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 paper 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 like size, molar mass (MM), chemical composition, degree of branching, and their respective distributions
to understand their behaviour. 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 semi-permeable 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 arises from its ability to separate analytes
over a broad size range (0.001 to 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 practised 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 co-elution of small and large analytes observed in SEC for highly branched polymers are attributed
to effects caused by the column packing material (4).
In practice, FFF offers users additional benefits. Prior to 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 elute in
the void peak or can be removed on-line through a semi-permeable membrane used in some FFF techniques.
Separations in FFF are dependent on the strength of an externally applied field which 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 multi-angle light scattering (MALS), differential refractive
index (dRI), and mass spectrometry (MS) detectors. For those interested in FFF, building a simple homemade system requires
a 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.
Sample Prep Perspectives | Ronald E. Majors: LCGC Columnist Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments in existing technology lines.
History of Chromatography | Industry Veterans: With each installment of this column, a different industry veteran covers an aspect of the evolution and continued development of the science of chromatography, from its birth to its eventual growth into the high-powered industry we see today.
MS — The Practical Art| Kate Yu:
Kate Yu is the editor of 'MS-The Practical Art' bringing her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers.
LC Troubleshooting | John Dolan: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan, Vice President of LC Resources and world renowned expert on HPLC, is able to highlight common problems and provide remedies for them.