Field-flow fractionation (FFF) is a family of techniques that is increasingly used for separating and characterizing macromolecules.
This review discusses recent advances in the characterization of biological, natural, and synthetic polymers. Applications
of FFF are contrasted with size-exclusion chromatography to illustrate practical considerations when characterizing macromolecules.
The use of different FFF fields allows separations based on size, mass, composition, and architecture. The open channel design
and subsequent low shear rate is well suited for analyzing weakly bound complexes, highly branched polymers, high molar mass
analytes, and aggregates. Other benefits of FFF that are highlighted in this article include simplified sample preparation,
flexibility in carrier fluid choice, and on-line removal of low-molecular-weight contaminants.
Macromolecules are ubiquitous in many areas of science and technology. Depending on the macromolecule, it is important to
analyze properties such as size, molar mass (MM), chemical composition, degree of branching, and their respective distributions
to understand their behavior. However, because of the complex nature of polymers, current separation techniques are not always
capable of comprehensive analyses. Size-exclusion chromatography (SEC) is widely regarded as the workhorse for polymer characterization,
but is limited by high molar mass (HMM) macromolecules, weakly bound complexes and aggregate species, and highly branched
polymers. Field-flow fractionation (FFF) is a versatile family of techniques that complements SEC with additional separation
capabilities based on analyte size, mass, composition, or architecture depending on the field used (Figure 1).
Figure 1: Types of FFF separation. (a) In AF4 a crossflow passes through a semipermeable membrane and porous frit. (b) In
HF5 a cylindrical semipermeable membrane is used and a radial outward flow creates the perpendicular field. (c) In ThFFF a
temperature gradient (ΔT) is formed between a hot wall and a cold wall, and sample migrates towards the cold wall because
of thermal diffusion (DT).
The open-channel FFF design results in a soft separation mechanism that is well suited for analysis of high and ultrahigh
MM polymers and samples containing microgel. Some key advantages of FFF over SEC arise from its ability to separate analytes
over a broad size range (0.001–100 μm) using a single channel, and the absence of column packing, which greatly reduces shear
degradation. SEC of protein aggregates often requires the addition of cosolvents or preconditioning of columns to reduce adsorption
(1). However, addition of cosolvents may induce aggregation, dissociate aggregates, or cause sample specific adsorption (Figure
2) (2). Preconditioning columns is often practiced but not reported in the literature, and even when preconditioning is used
poor recoveries and sample specific adsorption have been observed (3). In FFF the ability to use formulation buffer allows
separations and measurements under solution conditions that are more representative of actual use. For polymer analysis, the
shear degradation and coelution of small and large analytes observed in SEC for highly branched polymers are attributed to
effects caused by the column packing material (4).
Figure 2: An IgG1 recombinant fully humanized monoclonal antibody was analyzed by FFF in two different carrier fluids: (a)
0.1% acetic acid containing 50 mM magnesium chloride and (b) 10 mM phosphate buffer pH 7.1. High molar mass aggregates (peak
at ~18.5 min) present in (a) are absent in (b) as a result of weak aggregate interactions stabilized by the magnesium chloride.
Adapted and reproduced with permission from reference 2.
In practice, FFF offers users additional benefits. Before SEC, filtering is often implemented as a sample preparation step
to remove large components and help prolong the life of the column. Sample filtering has been shown to remove soluble and
insoluble microgels leading to erroneous MM and polydispersity results (Figure 3) (5). Filtering is not required in FFF and
soluble polymers and microgels can be simultaneously characterized. Many syntheses require the addition of excess reagents,
which may interfere with subsequent product analyses. Such reagents or interfering low MM sample components either are eluted
in the void peak or can be removed on-line through a semipermeable membrane used in some FFF techniques.
Figure 3: ThFFF–MALS–dRI analysis of unfiltered (solid line, black symbols) and 0.5-μm filtered (dashed line, grey symbols)
microgel-containing poly(vinyl acetate). The lines and symbols represent the dRI fractograms and rg, respectively. Significant
polymer loss in the filtered sample is evident in the lower MM distribution. Adapted and reproduced with permission from reference
Separations in FFF are dependent on the strength of an externally applied field that can be easily adjusted. Therefore, resolution
and separation speed are readily controlled without the need to change channels. In addition, the open-channel design greatly
reduces the chance of contamination and inexpensive membranes can be replaced when contaminated. Finally, FFF is easily coupled
on-line with detectors frequently used for SEC analysis, including multiangle light scattering (MALS), differential refractive
index (dRI), and mass spectrometry (MS) detectors. For those interested in FFF, building a simple homemade system requires
an FFF channel and standard high performance liquid chromatography (HPLC) components common to many laboratories. The recent
advances in FFF over the last three years are highlighted in this review.