoted to the characterization of particles.
Marcia H a n s e n (FFFractionation) presented some practical examples of applications of both flow and sedi mentation FFF to size analysis of in dustrial particles. The emphasis was on p r o b l e m a t i c s a m p l e s , s a m p l e preparation requirements, and ex perimental procedures. Flow FFF is universally applicable to particles ranging from a few nanometers to al most 100 μπι and is independent of particle density, whereas sedimenta tion F F F requires a minimal particle mass for retention but is very selec tive on the basis of particle size. Henk Merkus (Delft University of Technology, The Netherlands) dis cussed the evaluation of sedimenta tion F F F for quantitative measure ments of particle size distributions. He emphasized t h a t although the separation of particles of different sizes is performed with high resolu tion and precision, the quantitative determination of the particle concen tration in the channel effluent by UV detection may, in some instances, be difficult because of large fluctuations in the extinction coefficient with par ticle size. He recommended using white light, a multiwavelength pho tometer, or a single particle counter instead of a monowavelength pho tometer. Yasushige Mori (Kyoto University, Japan) discussed retention deviation
"One advantage of FFF methods over LC methods, when they can be applied to the same samples, is that FFF channels have much lower surface areas of contact with solid materials." t of submicrometer particles from the values predicted by t h e classical Brownian mode retention equation. The deviation is attributable to elec trostatic, van der Waals, and hydrodynamic interactions of the particles with the channel wall. The experi mental retention times of particles < 0.5 μπι were accurately estimated, using the D e r y a g u i n - L a n d a u Verwey-Overbeek theory to account for electrostatic and van der Waals interactions.
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For particles larger than - 0.5 μπι, retention is influenced by hydrodynamic lift forces exerted on the parti c l e s , a n d p r o p e r c a l i b r a t i o n is required for particle size character ization. Myeong Hee Moon (FFFRC) described applications to various types of particulates of a density compensation method previously de veloped for this purpose. However, because the method operates at a field strength exceeding the capabili ties of most sedimentation FFF in struments, it does not work for very heavy particles such as gold. Moon showed that the problem was solved by fraction collection followed by mi croscopic analysis. Jenq-Thun Li (University of Utah) described the application of flow and sedimentation FFF and electron spin resonance to determine the charac teristics of particles coated with triblock copolymers and their related protein repellent qualities. Li found that the curvature (size) of the parti cles strongly influences the thickness of the adsorbed copolymer layer (de termined by flow FFF), as well as its surface concentration (determined by sedimentation FFF). Both the sur face composition and the dynamics of the polymer chains are important factors in establishing a proteinrepelling surface. Presentations of other F F F appli cations for particle characterization included the analysis of cell lysates by a combination of sedimentation and flow F F F (Kim R a t a n a t h a n a wongs, FFFRC); the use of a SPLITT cell with a gravitational field for fractionation of diamond abrasive powders, according to a selected cut off size (Fei-Yan Ren, FFFRC); the use of a SPLITT cell with a gravita tional field for rapid particle size analysis in the 5-75-μπι size range (Bor Fuh, FFFRC); and the use of flow FFF with a single porous wall (asymmetrical flow FFF) for fast size characterization of 10-200-nm silica sols by Bengt Olsson (University of Lund, Sweden). Although sedimentation and flow FFF are the traditional methods for characterizing particulate materials, it was recently observed that submi crometer- and micrometer-sized par ticles can be fractionated according to their size by thermal FFF in both aqueous and nonaqueous suspen sions. Paul Shiundu (FFFRC) dis cussed some factors affecting particle retention, such as cold wall tempera ture, temperature gradient, and com position and size of particles. Reten tion is also influenced by the nature of the suspension medium and its
ionic strength, which controls the interaction of the particles with the accumulation wall. Polymer analysis Polymer analysis is a n o t h e r a r e a well suited for FFF, especially thermal and flow FFF. Generally, thermal F F F is used for organophilic polymers and flow F F F is used for water-soluble polymers. Marcus Myers (FFFRC) discussed the influence of experimental factors on the separation and evaluation of polymers. These factors include strength and nature of the applied field; flow rate; sample size; and solvent characteristics, such as boiling point (thermal FFF) and ionic strength (flow FFF). Seungho Lee (3M) described the characterization of high molecular weight intraocular lens materials by thermal FFF coupled with a multiangle laser light scattering detector. This detector allows determination of the absolute molecular weight and the radius of gyration of a polymer, provided the refractive index increment and the second virial coefficient are known. The results depend on the accuracy of these parameters. Don Rosenhamer (BP America) described the advantages of thermal FFF over gel permeation chromatography for the analysis of microgels in elastomers. Thermal FFF provides similar accuracy and better precision than does gel permeation chromatography but does not require a filtration step for sample p r e p a r a t i o n , which can cause problems because of shear degradation of polymers. In addition, because of its open channel feature, thermal FFF does not suffer from perturbation of adsorption effects. Rosenhamer suggested using an evaporative light-scattering detector for the wide variety of samples analyzed by thermal FFF. Thermal FFF can be applied not only to homopolymers but also to copolymers. The characterization of the separation requires an understanding of the influence of the copolymer structure on the thermal diffusion coefficient, which is a measure of the rate of polymer migration per unit of thermal gradient. Mauricio Hoyos (Ecole Supérieure de Physique et Chimie Industrielles, France) showed that the thermal diffusion of statistical vinyl chloride-vinyl acetate copolymers is independent of the chemical composition. Chad Rue (Boise S t a t e U n i v e r s i t y , ID), h o w e v e r , found that the thermal diffusion of block copolymers depends on the relative thermophoretic mobilities of the polymer segments segregated in
the inner and outer regions of the molecule. Because of the lack of an absolute thermal diffusion theory, the determ i n a t i o n of a polymer molecular weight distribution by thermal FFF r e q u i r e s a calibration procedure. This can be performed by using a series of standards with narrow molecular weight distributions, as shown by Peter Chen (FFFRC). Alternatively, Ron Beckett d e m o n s t r a t e d t h a t when such standards are not
"The lack of available commercial instruments has, for many years, hindered the use of FFF in industrial environments. However, the situation I is changing." available, multiple broad molecular weight standards can be used. Jianzhong Lou (FFFRC) showed t h a t separation of polysaccharides dissolved in dimethylsulfoxide based on molecular weight by thermal FFF can be monitored by an evaporative light-scattering detector. He also reported on the analysis of ultrahigh molecular weight polymers by thermal FFF. Retention of these polym e r s was influenced by lift forces acting on the macromolecules. In a similar study, our group found that the initial relaxation period following sample injection has a significant effect on the retention. We also showed that periodic oscillations of the elution profiles of the fractograms of these polymers are attributable to hydrodynamic instabilities that occur during migration of these highly viscous samples. Biological applications Various FFF techniques are suitable for the separation of biological species. The most versatile method is flow FFF, which can be applied to macromolecules of relatively low molecular weight and to micrometersized biological particles. Several examples of the use of flow F F F for rapid separation of proteins, viruses, virus aggregates, plasmids, polysaccharides, and unicellular algae were
shown by K a r l - G u s t a v W a h l u n d (University of Lund), who reviewed the present status of the asymmetrical version of flow FFF. This technique uses a single permeable wall rather than the two permeable walls used in symmetrical flow FFF. In the asymmetrical channel, a single flow inlet port is used; two flow outlet ports allow a fraction of the carrier liquid flow to elute at the downstream end of the channel, together with the sample species, while the complementary fraction permeates through the membrane. The symmetrical channel, comprising two inlet and two outlet ports, allows the independent introduction of the axial and the transversal carrier flow streams. Wahlund's lecture prompted a lively discussion about the respective advantages of the two versions. On the basis of this discussion, it appears that the symmetrical mode allows easy sample characterization from retention data, whereas t h e asymmetrical system provides fast and efficient separations. As an example, Anne Litzén (University of Lund) described the separation of monoclonal antibody aggregates. Min-Kuang Liu (FFFRC) pointed out that the symmetrical flow FFF system can be used to characterize the diffusivity of both single- and double-stranded linear and circular DNA molecules. Ping Li (FFFRC) added that the system also allows for the separation and characterization of various protein complexes, protein aggregates, and biological cells and for the determination of the lipoprotein profile in blood plasma samples. Sedimentation F F F can also be used for biological applications. Birgit Langwost (University of Utah) d e m o n s t r a t e d t h a t this technique allows q u a n t i t a t i v e monitoring of solid-phase immune reactions. An antigen (human IgG) was nonspecifi cally adsorbed onto polystyrene latex particles to form a monolayer that, in a second adsorption step, specifically adsorbed an antibody (rabbit antihuman IgG). Sedimentation FFF allows high-precision quantitation of substances adsorbed in these two steps. Chromosomes as well as blood cells and E. coli strains were separated by sedimentation FFF, but a particular wall material (polymethyl methacrylate) had to be used to avoid strong adsorption on the wall, as shown by Yong Jiang (FFFRC). One-gravity sedimentation F F F provides a field strength sufficient for retaining and separating cellular materials. Maciek Zborowski (Cleve-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993 · 317 A
FOCUS land Clinic Foundation, OH) used this method to determine the degree of cell viability. Viable and nonviable cells elute as separate peaks. In an attempt to screen potential anticancer agents, James Bigelow (University of Vermont) used this technique to identify a fraction of soil microorganisms, obtained from caves, that consistently exhibit marked cytotoxicity against cultured tumor cells. Philippe Cardot (Université de P a r i s Sud, France) showed some examples of applications of the technique for the isolation of parasites and discussed its potential use in pathological diagnosis. Another technique t h a t may become prominent in future biological applications is SPLITT fractionation. Bor Fuh showed how this method can be applied for continuous preparative fractionation of a three-protein mixt u r e , u s i n g a n electrical d r i v i n g force. C u r r e n t l y , t h i s method r e quires a minimum difference of two pi units for protein separation. The t h r o u g h p u t was approximately 15 mg/h. Fuh also discussed examples of fractionating h u m a n blood by a c e n t r i f u g a l S P L I T T s y s t e m . Although the number of outlet streams in such a continuous separation device is limited to two or three, he pointed out t h a t in principle it is possible to arrange several SPLITT cells in series to separate relatively complex materials.
Methodology Obtaining molecular weight or particle size distributions from fractograms requires appropriate data treatment. For highly accurate determ i n a t i o n s , t h e influence of b a n d broadening in the channel (because of flow inequalities) should be taken into account. Francesco Dondi (University of Ferrara, Italy) presented the method he has applied to polydisperse p a r t i c u l a t e samples. The validity of the method was verified by electron microscopy. One advantage of F F F methods over LC methods, when they can be applied to the same samples, is that F F F channels have much lower surface areas of contact with solid materials. Still, surface area effects are not absent in FFF, especially when high field strength pushes the sample species close to the accumulation wall. More or less irreversible adsorption on this wall will limit sample recovery. Kim R a t a n a t h a n a wongs investigated this aspect, defined different recovery categories, and recommended various strategies for improving sample recovery.
Theory Thermal F F F is an efficient method for polymer separations. However, unlike sedimentation and flow FFF, retention in thermal FFF cannot be predicted a priori because the underlying t h e r m a l diffusion process in liquids is poorly understood. Thermal FFF can be used as a physicochemical tool to expand the database on thermal diffusion. Conversion of the retention data into thermal diffusion parameters is complicated by the fact t h a t relevant transport parameters cannot be considered as constant because of the temperature variation in the channel thickness. Arian v a n Asten (University of Amsterdam, The Netherlands) investigated the influence of these temperature dependencies on the derived thermal diffusion parameters. Martin Schimpf (Boise State University) summarized what has been learned about thermal diffusion through the application of thermal F F F and presented a model for thermal diffusion based on the t e m p e r a t u r e dependence of the surface tension. Even when perturbation of particle wall interactions can be avoided, retention in F F F may be affected by the occurrence of particle-particle hydrodynamic and/or electrostatic interactions when samples of finite sizes are injected. To account for this effect, I presented a generalization of the classical retention model of the Brownian mode of FFF, based on the state equation of hard spheres and dense fluids, and discussed the application of this model to real particles. In the Brownian mode of retention, large particles elute after small ones. However, when the particle size is large enough, retention becomes influenced by steric hindrance near the accumulation wall and by the development of hydrodynamic lift forces repelling the particles from this wall. When these steric or lift modes are predominant, large particles elute before smaller ones do. Accordingly, an inversion in the elution order occ u r s a t some p a r t i c l e d i a m e t e r , which complicates separation and characterization. Giddings presented a general theoretical treatment of methods to manipulate the value of the inversion diameter by proper selection of the operating parameters. In addition, he suggested the possibility of using this inversion phenomenon to measure particle densities, shapes, and distributions. The F F F analysis of polydisperse particulate samples is generally per-
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formed by using a programmed decrease in field strength. A power program was previously developed for the Brownian mode of retention in such a way that the relative degree of fractionation is independent of particle size. Stephen Williams (FFFRC) discussed the extension of such a program when the steric effect influences the retention of particles, and he presented general guidelines for selecting an optimum program.
Instrumentation Sedimentation, flow, and t h e r m a l F F F are the primary technologies used for macromolecular and particulate analysis, as evidenced by most symposium presentations. Developments in instrumentation for all F F F methods were also chronicled. Anne Litzén discussed optimization of channel geometry and flow r a t e s in a s y m m e t r i c a l flow F F F , which is more complicated than the symmetrical version of the method because of the variation in axial flow velocity. Another possible improvement for flow FFF is the use of porous hollow fibers. J a n A k e Jônsson ( U n i v e r s i t y of L u n d ) d i s c u s s e d advances in this technique and mentioned that hollow fibers with sufficiently homogeneous pore distributions improve separations. Wim Kok (University of Amsterdam) used this technique to investigate the separation of polystyrene sulfonates. In the past, flow FFF was performed with aqueous carriers; recently, chemically resistant membranes have been used to perform flow FFF with nona q u e o u s s o l v e n t s , a s s h o w n by Michael Miller (FFFRC). Electrical F F F , conceived more t h a n 20 years ago, has not been used often because of poor performance associated with the presence of membranes in the FFF channel used to isolate the electrodes from the sample species. Karin Caldwell (University of Utah) presented results obtained with a new system that uses impervious polished graphite bars as electrodes, simplifying channel construction. The system was used to separate particles according to size, and a steric transition was observed. Although most F F F separations are carried out at or near room temperature (cold wall temperature for thermal FFF), it is possible to operate at high temperature. Frank Yang (FFFractionation) presented a hight e m p e r a t u r e t h e r m a l F F F system, p r i m a r i l y designed for polyolefin analysis, in which the cold wall temperature may reach 150 °C or more. Yuehong Xu (FFFRC) performed sed-
imentation FFF at elevated tempera tures and obtained improved separa tions of particulate standards as a result of reduced band broadening from enhanced sample diffusivity. In an attempt to use FFF for pre parative purposes, Cornelius Ivory (Washington State University) tried to extend t h e continuous bidimensional system (devised 15 years ago by Giddings a n d Myers) for onegravity separations to centrifugal separations. However, t h e develop ment of hydrodynamic instabilities due to Coriolis and centrifugal forces has prevented the successful opera tion of this rotating system. Conclusion In addition to the lecture and poster sessions, there were three discussion sessions devoted to a p p l i c a t i o n s strategies, bridging theory and prac tice, and technical needs and devel opments. Lively a n d i n t e n s e e x changes a m o n g t h e p a r t i c i p a n t s ensued. As Giddings said, the size of this audience was probably close to optimum for promoting discussions among t h e conferees. FFF '92 was the third FFF meeting held in three
and a half years, and many partici pants had attended one or both of the previous symposia. This type of par ticipation greatly contributes to cre ating an international FFF commu nity and reinforces the links among different groups involved in the de velopment or application of FFF. Certainly, most FFF developments have occurred at the FFFRC. Indeed, 28% of the lectures and 48% of the posters were presented by the cen ter's researchers. Over the years, an increasing number of advances have come from other institutions, b u t only a few presentations were given by industry. However, it was obvious from the discussions that the indus trial participants were extremely in terested in the high-resolution capa bilities of FFF compared with other methods that are used to character ize and separate macromolecules and particulate materials. The lack of available commercial i n s t r u m e n t s h a s , for many years, hindered the use of FFF in industrial environments. However, t h e situa tion is changing. It is expected that as t h e number of applications i n creases and as instruments for the
INDUSTRlAL & ENGINEERING =====
three main F F F techniques (sedi mentation, thermal, and flow) b e come commercially available around the world, the use of this methodol ogy for solving problems in industry will steadily grow.
Michel Martin received his Ph.D. in 1975 from the Université Pierre et Marie Curie (Paris) under the direction of Georges Guiochon. Following post-doctoral study at the University of Utah, he pursued research at the Ecole Polytechnique. In 1988 he joined the Ecole Supérieure de Physique et Chimie Industrielles in Paris. His research focuses on the physicochemical and hydrodynamic aspects of separation methods, with emphasis on the theoretical and experimental developments in FFF and chromatography.
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