Asymmetric-Flow Field-Flow Fractionation (AF4) is an advanced technique for achieving analytical and semi-preparative separations, applicable to a wide range of analytes including: proteins, polymers, nanoparticles, colloids, and complex fluids. Covering an extraordinary range of sizes from 1 nm to 10 µm, AF4 offers programmable separation power yet negligible shear. It does not disrupt delicate macromolecular assemblies or loose protein aggregates.
Even the most complex macromolecular solutions and colloidal particle suspensions can be analyzed utilizing multiple online detection methods after separation by AF4, from light scattering to ICP-MS. Hollow-Fiber Flow Field Flow Fractionation (HF5), closely related to AF4, performs low-volume analytical separations in convenient disposable cartridges with nanograms of analyte.
Wyatt Technology offers both complete solutions for FFF and upgrades to existing HPLC or SEC systems for FFF capabilities.
Understanding Flow Field-Flow Fractionation
Asymmetric Flow Field-Flow Fractionation is a one-phase chromatography technique, implemented in Wyatt's Eclipse® DualTec™ and Eclipse® AF4™ systems. High-resolution separation is achieved in a thin, ribbon-like channel by applying two flow streams. One in the longitudinal direction towards the outlet and a second, cross-flow in the perpendicular direction, which permeates through the porous bottom wall, consisting of a nano-porous membrane. The cross-flow acts as a force field, concentrating the sample against the bottom wall. Brownian motion represents a counter force working against the cross-flow. In a stationary equilibrium, an exponential concentration profile up into the channel is established with the half-width depending on the diffusion coefficient of the sample only. The average height of the sample components above the bottom wall result in different transport velocity towards the channel outlet. Smaller particles or macromolecules will elute first, followed by the larger components.
Asymmetric-Flow Field-Flow Fractionation (AF4)
Asymmetric Flow Field-Flow Fractionation is a one-phase chromatography technique, implemented in Wyatt's Eclipse® Dualtec™ and Eclipse® AF4™ systems. High-resolution separation is achieved within a very thin flow against which a perpendicular force field is applied. The flow and sample are confined within a channel consisting of two plates that are separated by a spacer foil; the plates are then bolted together. The spacer foil has a typical thickness of 100 – 500 µm.
The upper channel plate is impermeable. The bottom channel plate, on the other hand, is permeable, and made of a porous frit material. An ultra filtration membrane with a typical size barrier of 10 kDa covers the bottom plate to prevent the sample from penetrating the channel.
Within the flow channel a parabolic flow profile is created because of the laminar flow of the liquid: the stream moves slower closer to the boundary edges than it does at the center of the channel flow. When the perpendicular force field is applied to the flowing, laminar stream, the analytes are driven towards the boundary layer the so-called "accumulation wall" of the channel.
Diffusion associated with Brownian motion, in turn, creates a counteracting motion. Smaller particles, which have higher diffusion rates, tend to reach an equilibrium position higher up in the channel, where the longitudinal flow is faster. Thus, the velocity gradient flowing inside the channel separates different sizes of particles.
The smaller particles are transported more rapidly along the channel than the larger particles. This results in the smaller particles eluting before the larger ones – the opposite of a Size Exclusion/Gel Permeation Chromatography (SEC/GPC) separation in which the large molecules elute first.
With AF4 separation there is no column media to interact with the samples, so for very high molar mass polymers, there is no need to worry about shearing forces being applied. The entire separation is gentle, rapid, and non-destructive without a stationary phase that may interact, degrade, or alter the sample.
The separation process requires three steps: During the first two steps, injection and focusing, the main flow is split, enters the channel from both ends and is balanced to meet under the injection port. At this point the flow will move only down and permeate through the membrane. When the sample is injected it is focused in a thin band and concentrated towards the membrane. After complete transfer of the sample volume the injection flow is stopped and one typically allows for another minute of focusing before the flow pattern is switched to the elution mode. Now the flow enters only from the inlet port and exits at the outlet which is connected to the detectors. Sample constituents elute separated according to size and are monitored by the array of detectors.
Hollow-Fiber-Flow Field-Flow Fractionation (HF5)
Hollow-Fiber-Flow Field-Flow Fractionation (HF5)
The principle of operation of the hollow-fiber FFF channel is very similar to that of AF4, except there is no longer an upper plate - the membrane has been rolled into a tube. This allows for very small channel volumes, resulting in high sensitivity and very fast run times.
A new dimension in FFF
Standard field-flow fractionation using AF4 is a versatile, size-based separation technique that can be tuned to adjust resolution and range. With no packed stationary phase, AF4 is free of shear, and adverse surface interactions are minimal, making it an ideal technology for complex and difficult analytical fractionation tasks.
The Mobility adds the ability to modulate separation by charge. Depending on the particle’s charge and polarity, its motion under the electric field combines with AF4 cross flow and parabolic channel flow to produce later or earlier elution. Hence analytes with the same size but different charge leave the EAF4 channel and reach the detectors at different times. Each fraction is characterized separately by the detectors.
Combining electrical fractionation with AF4 in a single separation channel in EAF4, the Mobility is the most powerful and versatile separation technology available for macromolecules and nanoparticles from 1 nm to 10,000 nm.
EAF4 combines conventional asymmetric-flow field-flow fractionation with an applied electric field to separate particles by size and charge.
Key Benefits of FFF
- Robust separation of macromolecules and nanoparticles
from 1 nm - 1000 nm with excellent resolution
- Typical analysis times 10 - 30 minutes
- No stationary phase - open channel separation with minimal shear and surface interactions
- Versatile choice of separation channels, permitting sample loads from nL to mL, including semi-preparative FFF
- Optimize separation methods to enhance resolution or size range simply by adjusting flow ratios, even programmatically during a run
- Fractions can be collected and used for off-line analysis (with electron microscopy, MS, ICP-MS, ELISA, etc.)
Key benefits of Wyatt Technology Eclipse FFF
- Seamless integrated and automated system
- Choose between any two of SEC, AF4 or HF5 mode separations with a single HPLC and detector set
- Make use of the highest quality HPLC pumps and autosamplers from Agilent, Shimadzu, Thermo, or Waters
- Integrate with your favorite HPLC software from Agilent, Dionex or Shimadzu
- Couple to ICP-MS for elemental analysis of nanoparticles per size bin
- Sophisticated data processing of molar mass, size, shape, conformation, charge and charge distribution
- Software supported method development
- Gold or Silver Service Contracts available — comprehensive service contracts to protect your investment, save you money and keep your instruments running at their best
Eclipse FFF systems add to, and complements, size exclusion chromatography (SEC) for macromolecular separations. Separation occurs without shear or adverse, non-ideal column interactions. The Eclipse DualTec allows you to take full advantage of your favorite conventional SEC and AF4/HF5 separations in one convenient package.
The DualTec lets you use the same HPLC pump and autosampler, and the same MALS and DLS light scattering detectors, to characterize macromolecules for molecular weight, size and conformation by SEC or FFF. Switch between the two separation techniques at the click of a mouse.
Common solution-based methods for determining the size and size distributions of nanoparticles, such as batch dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), cannot offer the resolution, statistical confidence and absolute accuracy of a true separation-based technique that incorporates MALS and DLS downstream detection.
FFF-MALS and FFF-DLS performed with an Eclipse DualTec, DAWN HELEOS II and an embedded WyattQELS DLS detection module offer excellent resolution and accuracy in analysis of size distributions. Particle counts and sizes of viruses and virus-like particles estimated by FFF-MALS match closely TEM-based counts.
The Eclipse® DualTec supports both Hollow-Fiber Flow-FFF (HF5) and Asymmetric Flow FFF (AF4) in one instrument. Utilizing one HPLC stack and one set of Wyatt detectors, the DualTec permits automated switching between any two of SEC, AF4 and HF5 separation followed by MALS and/or DLS characterization.
DAWN® HELEOS® II
The most sensitive MALS detector available, anywhere. Incorporates detectors at 18 angles to determine molar masses from 200 Da to 1 GDa and radii from 10 – 500 nm. The WyattQELS module adds dynamic light scattering (DLS) capabilities to a DAWN, miniDAWN or μDAWN MALS detector. DLS determines the sizes of macromolecules and nanoparticles as Rh, the hydrodynamic radius, calculated from the diffusion coefficient.
A unique on-line differential refractometer for measuring concentration of any macromolecule, regardless of chromophores. The high-concentration Optilab accommodates protein concentration up to 180 mg/mL.
The Eclipse family of flow field-flow fractionation controllers and separation channels offers multiple options to meet the most demanding separation needs.
Eclipse DualTec The 5th generation AF4 controller incorporates novel tip injection technology to support HF5.
Eclipse AF4 The 4th generation AF4 controller supports semi-preparative and frit-inlet channels
Analytical Separation Channels Two sizes to optimize separation power and sample quantity in the microgram range
Mobility Separation Channel A new hardware design allows for applying the electrical field directly inside the channel. The connection is made by magnetic contacts without any wires to be attached manually.
Mobility Control Unit Aside of the high precision power-supply to generate the electrical field inside the channel the control unit also contains a Eclipse® conductivity detector and a pH sensor. It constantly provides these data to the analysis software to be displayed in the user interface.
HF5 Cartridges Disposable channels for nanogram separations
Semi-preparative Channel Fractionate milligrams of sample
Frit-inlet Channel Suitable for aggregation-prone samples