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)
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.
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® 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.
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 microDAWN 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
HF5 Cartridges Disposable channels for nanogram separations
Semi-preparative Channel Fractionate milligrams of sample
Frit-inlet Channel Suitable for aggregation-prone samples