Asymmetrical Flow Field-Flow Fractionation (AF4) Theory

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Asymmetrical flow field-flow fractionation is an orthogonal method to size exclusion chromatography (SEC) and sedimentation velocity analytical ultracentrifugation (SV-AUC). AF4 is applicable to a wide spectrum of uses, including the detection of soluble aggregates, characterization of species via multiangle laser light scattering (MALLS) and determination of particle size distributions. Follow the link for more information.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) method is the established standard experiment when it comes to AUC. It is carried out at high rotational speed and the easiest way to obtain sample composition and species characteristics (e.g. molecular weight and frictional ratio). Follow this link to get more information about SV-AUC theory.

While sedimentation velocity is the standard AUC experiment, sedimentation equilibrium analytical ultracentrifugation (SE-AUC) nonetheless offers valuable information on an analyte.  SE-AUC is a first principle method for the determination of the molecular weight. Therefore, it is the method of choice to determine the molecular weight of your API in solution. Follow this link to get more information about SE-AUC theory.

Light scattering is a well defined effect. The physical properties of light scattering are used for several applications, such as dynamic and static light scattering. Please follow the link to a detailed description of light scattering theory. 

In the last couple of years, the interest in the determination of predictive parameters within formulation development has grown. Interactions parameters provide valuable information for the prediction of molecular stability. These values are used in a “design of experiment” (DOE) approach to assist formulation developments. Please follow the link for more information.

ZentriForce Pharma is not merely an expert in analytical development. Our scientist are actively involved in further advances in the theory of hydrodynamic techniques. Follow the link to a detailed description of the theory behind our analytical services, including some of our own research


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Asymmetrical Flow Field-Flow Fractionation (AF4) Theory

The current portfolio of field-flow fractionation (FFF) techniques looks back on nearly 60 years of history. The first prototype of the FFF instrumentation was developed by Professor J. Calvin Giddings et al. in the 1960’s. Although the FFF   launched at the same time as high-pressure liquid chromatography (HPLC), it took a much longer time until reliable and robust commercial products were available. The most established FFF technique is the asymmetrical flow field-flow fractionation (AF4), which is widely used in biopharmaceutical development as well as for nano particle detection.

Field-Flow Fractionation (FFF) Techniques

All FFF techniques apply an external force field perpendicularly to the direction of the elution flow through a thin, ribbonlike channel (the channel thickness is variable and lies between 100 and 700 μm). Elution flow is created by solvent being flushed through the channel, which is narrow enough that a laminar parabolic flow profile results A laminar flow is characterized by a flow velocity that grows with increasing distance from the channel wall.

Depending on what type of FFF is used, the applied force can vary and constitute, e.g. an electrical field, a temperature gradient, a centrifugal field or a flow-induced force. The force drives the sample toward the accumulation wall, where the flow velocity is near zero. A counteracting force develops due to the concentration build-up at the wall and drives the solute back towards the center of the channel. When the forces are in balance, steady-state equilibrium is reached, and an exponential solute concentration profile is formed.

Depending on the scale of the counteracting force, the elution time of different species can vary, from which a separation of species results.

Asymmetrical flow field-flow fractionation (AF4) Frit Inlet Channel
Frit-Inlet AF4 Channel used for focus-stress-free AF4 measurements

Asymmetrical Flow Field-Flow Fractionation (AF4)

In asymmetrical flow field-flow fractionation (AF4) the force field is created by a crossflow of solvent. The crossflow is directed through a membrane at the bottom of the channel. Solutes are separated due to their size (diffusion coefficient), with larger species located closer to the membrane at steady-state conditions than small species. The technique allows the separation of solutes in a size range between 1 nm and 1000 nm. In theory, even larger species could be tested, although in this case the separation mechanism is steric and thus differs from the classical separation model. Therefore, an overlay with other species might be observed. A classical AF4 measurement includes several steps. First, the sample is focused on a very small region at the beginning of the channel until all solutes reach the steady state condition that is created between the force of the crossflow and the back-diffusion of the solutes into the channel. When equilibrium is reached, the second step is the start of elution, which marks the beginning of separation by elution velocity. Larger solutes travel the channel length close to the accumulation wall and therefore show a slower elution than smaller solutes. In AF4, elution behavior is inverse to that which is observed in size exclusion chromatography.

In most cases, AF4 is used as a method to separate species that are then immediately and individually characterized by multiangle laser light scattering (MALLS). However, AF4 itself has the potential to be a valuable tool for characterization.  As separation within the AF4 channel is due to diffusion behavior that differs for each individual species, it is possible to calculate the diffusion coefficient distribution from the resulting fractiogram

Asymmetrical flow field-flow fractionation is an orthogonal method to size exclusion chromatography (SEC) and sedimentation velocity analytical ultracentrifugation (SV-AUC). AF4 is applicable to a wide spectrum of uses, including the detection of soluble aggregates, characterization of species via multiangle laser light scattering (MALLS) and determination of particle size distributions. Follow the link for more information.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) method is the established standard experiment when it comes to AUC. It is carried out at high rotational speed and the easiest way to obtain sample composition and species characteristics (e.g. molecular weight and frictional ratio). Follow this link to get more information about SV-AUC theory.

While sedimentation velocity is the standard AUC experiment, sedimentation equilibrium analytical ultracentrifugation (SE-AUC) nonetheless offers valuable information on an analyte.  SE-AUC is a first principle method for the determination of the molecular weight. Therefore, it is the method of choice to determine the molecular weight of your API in solution. Follow this link to get more information about SE-AUC theory.

Light scattering is a well defined effect. The physical properties of light scattering are used for several applications, such as dynamic and static light scattering. Please follow the link to a detailed description of light scattering theory. 

In the last couple of years, the interest in the determination of predictive parameters within formulation development has grown. Interactions parameters provide valuable information for the prediction of molecular stability. These values are used in a “design of experiment” (DOE) approach to assist formulation developments. Please follow the link for more information.

ZentriForce Pharma is not merely an expert in analytical development. Our scientist are actively involved in further advances in the theory of hydrodynamic techniques. Follow the link to a detailed description of the theory behind our analytical services, including some of our own research

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