Virtual Method Optimization and Size Analysis for FFF
Flow-field flow fractionation (flow-FFF) offers highly versatile separations for the analysis of complex fluids, covering a size range of macromolecules and particles from 1 nm to 10,000 nm. However, flow-FFF is often perceived as a difficult technique to learn because of the multiple parameters available for adjustment. Recent advances in software for simulating flow-FFF overcome this obstacle, enabling the virtual optimization of flow-FFF methods and opening up the power of flow-FFF separations to non-experts. An added benefit is the ability to easily analyze particle size distributions by elution time from first principles.
Flow-field flow fractionation (flow‑FFF) is a proven technique for analytical separations and is used to characterize biopharmaceutical aggregates, drug and gene delivery colloids, polymers, nanomaterials, and others (1–6). Typically used in conjunction with on‑line multi‑angle light scattering (MALS) (7) and dynamic light scattering (DLS) for the measurement of molar mass and size distributions, in many instances particle sizes can also be calculated directly from elution time according to first-principles FFF theory, which relates channel-flow rate, cross-flow rate, and particle size to elution properties. The resolving power is broadly tunable by simply changing a flow-rate ratio or channel spacer.
Flow-FFF is quite versatile and can provide excellent resolution over a very large size range—from 1 nm to 10,000 nm—with minimal shear and surface interactions. However, it has not seen widespread adoption on the scale of gel permeation chromatography (GPC) or ultracentrifugation. One of the primary obstacles to the extended adoption of flow-FFF is its perception as a complicated technique, with a steep learning curve for method development. The technique’s great versatility is both a blessing and a curse: which initial parameter values should be chosen, and how should they be modified to improve the separation of a given sample?
Flow-FFF is often compared to size‑exclusion chromatography (SEC). Like flow-FFF, SEC separates molecules and particles according to hydrodynamic size (although the elution order is reversed, making flow-FFF more effective in characterizing aggregates and widely spaced sizes). Given a set of columns, SEC has no adjustable parameters that have any significant impact on resolution: the sample is transported at a constant flow rate under isocratic conditions, with minimal dependence of the separation on flow rate. The column calibration curve, which relates the log molar mass to elution volume, essentially maintains a constant slope between the void and total column volumes, so plate numbers are fixed. Therefore, SEC method development focuses on selecting a mobile phase and column chemistry that achieve purely steric sample–column interactions, and choosing conditions that avoid overloading and degradation.
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