Fractionation A Versatile Method for the Characterization of Macromolecular and Particulate Materials It gives me a sense o f the fleetness of time to realize that the concept offield-flow fractionation (FFF) has been occupying us continuously for 15yearsasofthisyear (1980), presenting incredible challenges and frustrations mixed with intervals of buoyant success. The bursts o f success are idyllic moments in any scientist’s life, and l a m happy to share this moment of recognition with some of those present today who have suffered and succeeded with me, especially Marcus Myers, who has worked with me on FFF since its inception, and Karin Caldwell, who has spent eight years helping develop FFF. Also here today are Frank Yang and Michel Martin, both of whom have made significant contribu tions. My other important coworkers are listed at the end of this paper. May I also acknowledge unsung heroes Robert Melville and his colleagues at NIH, who believed in our work through thickand thin, and kept our program alive during 15 of the 23years o f continuous NIH support.
1170A * ANALYTICAL CHEMISTRY,
Since the time Henry Eyring introduced me to chromatography some 21 years ago ( I ) , I have been fascinated by the forces driving.molecules in and out of the stationary region (phase) of a chromatographicsystem. At the beginning of the 19608, I wondered if centrifugal forces could he used to drive molecules into quiescent regions of a column, thus creating a kind of chromatography (which I called centrifugal chromatography) without a stationary phase. I did lengthy theoretical calculations to check the feasibility of the idea. I calculated the spin forces needed to push small molecules and isotopic species (which I was then interested in) over the short distances needed for effective chromatographic operation, and found that the most powerful ultracentrifuge was far too weak. I put aside my notes on the matter (see Figure 1)and forgot about it, a t least at the conscious level. A few years later, I started thinking about the special challenges of separating macromolecules by chromatography, and I eventually published a paper on the subject (2).In a completely different area, I considered applying my nonequilibrium theory to thermal diffusion columns. In 1965, all these ideas came together. In the course of a trip to Wyoming that year, I had occasion to spend the night in a motel in Evanston just north of the Utah border. Noises kept me awake, and I pondered anew the question of how to restrain molecules in quiescent regions of a column without using a stationary phase, which had certain limitations for the goals I had in mind. I toyed again with the idea of using external fields. In a flash it occurred to me that a thermal gradi-
VOL. 53. NO. 11, SEPTEMBER 1981
ent might do everything I wanted. Not only would species be forced by thermal diffusion into a distinct region of the conduit, but the temperature drop itself would, by virtue of its effect on viscosity, create an effective stationary phase a t the high viscosity extreme of temperature. It was an exciting moment, followed by a sleepless night of speculation. I soon realized that laminar flow in a channel, without any temperature gradient at all, creates a kind of stationary region near the channel wall due to the viscous drag of the surface. The concept became very general; we could use any kind of external field or gradient (including centrifugal and electrical) that could push molecules or particles toward the wall. I soon wrote a short paper outlining many of the apparent variations and advantages of the new “field-flow fractionation” technique (3).I predicted that FFF would “exhibit a distinct advantage in separating macromolecules and colloids, because of its essentially one-phase nature.” Indeed, FFF is most advantageous with macromolecules and colloids, but its fractionating power apparently extends far in either direction, presently from 30-pm particles, far beyond the colloid range. The effective mass or molecular weight range is -103-1017, an unparalleled 1014-foldrange of continuous separation. We will add several more orders of magnitude to this range before we we through. Figure 2 shows the resolution of several small polystyrene latex beads by FFF. No other method shows such high resolving power for colloidal particles. 0003-2700/81/A351-1170$01.00/0
0 1981 American Chemical Society
J. Calvin Glddings Department 01 Chemistry
University of Utah Sail Lake City, Utah 841 12
Part of initial page of a der& ng from about 1960 describ
ifugal chromatography." a of sedimentation fieid-flow on
The study of macromolecules and particles has assumed increasing importance in recent years as increased efforts have been made to understand the vital role of these species in the organization and functioning of living systems, the formation of the sedimentary structures of our earth, the recovery of natural resources, the contamination of our environment, and in the performance of myriads of industrial products. The characterization of these entities presents enormous challenges, which have not yet been sufficiently addressed by the careful methodologies of analytical chemistry. In the following sections I will attempt to show that FFF has considerable potential in this important area.
Prlmlplesof FFF In its present form, FFF is carried out in a thin (50-500 pm) ribbonlike flow channel, with an external field or gradient applied acrosa the channel faces (see Figure 3). The field serves to force molecules or particles toward one wall, where their downstream velocitv is diminished because of the slug&h flow of carrier fluid caused by the drag of the wall. Components that interact strongly with the field are pushed into layen against the wall,
which may extend no more than 1-10 pm into the flow stream. Such thin layers may he carried along a t velocities only a few percent of the mean velocity of the carher in the flow channel, because of extreme flow retardation so close to the wall. The increasing compression of the layers, along with increasing interaction with the field (which leads to increasing retention or retardation), is responsible for the differential migration and thus seDaration of comwnents. as illustrated in Figure 2. While I will avoid the detailed development of theory, I would like to point out the important analytical role played by that theory in FFF. Because the FFF channel is open (unpacked) and geometrically simple, and its flow well characterized, both retention and peak broadening can be rigorously described by theory. Theory thus becomes a tool for optimization, for checking the proper operation of FFF devices, and, most important analytically, for characterizing (and thus identifying) the emerging components by using the information reflected in observed elution times and oeak broadening. The generation'of analytical information by FFF will be addressed more completely in the last sect ion. ~~~
~~
~
~
~~
~
Theory shows that the retention of a component is related to the mean layer thickness e of that component and to channel thicknesa w bv
R = E [coth W
(E]- $1
(1)
where retention ratio R is the ratio of the component velocity to the mean carrier velocity (4).When component layers are compressed enough so that R < 0.5, Equation 1can be accurately approximated by the simple form
R
= Selw
(2)
Layer thickness e is given by
e = BTIF
(3)
where 5 7 is the gas constant, T the absolute temperature, and F the force imposed by the field on Avogadro's number of component particles. The substitution of Equation 3 into Equation 2 gives
R = SBT1Ftu
(4)
which is an extremely simple but usuallv accurate exmession relatine observed retention ratio R to the k e r a c tion force F between the component particles and the field. Force F refers to different proper-
ANALYTICAL CHEMISTRY. VOL. 53. NO. 11. SEPTEMBER I981
1171 A
The scope of FFF, the newer technique, is expanding rapidly. FFF methods are presently applicable to hoth aqueous and nonaqueous systems. They apply to diverse materials of biological, industrial, and environmental origins. The mass range of particles to which FFF is already applicable is -10’4, as noted earlier. This range is shown in Figure 4 in comparison with the range of chromatography, which is much more limited. (Hydrodynamic chromatography is not included because it is not truly chromatographic, lacking a stationary phase, and it is not very selective.)
Advantages of FFF
Figure 2. Separation of three sizes of colloidal particles (polystyrene latex beads) by sedimentation FFF. Particle diameter is shown
ties of the components, depending on which field is used. If an electrical field is applied (electrical FFF), F relates to the particle charge or mobility (and the field strength). If a centrifuge is used (sedimentation FFF), F relates to the effective mass of the particles. A thermal gradient (thermal FFF) ties in with thermal diffusion parameters. A cross-flow of carrier liquid (flow FFF) leads to particle size. The exact way in which these various parameters control retention will be shown in the last section. Relatlve Scope of FFF and
Chromatography FFF is dynamically much like chromatography hecause it is a flow system in which species “partition” between a relatively stationary region (near the wall) and a relatively mobile region (away from the wall). However, the partitioning occurs in one phase, not between phases, so the method is not technically chromatographic. With some license FFF can be termed “one-phase chromatography” (5). Chromatography is, of course, an extremely diverse technique, utilizing many variations in scale, flow, phases, and programming, to gain command of refractory analytical problems. However, in terms of basic methodological variahility, FFF appears to be as diverse as chromatography. Table I compares the intrinsic diversity of the two, showing them to be essentially comparable. FFF is also subject to an enormous variety of experimental manipulation to optimize the control of retention, 1172A
resolution, speed, sample capacity, analyte type, analyte mass, and mass range. Table I1 shows many of the experimental variables subject to manipulation, alongside analogous chromatographic variables. The major separative parameters affected by each kind of manipulation are shown on the right side of the table. Again, FFF and chromatography appear comparable in experimental scope. FFF and chromatography are largely complementary in applicability. Chromatography works best with lowmolecular-weight materials, FFF with high. There is, however, a substantial region of overlap in which relative advantages will have to be worked out.
Stated succinctly, FFF has the triple assets of simplicity, effectiveness, and versatility. With respect to the latter, it appears likely that FFF will eventually prove applicable to almost any complex mixture of soluble or suspendable molecules, macromolecules, or particles. The 10’4-fold mass range of applicability has been noted. The potential for high resolution and speed over this entire mass range is excellent, especially when compared to other methods applicable to macromolecules and colloids. The favorable position of FFF as an analytical separations tool relates to several specific characteristics including: FFF is a continuous-flow separation method (like chromatography) in which eluted fractions can be conveniently detected and collected as they emerge. The collected samples can be identified and/or subjected to suhsequent analytical steps using spectroscopic, electron micrhscopic, biological, or other FFF methods. As noted earlier, FFF is theoretically “clean” because of the well-defined geometry, field strength, and flow pro-
Table 1. Enormous intrinsic Diversity of Chromatograoh Probably Matched by Diversity of FFF Chranalwraphy
Core melhodr
Maw v a r l m k 1. AHeredllow
Thermal FFF, electrical FFF. dimentation FF magnetic FFF. FFF wII other fields
Thla layer chmmatosraphy
Flow FFF (solvent
Size exclusion chromatog
thermogravltationalFFF Steric FFF
exchange),
mechanics 2. AHered basts of
retention 3. Program variables 4. M e r
ANALYTICAL CHEMISTRY, VOL. 53, NO. 11. SEPTEMBER 1981
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Temperature, flow, solvent strength Continuous operhtion.
Field strength, flow. carrier
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ANALYTiCAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
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.
-component
i ’~ayers----/
Figure 3. Side view 01 ribbonlike FFF llow channel. The lie iradient applied across the thickness of the channel compresses solutes into layers of different thickness, depending on how strongly they interact with the field. The thinner the layer. the slower its motion because of the “low flow” condition caused by Irictional drag near the wall of the channel. Zones A. 8. and C are, of course. diffuse at the top and the edges: this figure shows only the relative compression and associated downstreammotion
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file. Thus the physicochemical properties noted above (charge, mass, size, etc.) can be accurately related to elution characteristics, in support of analytical goals (see next section). The experimental apparatus for FFF i s very “elastic,” which means that many kinds of fields can be applied, different channel dimensions employed, and various surface geometries and chemical properties utilized, each bringinginto focus different characteristics. Retention i s controlled by external
MW-
I
d ( m ) -0oo01
fields and gradients that can he altered quickly, and almost infinitely, to whatever levels are best for a given separation. Fields can be turned completely off b flush out residues. Field strength can be varied in any desired manner during a run to create a versatile programming system to extend the range of separation. A new separation problem does not generally require a new flow channel; it only requires a new field strength or field program. FFF has an intrinsically high resolving power, particularly when com-
103
10‘
109
10’2
10’5
10’8
0001
001
o1
1
10
100
I
Chromatography
.I.
(Overall Range)
1111
......
Sedimentation FFF
Thermal FFF
..
Steric FFF 1 1 1 1 .
-..I.
Flow FFF
I...
Electrical FFF
. . . . . I . . I . . . . . . .
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Figure 4. Range of chromatography and FFF on approximate scales of molecular weight (MW) and particle diameter (a.Solid lines represent known capabilities: dashed lines are speculative or else tried but poorly performing. End Doints are somewhat arbitrary
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
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1175A
analyte mass, sam 3. Field combinati
Analyte type. rete Speed, resolution Retention. resolut Retention, analyte ty
9. Channel brea 10. Channel thickn
Resolution. sample ca
Column diamet
Particle diameter Temperatwe progra
Speed, resolution, samp
Resolution. mass range.
13. Carrier progra 14. Surface relief 15. Wail permeab 16. Second dimen
e mass. solvent exchang
pared with-othermacromolecular separation methods (6). Clear directions exist for generating information very rapidly hy FFF, despite the fact that the transport processes underlying the separation of macromolecules and narticles me verv sluggish (7). The one-nhase nature of FFF means that there are minimal interfacial influences to complicate component migration and the interpretation of elution data. Different surfaces can be used to further reduce interactions with complex macromolecular/colloidal materials. The FFF technique operates without the use of abrupt forces, interfacial transport, and strong shear gradients (it altogether lacks extensional shear), all of which can be disruptive to high-molecular-weight polymers, weakly bound molecular and particulate aggregates, delicate biological macromolecules, and even living cells. I will now proceed to show how analytical information can be extracted from FFF, based largely on the above advantages.
Table 111. Particle Parameters (RightBe Obtained from FFF Retention Me Retention Equations In the Center Column’ Prhclpal p.rtlc*
~I
FFF and Analytical Information Creating information is the core business of analytical chemistry. FFF is a powerful information-producing tool, all the more promising because it spans such a wide and difficult range of complex materials. The high quality and yield of information is generally related to the advantages of FFF cited above. For example, high resolution and speed, along with all the advantages contributing to versatility (and 1176A
ANALYTICAL CHEMISTRY. VOL.
F ff nmlhed
Sedimentation FFF
-urn
R=
6% T
M = molecuku weight
w 1 - PlP*)W
ps = density
Electrical FFF Flow FFF
thus wide applicability), aid information flow. Of special importance, however, is the theoretical exactness of FFF, which aids the acquisition of accurate information with minimal needs for calibration. The time needed to elute a particle can be rigorously related to that property of the particle interacting with the field. In the case of sedimentation FFF, it is the mass of the particle (less the buoyant mass) that couples with the centrifugal field to produce displacement toward the channel wall. This mass (along with the density difference controlling buoyant forces) is 53,
NO.
11, SEPTEMBER 1981
pm.nuh.n churl.*.M by mlmllon
R.l&
= diffusion coefficient p = mobility D = diffusion cmfficient
(a = Stokes radius)
therefore reflected in the measured elution time or volume. We now believe that we can measure mass parameters to almost l%accuracy in favorable cases ( 8 ) .In this way, we can measure the molecular weight of particles such as viruses and virus clusters (9, IO),information much needed hv viroIo&ts. Different subtechniaues of FFF yield different particlLparameters, as shown in Table 111. A single retention measurement, of course, yields but a single particle parameter. This parameter may relate to a host of other parameters, but to get them all we need
i
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When used as a syringe-loading injector. the syringe calibrations determine sample volume. You can inject as little as 1pl with zero sample loss. When used as a loop-loading injector, the loop size determines sample volume. You get precise repeatability with any of eight sample loops from 1O p l to 2ml.
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1177A
counts.
other measurements. Fortunately, many of these additional measurements often stem from FFF elution diagrams obtained under different conditions. For example, while a single measured retention volume in sedimentation FFF is a function of both particle mass and density, measurements of retention in carriers of two or more densitiesyield both mass and density, so that both are specified by the two (or more) measurements (11). Retention measurements are not alone in yielding particle parameters. Zone broadening yields the diffusion coefficient D.Since D relates to the size (e.g., the Stokes radius a ) and shape of particles, this measurement can be used to check the self-consistency of retention-based parameters, such as molecular weight M. For nonhomogeneous or polydisperse particle collections-perhaps the greatest analytical challenge-we can get detailed parameter distributions (such as molecular weight distributions or particle diameter distributions) because FFF separates as it measures. Narrow cuts can be collected and subjected to additional analytical measurements, including, if desired, measurements by subsequent FFF procedures. For example, a plot of zone spreading (as measured by plate height) vs. velocity for the cut will yield the diffusion coefficient and related particle size parameters. If the cut from a sedimentation FFF fractogram contains particles of different densities, the zone configuration resulting from rerunning the cut with a new carrier of a different density will reflect the density (and mass) distribution within the cut. The possibilities are nearly endless. Also, we now believe that particle density and shape information for polydisperse samples will he forthcoming from the steric transition, the point at which the elution diagram folds back on itself as a consequence of the transition from normal to steric FFF. This latter phenomenon is under active investigation in our laboratory. Despite our 15 years of effort, I believe we have harely begun to exploit the analytical potential of FFF, or even to envision some of the major ways in which analytical information might arise from its use. I owe more than I can say to my coworkers, who have not only made FFF workable, but have provided an amiable working environment. I t is an ex. ceptional group of which I am proud. Following the format used by the preceding Fisher Award winner, Velmer Fassel(12), I acknowledge below all co-workers with whom I have jointly published FFF papers. They are listed in order of decreasing number of joint FFF publications, the number being
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shown in parentheses. I regret that this leaves out non-FFF co-workers from the list and non-FFF papers from the numbers compiled for some eo-workers. Marcus N. Myers (49), Karin D. Caldwell (21), Frank J. Yang (12), Michel Martin (7), George Karaiskakis (6),LaRell K. Smith (5),Gwo-Chung Lin (4), Gary H. Thompson (4), Margo E. Hovingh (3), Susan R. Fisher (Z), Kathy A. Graff (2), Josef Janca (Z), Laya F. Kesner (21, John F. Moellmer (2). Thanh T. Nguyen (2). Young Hee Yoon (Z), Steven L. Brimhall (l), Thomas H. Dickinson (l),Eli Grushka (l), Joseph Pav (1). and Horace M. Mazzone (1). References (1) Giddings, J. C. In “75 Years of Chromatography-A Historical Dialogue”; Ettre, L. S.; Zlatkis, A,, Eds.; Elsevier: Amsterdam, 1979.87-98. (2) Giddings, J. C. J. Gas Chromatogr. 1967,5,413-19. (3) Giddings, J. C. Sep. Sei. 1966,1,123-5. (4) Grushka, E.; Caldwell. K. D.; Myers, M. N.; Giddin s, J C Separation and Purification dethbdi 1973.2.121-51.
(5) . . Giddines. J. C. J. Chromatoer. 1976. I
125,3-16: ’ (6) Giddings, J. C. Pure & Appl. Chem.
1979,51,1459-71. Martin, M.; Myers, M. N. J. Chromatogr. 1978,158,419-35. (8) Karaiskakis,G.;Myers, M. N.; Caldwell, K. D.; Giddings, J. C. Anal. Chem. 1981.53.1314-17. (9) Catdwell. K. D.; Nguyen, T. T.; Giddings, J. C.; Mazzone, H.M. J. Virologicof Methods 1980.1. 241-56. (10) Caldwell, K. D.; Karaiskakis,G.; Giddings, J. C., submitted for publication in J. Chromotopr. (11) Giddings, J. C.; Karaiskakis,G.; Caldwell, K. D. Sep. Sci. Technof. 1981.16, in press. (12) Fassel, V. A. Anal. Chem. 1979,51, (7) Giddings, J. C.;
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These inveati stions were supported by NIGMS Grant E M 10851-24. National Institutes of Health.
J . Calvin Giddings is professor of chemistry at the Uniuersity of Utah, where he earned his PhD in 1954. His research interests include field-flow fractionation, macromolecular separations, the theory ofseparations, high-pressure chromatography, the theory of chromatography, enuiron. mental science, and world population.
