FIELD-
FLOW FRACTIONATION Karin D. Caldwell Department of Bioengineering Center for Biopolymers at Interfaces University of Utah Salt Lake City, UT 84112
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 (i). 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 1 outlines 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/$01.50/0 © 1988 American Chemical Society
The well-defined geometry of the channel and the ability to easily and accurately control the magnitude 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 be 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.
INSTRUMENTATION Reservoir
Computer
Recorder
Figure 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 during passage, and elution times are governed by the layer thickness £ characteristic for each zone, so that £3< £2< £\. The system's computer regulates both field and flow and processes the detector signal. ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988 · 959 A
Figure 2 . Mass s e l e c t i v i t y range for c o m m e r c i a l l y a v a i l a b l e FFF i n s t r u m e n t a t i o n . For each technique, maximum selectivity is reached at a retention of 10 column volumes and is main tained until the (field-dependent) onset of steric effects; for flow and thermal FFF, its value ranges from 0.33 to 0.65, depending on molecular conformation. Su curves for the two sedimentation systems reflect operation at maximum field; sample and solvent densities are 1.50 and 1.00 g/mL, respectively. A repre sentative value for size-exclusion chromatography is shown for comparison.
Nature of field Sedimentation (known density)
Thermal
Flow
Electrical
The recent HLPC '88 conference in Washington, DC, devoted a day-long session to FFF, and contributions from several European and U.S. laboratories testified to a growing interest in the technique. In addition to much experi mental evidence for the soundness of retention and plate-height theory in their present state, the session also in cluded a Monte Carlo simulation of the sedimentation FFF process (4), which generally confirmed the analytically derived theory. The relationships be tween the retention volume observed at a given field strength and the molec ular weight M or size dp of the sample have been discussed in detail elsewhere (2,5); Figure 3 summarizes the particu lar sample characteristics that are ac cessible with the different subtech niques. The ability to model a separation process from first principles is particu larly valuable in characterization work because it eliminates the need for elab orate calibration procedures. Although standard samples of given molecular weight and polydispersity are fre quently used also in FFF, their purpose is more to verify the system's perfor mance than to fill a calibrating func tion. 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 mod els that describe retention and zone broadening for the various FFF subtechniques allows the operator to select optimal conditions for rapid resolution of any pair of samples with known properties. In the case of multicomponent mixtures, this 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 description
M,dp D/Dr
