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Karln D. Caldwell Department of Bioengineering Center for Biopolymers at Interfaces university of Utah Salt lake City, UT 841 12
Recent years have seen an explosive growth in methodology, instrumentation, and data handling for liquid chromatography of macromolecules. New packing materials are steadily emerging that allow the user to perform highly efficient separations with low nonspecific adsorption and minimal hydrolysis of the stationary phase, even under chemically harsh conditions. Matrices for size-exclusion chromatography (SEC) are becoming even more porous and therefore capable of processing polymers of ultra-high molecular weight and even fine colloidal particulates. Parallel to this activity, although somewhat less visible, has been the development of field-flow fractionation (FFF) into a set of rapid and highly selective separation tools suitable for processing samples that range over 15 orders of magnitude in molecular weight (1). This family of techniques may be described as a one-phase analogue of chromatography in which samples, instead of being differentially partitioned into some stationary phase, are being partitioned into regions of different carrier velocity in an open, unobstructed channel. Figure 1outlines the principle of the method and gives an overview of the experimental setup. The channel is generally a thin duct of rectangular cross section whose smooth, parallel walls confine the carrier to laminar flow. The partitioning, in turn, is accompanied by an externally applied field that acts in a direction perpendicular to the flow. The nature of the selected field confers on each FFF subtechnique its special selectivity (see Figure 2). 0003-2700/88/0360-959A/$O 1.50/0
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1988 American Chemical Society
The well-defined geometry of the channel and the ability to easily and accurately control themagnitude of the applied field have permitted the development and experimental verification of theoretical models that describe both retention and zone broadening (2, 3). Most of the theoretical development can he credited to J. C. Giddings
at the University of Utah, who was the first to postulate the new separation principle in 1965. Since that time, ample experimental evidence has accumulated in support of the early models that were established for the four types of fields used most commonly to date: sedimentation, thermal, hydraulic ("flow"), and electrical.
Flgura 1. Schematic of the experimental arrangement: Laminar flow through the thin channel (thickness w) transports sample zones from Injector to exit port. The zones maintain their field-induced equilibrium distribution duing passage, and elution t i m a we gwerned by me layer thickness characteristic for each zone. w that < he system's computer regulates bdh field and flow and processes me detector signal.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
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Flgure 2. Mass selectivity'range for commercially available FFF instrumentation. For each technique. maximum selenivity is reached at a retention of 10 wlumn MIW and is maintained until the (fielbdependent)onsel of sieric elf&; fa flow and " a 1 FFF. Its value ranges hm 0.33 lo 0.65, depending on molewlar mfametlon. $r curves fathe two sedlmenfationsystem reflect operation at maximum field sample and solvent denskies are 1.50 and 1.00 p/mL, mspctively. A repre s"live value la sIze-exd1181on chomaiographyis shown tor mmparison.
The recent HLPC '88 conference in Washington, DC, devoted a day-long segsion to FFF, and contributions from several European and US.laboratories testified to a growing interest in the technique. In addition to much experimental evidence for the soundness of retention and plate-height theory in their present state, the session also included a Monte Carlo simulation of the sedimentation FFF process (4), which generally confirmed the analytically derived theory. The relationships between the retention volume observed a t a given field strength and the molecular weight M or size dp of the sample have been discussed in detail elsewhere (2,5);Figure 3 summarizes the particular sample characteristics that are accessible with the different subtechniques. The ability to model a separation procesa from first principles is particularlv valuable in characterization work because it eliminates the need for elaborate calibration procedures. Although standard samples of given molecular weight and polydispersity are frequently used also in FFF, their purpose is more to verify the system's performance than to fill a calibrating function. This is not true for thermal FFF, where the lack of a good physical model to describe the phenomenon of thermal diffusion necessitates using standards to convert observed retentions into sample molecular weights or hydrodynamic radii. The availability of theoretical models that describe retention and zone hroadenine for the various FFF subtechniquesallows the operator to select optimal conditions for rapid resolution of any pair of samples with known properties. In the case of multicomponent mixtures, chis will often suggest the use of gradient elution, which in FFF is accomplished through a gradual reduction of field strength. With field and flow under microprocessor control it is therefore possible to rapidly achieve sample resolution.
Apparatus and techniques: general
..
J on subtech D, to measured retention volume. V,, expresses different characteristics of the sample (e.g., molecular weight M, particle diameter 4,diffusivity D, thermal dlffusivlty Dr. or electrophoretic mobility, p).
Exponent "a" varies wllh mlemlar mformailan:Its value ranges between 0.33 and 0.65.
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ANALYTICAL CHEMISTRY. VOL. 60, NO. 17, SEPTEMBER 1, 1988
description System. An overview of the experimental arrangement is given in Figure I. In the center the column is outlined as a thin flat duct with tapered inlet and outlet. Although it is possible to perform separations under the coupled influence of field and flow also in channels of other geometries, such as the cylindrical fibers used by Lightfoot in his polarization chromatography (6). m o a experimental results published to date have originated in thin columns of parallel place geometry. To minimize zone-broadening effects caused by sluggish sample transport along the side walls of the channels, their breadth-to-thickness ratios are gener-
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A mischievous mixing cavity often occurs where LC tubing connects to a sample injector port. Though tiny, the cavity can disperse sample enough to degrade resolution. We've found effective ways to fight such cavities. And we tell you about them in our Technical Note 9. It discusses mixing cavities and other sources of extra-column
dispersion. Shows how to minimize dispersion. And provides guidelines for choosing the bore and length of connecting tubes. If you need very low dispersion-as when using 1-mm or 2-mm bore columns -you'll find Technical Note 9 particularly helpful. Because it also describes our minimum-dispersion injector, Model 8125, and the unique CIRCLE 140 ON READER SERVICE CARD
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
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ally kept in excess of about 50. Because resolution increases with a decrease in the channel thickness w (7), there is a continuing effort to design channels thinner than those with a LL‘ of around 250 pm, the size used most extensively to date. In reducing w , however, one must pay increased attention to the smoothness of the major walls as well as to their structural integrity. Roughness and bowing will lead to progressively larger departures from ideal behavior, and therefore to reduced accuracy in the retention-derived sample characteristics. The open-channel geometry causes only moderate resistance to flow, and no extraordinary demands are placed on the pressures to be generated by the pumping system responsible for carrier delivery. However, pulsing flows are poorly dampened by these channels, and without additional dampeners, peristaltic and other pulsing pumps should be avoided in high-resolution FFF work. Detectors and recorders are the same as those used in regular high-performance liquid chromatography (HPLC) systems. As more and more FFF systems are computer controlled, there is a tendency to adapt the configurations used in modern HPLC, which entails substituting the recorder with a video monitor for real-time display of the course of the separation and a printer for producing a hard copy of the frectogram. In addition to its data display function, the computer normally processes the detector signal interactively to give particle diameter and molecular weight information for selected positions in the fractogram. The strength of the applied field is inversely related to the zonal layer thickness (see Figure l),which determines the retention time for a given sample. The introduction of computerized field control has, more than any other FFF improvement, enabled the operator to perform highly reproducible fractionations under optimal conditions of analysis speed and component resolution. Although much of the methods development has been done using a constant field, sometimes referred to as “isocratic” conditions, it was realized early on (8) that processing of complex sample mixtures, whose components spanned a large mass range, might profitably involve a programmed reduction in field strength, as shown in Figure 4 (9). Recent theoretical analysis by Williams and Giddings (10)has led to the formulation of some general guidelines for the choice of program form, which can be easily implemented through the computer-guided operation. Sample. Injections into the FFF channels, whose volumes tend to range from 1 to 5 mL, are performed either
r w r e 4. Thermal FFF of a nine-component mixture of polystyrenes in ethylbenzene. After a 1-h perid at constant field, characterizedby a hot wall temperature. 7, of 87 OC.the field was re. ducedto zero over a 0-h period using a parabolic decay program. The cold wall was maintained at 27 "C throughout.
via an injection valve or with a syringe pierced through a septum-containing injection port. Sample volumes tend to range from 1to 500 p L , and concentrations rarely exceed 1%.After the injection, flow is generally interrupted for a period of time to allow the sample to relax into its equilibrium distribution. The times needed for relaxation will vary with the sample as well as with the type and strength of the applied field, and are directly proportional to the thickness of the channel. Typically, relaxation of high molecular weight polymers in the thin thermal FFF channels is accomplished in several seconds, whereas latex particles forced to relax under a weak sedimentation field in a carrier whose density is close to their own may require an hour or more to reach equilibrium. Giddings ( 1 1 ) recently proposed the use of split flow injection to reduce the times needed for relaxation. In this mode of operation, the carrier is entering the channel through the top and bottom walls a t the tapered inlet. The ratio of the two flows is such that only a minor portion enters through the accumulation wall, which is the chosen site for the injection. As the sample enters and becomes exposed to the field, it is already confined to the vicinity of its equilibrium position, and the relaxation process is therefore considerably shortened. The fact that the zone moves as a thin band along the analytical wall, propelled forward by the flow of (relatively speaking) large amounts of free sample carrier, has suggested a splitting of the flow also at the channel outlet ( 1 1 , l Z ) . By collecting only a minor fraction of the effluent in the vicinity of the analytical wall, it has been possihle to significantly enhance detectability, as shown in Figure 5 (12).The ability to work with smaller sample loads will lead to increased accuracy in the reten-
tion-based assignments of molecular weights or diameter, particularly for samples that tend to aggregate at high concentration. Although the overall configuration of available FFF instrumentation is quite similar from subtechnique to suhtechnique and from manufacturer to manufacturer, there are important differences in certain details of the various designs. In the following section, existing instrumentation will be examined on a subtechnique hasis beginning with sedimentation FFF, which, according to Figure 2, is the most highly selective of those developed to date.
Wlmentation instrumentation a n applications Sedimentation FFF. In 1986 Du Pont introduced the Model 1000SF3particle fractionator. Although this was the first commercially available FFF unit, much development work had occurred previously, both at Du Pont by Kirkland, Yau, and their colleagues (13)and a t the University of Utah, where Giddings and associates (14)were the first to demonstrate the significant analytical potential of the technique. The Utah design, introduced at this year's Pittsburgh Conference, is commercially available from FFFractionation, Inc. (Salt Lake City). This corporation also markets the thermal and flow FFF systems described below. As the name implies, the operating field in this subtechnique induces sedimentation of injected particles in the channel. For particles of 1-2 pm, such settling is easily accomplished in a flat channel under the influence of gravity alone. However, smaller particles require the application of a centrifugal field, and for this purpose the thin channel is curved to fit inside a rotor basket that can he spun to generate a desired gravitational acceleration. The most important difference be-
Flgure 5. Principle of flow splining and application to flow FFF of sulfonated
polystyrene Ths lower trace was recordeo as ad eHl.em was forced to ex11Inrough the .pper pan A cons oelao1e signal ennancBmOn1 ("Ope, 1mc4 wa* oota ned by sp nsng ltw dlibeot so lhat 1/4 1 01 !ne total was aiiowed to exit tnrodgh tne owel pori 1st me accumuiation wail)
tween Du Pont's SF3, now available in the upgraded Model 2000 version, and the FFFractionation design resides in the construction of the seals that link the spinning channel to the stationary pump and detector. The Du Pont unit uses a face seal, in which one hard disk (tungsten carbide) is butting a soft disk of composite polymeric material (consisting of Teflon, polyimide, and graphite). This arrangement allows the flow of carrier through the system even at high spin rates, and field strengths of about 40,000 gravities are entirely within reach. Indeed, a prototype model has performed well at 100,000 gravities (13).At maximum field, this unit
ANALYTICAL CHEMISTRY. VOL. 60. NO. 17, SEPTEMBER 1, 1988
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can resolve samples with molecular weights down to ahout 5 X 105,the exact value being a function of sample density. An example of baseline resolution of DNA samples in the 106 dalton molecular weight range is shown in Figure 6 (15). The FFFractionation design is based on seals whose spinning, self-luhricating rubber O-rings form tight fits around the stationary, highly polished tungsten carhide coated shafts that are the conduits for the carrier. Here, the maximum field strength sustainable for any length of time is 1600 gravities, and the lower limit of resolvable molecular weights is about 5 X lo7. The Du Pont design has a smaller rotor radius than its FFFractionation counterpart (9cm vs. 15.5 cm), and the channel is therefore shorter. As in other elution chromatographic systems, resolution increases with the square root of the channel length, and under otherwise identical conditions, the FFFractionation system will show somewhat higher resolution than the SF3.Currently neither system is suitable for extended operation in nonaqueous carriers. However, two units of the FFFractionation type have been successfully operated in ethanol ( 1 6 , I n . Both systems are under microprocessor control, which allows the operator to select operational parameters such as field strength with or without a programmed decay. The Du Pont unit limits its programming options to exponential field decays of the type shown in Figure 7 (IS),whereas the FFFractionation system offers a somewhat broader array of functional forms for its field programming. In addition, this system will soon contain an option for programming the carrier flow. Although this mode of programming has been less explored because of experi-
mental difficulties, it shows some theoretical advantages (19) over the more easily implemented field programming in terms of speed of resolution. Aside from controlling field and flow, the system's computer also stores the detector signal during a run for later processing into information on sample molecular weight or particle size. In analyzing samples of broad size distribution, such as the one shown in Figure 8 (20),the first step is generally to convert elution volumes into units of mass or size. This step requires knowledge of the solute and solvent densities as well as of the time evolution of field strength. In the example presented here, the field was held constant during the run and the densities were previously determined. The fractogram was therefore easily transformed into an uncorrected size distribution for the sample. Further transformation to yield a corrected distribution would require the application of a volume correction (21),which accounts for differences in selectivity along the elution volume axis. A second correction is needed when the detector response varies with particle size, as in the case of optical deteetion of colloidal materials. To this end, the Du Pont system offers software to correct for the Mie scattering of the eluate. The broad peak in Figure 8 represents the envelope of numerous partly resolved components of the distribution. By collecting fractions in the middle of the peak and reinjecting them into the channel under identical conditions of field and flow, it was possible to demonstrate the method's reproducibility as well as the relative uniformity in size within each selected cut (20). Although monodisperse particles can he accurately characterized by photon correlation spectroscopy
(PCS), this technique gives little information ahout the size distribution of polydisperse materials. However, in combination with sedimentation FFF, which provides fractions of rather uniform particle size, PCS gives precise diameter assignments to each fraction of sufficient concentration. Aside from being a valuable verification of the fractionation procedure, the PCS measurement is useful in cases where the sample density is unknown. By relating the PCS-determined diameter to the elution volume a t which the particular fraction is collected, one can use FFF retention theory to evaluate the density of the particle. This strategy is helpful in work with emulsions and other particulates whose densities are difficult to establish. It complements methods for density determination that are based on the collection of retention data in carriers of different density. Although variations in carrier composition allow determination of diameter and density characteristics for stable particles (2),the method introduces serious errors for particles susceptible to conformational changes with changes in their environment. The FFFractionation sedimentation FFF system can be obtained with a split outlet, allowing the separate collection of effluent from the upper and lower half of the channel. In addition to enhancing sample detection, this may he a useful option if one is faced with the task of determining size distribu-
Figure 7. RaDid fractionation of Dolyst
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Flaure 8. Sedimentation FFF of a DNA mixture. The Peak labeled 174 Virion" represents a single-stranded molecule: the second peak contains its double-stranded counterpart. me initial fleld of 69,600graviues was held m t a n l lor 12 min. tollawed by an e x m n t i i l decay with a time omstant of 12 mln. Calculated molecular weights were In soad agreement with literaturedate. (Adapted with permission hom Reference 15.)
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rene latex particles, using the '*timedelayed exponential" program develooed at Du Pont. - 7 - - -
The field was initially held at 10.060 gravities for 0.9 min and was then set lo decay expnentially With a t i cmstant of 0.9 min. (Adapted with permissionfrom Reference 16, courtesy of Msr-
wl Dekker.1
tions for mixtures of particles of different density (22).A carrier whose density is intermediate between those of the two particle types will force floaters and sinkers to accumulate and migrate along different walls, and eventually to leave the channel through different exit ports. Because the velocity profile in the channel is completely symmetrical around the central flowline, it makes no difference in the size evaluation whether a sample has accumulated at the inner or the outer wall, and accurate distributions can therefore be determined for both particle types. Thermal FFF Thermal FFF was the first suhtechnique to show evidence of fractionation (23).The sample in this early study was a mixture of linear polystyrene standards, separated under a temperature differential of 70 " C using toluene as a
carrier. Not much attention had been given to the smoothness of the accumulation wall in the first functioning unit, and because of significant instrumental band broadening, it took a full 16 h to accomplish the baseline separation of three components with molecular weights of 3600,51,000, and 160,000 daltons. In a later unit, a similar separation was carried out under comparable field strength in just over 4 min, as shown in Figure 9 (7); with a thinner channel, virtually the same result was obtained in less than 1 min (7). The production of highly polished channel walls is clearly a prerequisite for efficient operation of any FFF unit. The maximum mass selectivity of thermal FFF is somewhat lower than that of sedimentation FFF (Figure 2) and ranges between 0.33 for compact spheres to around 0.65 for linear polymers in good solvents. Unfortunately,
only a few samples experience thermal diffusion in aqueous systems (24);consequently, thermal FFF has been used almost exclusively with nonaqueous carriers. In these systems, however, even relatively small molecules undergo rapid migration to the cold wall of the channel, and the range of resolvable molecular weights is wider for thermal FFF than for the sedimentation FFF subtechnique. Only one thermal FFF system is commercially availahle today. Its core component, the "column," consists of two copper blocks, each with one face chrome plated and polished to a mirror finish. These blocks are held together around a 76-pm thick mylar spacer that defines the channel space (column lengths are generally around 50 cm and their widths around 2 cm). Two 1500W heating rods supply heat to the upper block, and the lower block is routed out for efficient heat exchange with a circulating coolant, which most often is running tap water. Thermocouples, permanently mounted in the blocks near the channel walls, record the two wall temperatures; their signals are fed to the system's microprocessor, which, through a feedback mechanism, controls the temperature of the hot wall. Under normal operation, temperature differentials of up to 80 "C, corresponding to gradients in excess of 1000 'Clem, are generated across the channels, whose cold wall temperatures (T,) are kept around 25 "C. Because samples tend to accumulate a t the cold wall, their solubility a t T,becomes a guiding factor in selecting this temperature. To generate sufficient temperature gradients on top of a high T, without causing the carrier to boil a t the hot wall, it is sometimes advantageous to pressurize the system, thereby increasing the boiling point of the carrier. This strategy was used successful-
Retention time (min) jure 8 . . - ---,-- -. a field of 86.2 gravities (upper trace). .. Cuts were coiiecti at the specified posilioni in the fractogram and subsequently reinjected under identical field and flow. FFF retentiontheory was used to establisha particle diameter scale from me obsewsd relentlon times. (Adaptedwlth wrmission from Reference 20.)
Flgure 9. Separation of linear polystyrene standards by thermal FFF. The channel length was 42 cm and a temperature differential of 60 ' C was ap-
plied across the 51-pm thick channel. (Adaptedwith permissionfrom Reference 7.)
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1. 1988
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Flgure 10. Separations of two polymer standards with different chemical composition.
comparative study (27)(see Figure 2). The situation is altogether different if the sample molecules vary not only in size but also in composition. Although SEC will resolve the sample constituents based on differences in Rc alone, separation in thermal FFF (TFFF) is governed also by the sample's thermal diffusivity, which varies with polymer and carrier composition. This difference in retention behavior with differences in sample composition, which is illustrated in Figure 10 (2% can be used to analyze the products of block copolymerization, whose function depends on composition as well as on molecular weight. Of the zone-broadening effects most commonly encountered in FFF, that caused by the apparatus itself is normally negligible in the highly polished modern TFFF system, leaving the velocity-dependent nonequilibrium effect as the major system-induced cause of zone spreading. This effect, which is progressively reduced a t high retention, can he quantified and deconvoluted from the elution curve of a retained polymer sample. The remaining zone broadening is attributable to polydispersity; for relatively uniform samples, the peak width can be directly translated into the ratio ( p ) of its weight and number average molecular weights, or to a standard deviation in particle diameter. Because of the system's high selectivity, p-values as low as 1.003 have been reproducibly determined (29). For highly polydisperse samples, the fractogram provides a detailed representation of the molecular weight or size distribution. A true distribution curve may be obtained by treating the fractogram with the scale correction described earlier, together with a recently developed deconvolution procedure to remove system dispersion (30). The low shears present in the FFF channels make the method particularly well suited for analysis of shear sensitive samples, such as linear polymers of high molecular weight (M). The fractogram of Figure 11represents a polydisperse polystyrene sample with a manufacturer-assigned (weight average) M of 25 X 106 daltons; the figure shows the high end of the distribution to contain molecules with M in excess of 50 X 106. Although reinjection analyses of linear polymers in SEC (31)have demonstrated scission of samples with M in excess of IO6, no such effects were noted in a TFFF reinjection analysis of the sample in Figure 11 (32).
me wo cc-elute during sizeexcIusIon chmmatcgraphy on a waters UlhaStyragel column (le" 30 cm. 1.d. 0.78 cm, flow rate 1.0 mLlmin1 indlwtlting mleCUlar dimensions. B~ causeof ditlerenm hm S m l dlllcelvny. D, (1.31 X iO-'cm2s-' K-'forpol~lmeWl"hacrykte) vs. 0.82 X lo-' lor polystyrene), the two are virtually ba681ine-resoIvec by. mermal FFF (AT= 41 'C w = 76 pm, Yo = 0.75 mL). (Adapted frm Referen- 28.)
Flow FFF This suhtechnique differs from all 0thers in that retention is a function of one single sample property (i.e., the diffusion coefficient) rather than a combination of two, as is otherwise the case (see Figure 3). Elution volumes can
ly in the analysis of a series of polyethylene samples in tetrachloroethylene, whose boiling point is 121 "C a t atmospheric pressure. With the channel pressurized to 15 atm, it was possible to accommodate a temperature differential of 86 "C on top of a Tcof 107 "C (25).
For a given polymer solvent system, retention data provide linear correlations with the sample's radius of gyration, RG,for linear as well as branched molecules (26). In this respect, the thermal FFF performance is similar to that of SEC, although its mass selectivity is higher, as was shown in a recent
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5
10
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25
30
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u 25102030
50 70 100
Mx 1 w
Figure 11. Thermal FFF of polymers with ultra-high molecular weight. The lractogramwas recwded la a polydisperse polystyrene sample in TW. Experimamal conditions were: AT = 8 OC. w = 254 um. V o = 2.4 mL.
therefore he directly translated into sample diffusivities without the need for calibration. As in SEC, however, any conversion of diffusivity data into molecular weights requires additional knowledge of the hydrodynamic properties of the samples. The "field" in the case of flow FFF is simply a flow of carrier, applied in a direction perpendicular to the regular channel flow that transports the sample from injection site to exit port. Application of the crossflow requires that the channel be equipped with at least one semipermeable wall where the sample can accumulate for subsequent downstream migration in the normal FFF mode. The selectivity range of flow FFF largely overlaps that of thermal FFF; the lower limit of resolvable molecular weights is set by the choice of membrane to serve as the analytical wall in the channel. For most applications in aqueous systems, this wall consists of a skin membrane of polysulfone (e.g., Millipore PTGC or Amicon PM) or cellulose (e.g., Amicon YM5) with a nominal cutoff limit (for glohular proteins) of 5000-10,ooO daltons. In the case of linear nolvmers. the cutoff occurs a t lower mofecular' weights, and current applications range from a low value of 1000daltons up to particle diameters of around 1pm, where steric effects dominate the separation. Although most published applications have involved aqueous carriers, the technique is by no means limited t o such systems, as
ChromNet: Okay everyone, here's the sample information. Autosampler: Let's inject the next sample. Integrator: I'm ready. Pump: I've changed the gradient and the flow is stable. Autosampler: I'm injecting the sample. Everyone start. ChromNet: Okay, I'll monitor everything. Yes, there's intelligent life here. With our new ChmmNet" System Manager, the modules in a Spectra-Physics LC system actually "talk"to each other. Even more remarkable, the samples control the system through ChromNet's unique sample information queue. A single keyboard and pop-up menus allow you to set up the sample queue, develop the method or monitor the system at any time. ChromNet works with ChromStation- and other Spectra-Physics systems. Make your LC system think for itself, so you can think about something else. Call toll free 800-424-7666. Spectra-Physics, Autolab Division, 3333 North First Street, San Jose, CA 95134. In Europe, Spectra-Physics GmbH Siemensstrasse 20, D-6100, Darmstadt-Kranichstein, Fed. Rep. Germany.
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shown in Figure 12 (33). A t this time, only one flow FFF instrument is commercially available (by special order). In this version, the channel (50 cm long, 2 em wide) is defined by a 2 5 0 - p m thick Teflon spacer, which is sandwiched between two hollow plexiglass blocks held together with bolts. Each block has one porous ceramic wall facing the channel; the one serving as the accumulation wall is covered by the skin membrane mentioned earlier. Both crossflows and longitudinal flows are delivered by ordinary HPLC pumps, and their relative magnitudes are regulated by the two pumps as well as by a restriction valve placed after the detector a t the column exit. A somewhat different system with comparable performance has been constructed by Wahlund ( 3 4 ) . This unit has only one semipermeable wall; the other major channel wall is a plate of solid glass. One pump is delivering carrier to the system, and by a set of flow restrictors, the carrier stream is divided so that only a small portion is allowed to exit a t the end of the channel and the major part is forced through the membrane a t the analytical wall. Although this asymmetrical delivery of crossflow gives different retention hehavior than its symmetrical counterpart for poorly retained samples, there is a virtual overlap in performance for retentions beyond two column volumes. The semipermeable membranes used in these systems have nowhere near the flatness of the polished metal surfaces serving as analytical walls in the sedimentation and thermal systems; hence, instrumental zone broadening is more pronounced in the flow FFF units. As with other FFF tecbniques, the velocity-dependent nonequilibrium zone broadening in flow FFF is suppressed by operation a t high retention. By setting the crossflow at an appropriately high level, one can therefore still perform high-resolution separations in relatively short times, as shown in Figure 13 (34). EledTkal FFF Despite high expectations, this suhtechnique has thus far failed to show significant resolution of complex samples. In an early study (39,a qualitative agreement was shown between the experimentally observed and theoretically predicted elution orders for mixtures of proteins with different isoelectric points. The system only appeared to perform well a t very weak fields, and at those field strengths that were needed for high selectivity and suppressed nonequilihrium zone broadening, the samples became virtually immobilized in the column. This effect, which could possibly he caused by membrane polar868A
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Flgure 12. Flow FFF fractogram of broad and narrow polystyrene samples in ethyl-
benzene. Experimentalconditions were: longltudinaitiow
