‘In 1965 I started off in a new direction: I developed the concept of a chromatographic-like system in which retention is established and controlled by an external field rather than by the stationary phase. This system, which we call field-flow fractionation (FFF) extends the range of chromatography upwards to include macromolecules and particles of almost every type and size, from 0.001 to 10 µm and beyond. […] I will add no more, for the story of FFF belongs more in the future than in the past.’

This is how J. Calvin Giddings, a Distinguished Professor of chemistry at the University of Utah, introduced his invention in the Journal of Chromatography Library in 1979. Though this technique is almost sixty years old, it remains underused. And researchers are missing out, says Alina Astefanei, assistant professor in analytical chemistry at the University of Amsterdam.

Milk, bear, wine, serum

Astefanei specialises in FFF and believes the potential of the technique is way higher than most people realise. ‘Essentially, FFF is a family of techniques that “fractionate” macromolecules under the influence of an external field’, she says. ‘The most widely used version, asymmetrical flow FFF [AF4, ed.], separates macromolecules based on size, just like in size exclusion chromatography. The difference is that AF4 doesn’t have a stationary phase: instead, the separation field is a cross-flow across a semi-permeable membrane. Very small molecules pass through while the analytes of interest are separated according to their diffusion under the influence of the flow.’

‘It’s not rocket science, it’s just science!’

It is a very gentle technique, Astefanei adds. ‘Molecules aren’t forced through a stationary phase. Instead, they reach a steady-state distribution between diffusion and the cross-flow, and are then carried to the detectors by the channel flow.’ Once they are eluted, the real fun begins. ‘You can couple FFF to all kinds of detectors, so the amount of information you can possibly gather is very rich. The sky is the limit here, because you can use a broad range of mobile phases, while injecting “real world” samples like milk, bear, wine, serum, you name it.’

Bigger picture

‘FFF can also act as an online clean-up step removing unwanted small molecules while retaining and pre-concentrating the analytes of interest. This makes it well-suited for dilute or complex samples’, Astefanei explains. ‘In mass spectrometry, we zoom in a lot. But in FFF, you zoom out to see the bigger picture’, she continues. ‘Everything in the sample stays intact, with minimal disturbance to molecular assemblies. Then, you can pick a specific fraction and zoom in on it using a coupled technique like mass spectrometry. I think it’s especially interesting for biologists.’

It’s not all sunshine and rainbows however. ‘The truth is that it is a complex technique; the hardware is more involved than a simple chromatography column, there is a lot going on. Without the theoretical understanding and proper training it can feel intimidating’, says Astefanei. ‘On the flip side, people don’t learn about this technique, which creates a negative spiral: people don’t learn about it and don’t see its value, and when they do encounter it, they often misuse or dismiss it. We can’t expect scientists to use complex techniques if they’re not trained beyond the basics. Analytical chemistry students should become at least acquainted with this technique so we can flip it into a positive spiral. It’s not rocket science, it’s just science!’

International network

To increase the visibility and use of FFF, Astefanei with an international team created the Young Scientists for FFF. ‘Our goal is very simple: education. We are building a platform, which we call INFLOW, to share knowledge, troubleshoot, develop theory and support new researchers who take up this technique.’ Just as important, they want to retain talent, support existing users through mentoring, and foster collaborations. In other words, a platform for everything you need to get started and keep going with FFF.

‘FFF is not the answer to every problem, but it can really be an asset’

The platform already brings together interesting contributors from academia, industry, and regulation. ‘We have someone from the US Food and Drug Administration who applies FFF to show that it helps to validate drugs for the market, for example; there is someone from biopharma and the vaccine industry as well; and the leader of the only FFF hub in the world in Toulouse, France, which houses every type of FFF instruments you can think of; and many more.’

When you’ve never heard of the technique but your curiosity is stirred, this platform is for you, says Astefanei. ‘We welcome everyone who is interested. For example, Joost Reek from the UvA came to me after reading the press release to explore the possibilities of FFF and discuss a collaboration. I think the more people that hear about it, the better. FFF is not the answer to every problem, but it can really be an asset for more researchers than just us.’