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Techniques & Tools Mass Spectrometry, Liquid Chromatography

In the FFF Hot Seat

How did it all start?

As noted by Wim Kok, Calvin Giddings invented FFF. He was a chemistry professor at the University of Utah (Salt Lake City, Utah, USA) and an expert in white water kayaking. According to legend, he had the idea for FFF when he noticed how different water currents penetrate each other as water flows along a river. His observations inspired him to design a separation technique using a force field applied perpendicular to a transport flow within a channel.

My personal involvement in the technique and with Calvin Giddings began in 1990, when I signed up for an FFF workshop at Pittcon. Attendance was poor because FFF wasn’t well known or popular. Nevertheless, I was attracted to FFF because of its ability to separate particles (which isn’t possible with size-exclusion chromatography) and I saw it as a potential tool to use with multiangle light scattering (MALS) from separated samples for particle characterization.

In 1995, I sold 15 symmetrical flow FFF instruments, but I didn’t sell any the following year because most customers were simply unable to use the system; it was too complicated and tedious. For example, replacing a membrane took almost a full day to do and it was an art to reassemble the channel without trapping bubbles or having leakages. The experience taught me that ease of use and robustness are the most important points to consider in developing FFF instrumentation. And, this user-friendly focus has led to the technological breakthroughs we have seen in the last decade in this field.

Why do you think FFF has suffered ups and downs?

FFF has been ‘down’ in the past because the excellent results published, mostly by the Giddings group, raised high expectations from scientists. Unfortunately, others could not reproduce similar results, mainly because commercially available instruments at that time were impossible to use. As a result, the general opinion about FFF was that it looks good in literature, but it cannot be used in practice. As always in such cases, it takes a long time to change perceptions and this is why it has taken years to convince people to try FFF using one of the new generation of FFF instruments.

I think the biggest challenge today is the complexity of the analytical problems that tend to need FFF as a solution. Easy samples are analyzed using column chromatography and only applications that cannot be managed using traditional techniques are considered for FFF separation. A growing number of excellent publications highlight the progress made in recent years. Applications that work exceedingly well with FFF include virus-like particles (VLP), vaccines and nanoparticle characterization for environmental applications.

How important is FFF today?

In my opinion, there is no doubt that FFF has its place in the analytical techniques toolbox for macromolecular and particle characterization. However, it is far from being a routine technique as it’s still at the pioneering stage with a relatively small user base of enthusiasts. These people produce high quality research and publications, which are relevant for other researchers in the same field. This is why I am confident that FFF will grow and become more widespread. Many others will accept the technique as a standard tool for a variety of applications.

How are people using FFF?

I would like to highlight the separation of virus like particles, which is achievable by flow FFF coupled with a MALS detector. As VLPs are becoming more important as vaccines, we are witnessing a growing interest for FFF instrumentation in the pharmaceutical industry and in research. The advantage of FFF is that its resolution is superior to SEC. Recoveries are typically very good and it is possible to separate VLP populations, which maintain their biological activity throughout the separation process. This allows further evaluation of fractions after separation with respect to their efficacy of stimulating an immune response.

Where does FFF stand within analytical science?

FFF is similar to liquid chromatography (LC) although it does not have a stationary phase. Therefore, it is possible to use it in high-performance liquid chromatography (HPLC) laboratories, because it needs a similar infrastructure. But most FFF applications, such as particle characterization, are not performed with HPLC. This is an interesting problem; the scientists from the particle characterization labs have a harder time operating an FFF system than their HPLC colleagues would have. However, the HPLC group does not look at particles, so a reorientation of perspectives, know-how and organizational structure is necessary in order to use FFF successfully. This is especially true of industry, which makes it difficult to implement FFF.

Are people missing opportunities to utilize FFF?

Yes, I think so. FFF is still considered as a replacement to SEC.

Moreover, the general expectation is that FFF should produce similar results to column separations. This is not the case because FFF has very different characteristics. For example, the sample is concentrated at the accumulation wall and shear forces are absent during separation. The species eluting from FFF can be different from that from an SEC column. This does not mean one technique is right and the other wrong or that there are artefacts – the fractogram will reflect the properties of the sample under such conditions. Therefore, FFF can be an excellent tool to study a sample’s behavior under high concentration conditions and this gives an opportunity to retrieve information about stability or aggregation behavior of proteins and other macromolecules.

What do you think are the most important recent developments?

Hollow-fiber-flow FFF (HF5) is an important new development. This technique employs a disposable channel that can be replaced in a few minutes. The system has a very low channel volume (below 100 microliters) which leads to less sample dilution and a 33 percent higher retention at the same cross-flow density. Applications are particularly interesting for nanoparticles, and HF5 has distinct advantages when coupled to ICP-MS.

Where do you think FFF will be in five years?

I believe that FFF will get closer to becoming a routine method within the next five years. There will be several applications, mainly for nanoparticles, required by EU legislation to classify a product as being ‘nano’ or not. Some of the new vaccine candidates in development today – that are characterized using flow FFF methods – will be on the market by then. FFF will be used for QC of some of these drugs. The instrumentation and software will be markedly improved and automated. Performing an FFF analysis will be almost as simple as using an automated coffee maker. The software will constantly monitor the system status and actively prompt the user to take service action or it will give instructions how to maintain perfect status and functionality of the FFF instrument.

Taking FFF to the Next Dimensionby Thorsten Klein, founder and CEO of Postnova Analytics, Landsberg, Germany.

AF4’s Time in the Sun?by Wim Kok, Faculty of Science, the Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, The Netherlands.

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About the Author
Christoph Johann

Founder and MD of Wyatt Technology Europe, the company that originally introduced field-flow fractionation (FFF) instrumentation to Europe’s analytical scientists, looks at the history of the technique and shares his involvement with the field.

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