Mass, Size, Composition: FFF for Biomolecules and Nanoparticles - - Chromatography Online
Mass, Size, Composition: FFF for Biomolecules and Nanoparticles

The Column
Volume 10, Issue 14, pp. 1721

"What size is my sample" is a fundamental question asked at various stages from research and development through to manufacturing. Over the past 15 years, field-flow fractionation (FFF) for the separation of sample mixtures has been developed to determine overall sample size distributions and answer this fundamental question. This article discusses the different types of FFF methods that have been developed to determine the mass, size, and composition of biomolecules and nanoparticles, and also focuses on characterization by light scattering and inductively-coupled plasma mass spectrometry (ICP–MS).

Several different types of field-flow fractionation (FFF) have been developed for simple to complex sample mixtures. One of the most widely adopted1,2 FFF methods is flow field-flow fractionation (AF4), where samples in solution (or suspension) are fractionated within a channel or a fibre by liquid forces. The molar mass, size, and elemental composition of the fractionated samples can then be determined using a variety of on-line detectors. Static and dynamic light scattering detectors are often used to determine size, radius, and potentially conformation information about samples. More recently mass spectrometers, and also inductively-coupled plasma mass spectrometry (ICP–MS), have been added to the end of flow pathways to determine elemental composition of the flow-fractionated samples.

Flow Field-Flow Fractionation: How Does It Work?

Fractionation takes place in a channel containing a very thin film of liquid sandwiched between two flat surfaces known as the depletion wall and the accumulation wall. In early FFF methods, the depletion wall was a porous frit and the force field of liquid was forced into the channel through this frit and migrated towards the accumulation wall, in a process known as symmetric flow FFF. In symmetric flow FFF the accumulation wall is a flat surface lined with a replaceable semi-permeable membrane resting on its own frit for support. Membrane porosity is selected to permit the fluid to pass through while retaining the sample of interest. An additional flow is then entered at one end of the channel along the length of the channel at a defined flow rate. Over the last decade there has been a shift to asymmetric flow FFF, where the liquid providing the force field is combined with the detector flow coming in at one end of the channel, eliminating the need for a frit on the depletion wall and a separate flow stream for this liquid. The thinness of this flow from one end of the channel to the other produces a laminar flow of non-uniform velocity. The highest velocity flow along the length of the channel is at the centre of the flow profile. The closer you get to the depletion or accumulation wall, the slower the flow because of drag and other considerations. Therefore, the liquid in the channel has a velocity gradient that can be used to fractionate molecules of different sizes depending on which flow zone they end up in.

Figure 1: Flat channel design for flow FFF. Red and blue flow: Mobile phase entering the channel separation zone during the focusing process. Purple flow: Sample injection. The combined flows pass through the membrane and exit from the channel (yellow flow path).
The sample enters the channel either through its own dedicated injection port or along with the mobile phase entering the channel at the dedicated inlet port, under special conditions that push the sample onto the accumulation wall surface as shown in Figure 1; the liquid flow is balanced to create a focal point where the opposing flow streams meet, then permeate through the membrane, and pass out the opposite side.

The sample accumulates within the focal point, which can be demonstrated by injecting a dyed compound like Blue Dextran or azocasein, and undergoes a process called "relaxation". The sample is then pushed by the carrier fluid towards the membrane, but diffuses back from the surface of the membrane at a rate that is proportional to its translational diffusion coefficient. Within a few minutes, a cloud layer forms in the vicinity of the focus point, with larger molecules closer to the membrane and smaller species progressively further from the membrane, but generally staying within 10 microns of the membrane surface.

Figure 2: Process of fractionation in an AF4 flat channel during sample elution with crossflow.
Once the process of sample accumulation and relaxation has been completed, fractionation of the sample occurs during its transportation from one end of the separation channel to the other. The sample diffuses away from the accumulation wall, meanwhile it is counteracted by fluid that permeates through the semi-permeable membrane and escapes the channel. Figure 2 shows how the flow moving laterally carries the sample along the membrane surface while additional fluid is permitted to exit through the membrane, producing the force field. This force field is adjustable and determines the overall efficiency of the fractionation of the molecules or particles of interest.


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