Focusing on Field-Flow Fractionation

insights from industryDr. Soheyl TadjikiManaging DirectorPostnova Analytics USA

In this interview, AZoM talks to Dr. Soheyl Tadjiki, Managing Director of Postnova Analytics USA, about the applications and benefits of Field-Flow Fractionation.

Welcome, Soheyl. Can you give a brief overview of Postnova Analytics, the work you do, and the industries you serve?

Thanks for having me! Sure. Postnova is the leading supplier of Field-Flow Fractionation (FFF) systems globally. The technique had been invented decades before at the University of Utah by Professor J. Cal Giddings. He commercialized FFF in the mid-80s with the company FFFractionation, which produced multiple subtypes of FFF. Postnova was founded in Munich, Germany, in 1997 and produced the first commercially successful asymmetrical flow FFF (AF4). In 2001, FFFractionation merged with Postnova to form Postnova USA. This connection with the inventor of FFF, and access to his original designs and patents, gives Postnova a very diverse platform of instruments. 

I have been with Postnova for almost 20 years, and I am the Managing Director of Postnova USA. Some of my responsibilities are to supervise demonstration analyses, provide both pre- and post-sale technical customer support for North America and to provide input into our global marketing.
With such a versatile technique as FFF, we have a lot of different customers! We primarily provide instruments for biopharmaceutical, polymer, nanotechnology, and environmental researchers. These researchers can be found in a variety of roles in academia, industry, and government.

Can you tell us more about your FFF platform?

As I mentioned, Postnova started out with AF4 and added more FFF subtypes. Now we have AF4, which uses a flow field to separate by hydrodynamic size; Electrical-AF4, or EAF4, which has the flow field and adds an electric field for determination of analyte surface charge; Centrifugal FFF, or CF3, which separates by mass, and Thermal FFF, TF3, which separates by size and chemical composition. We can also easily add size exclusion chromatography to any of our systems as a nice complement to FFF. Anyone of these techniques is powerful on its own, but having multiple orthogonal separation techniques provides more in-depth information about your samples.

The separation is important, but you also need the right detectors in-line. Postnova’s modular system design allows the use of a portfolio of detectors suited for the types of analytes separated by FFF and SEC. We provide multi-angle light scattering, dynamic light scattering, refractive index, UV/Vis, and fluorescence detectors. For elemental analysis, coupling with ICP-MS is possible as well.

Most analytical scientists are familiar with chromatography, but FFF is not as well known. How is it different from, for example, size exclusion chromatography?

Well first, I would say there are some similarities. We are separating analytes of interest prior to the detectors in both cases, and AF4 and SEC both separate by size but with different physical principles. Probably the biggest advantage of FFF is the open channel architecture. This allows separation using only a mobile phase and physical forces that are the channel flow and the field(s) perpendicular to the channel flow that drives separation. This can be a flow field, centrifugal field, et cetera. Chromatography columns are filled with a stationary phase that provides the separation. Larger particles, for example, virus aggregates or ultra-high molecular weight polymers can be too large to pass through the pores in the stationary phase. By contrast, the open channel design of FFF allows relatively huge particles to pass, even up into the micrometer range. This enables a very wide analytical range, allowing us to separate particles and dissolved molecules in a single run, meaning you don’t need to ensure your sample is fully dissolved in a given solvent before analysis. We typically see better sample recovery with FFF as well. Since the separation size range and resolution depend on the interaction of the channel flow and field, a single FFF channel can be used for just about every application from 1 nm to over a micron. There is no need to buy an assortment of different columns for all your different projects.

You mention different applications but what are the most common research areas in which FFF is used?

Right now, obviously, viruses and antibodies are hot topics! We have customers in those fields, where often the specific application is measuring the extent of biomolecule aggregation. When used directly as a treatment for illness, having large amounts of aggregates present can cause an unwanted immune response. Determination of aggregate content is one great example of when FFF may be the best choice for biopharmaceutical applications. 

Nanotechnology has been a strong research topic for the last couple of decades as well. Although many definitions state that a nanoparticle is 1 to 100 nm in diameter, in reality, most “nano” formulations contain particles larger than 100 nm. At around 100 nm in diameter, the use of the SEC suffers from poor recovery due to the loss of particles on the stationary phase. As a result, FFF is often preferable to get accurate size distributions for nanoparticles. Applications of FFF may be found in quality control of engineered silver or gold nanoparticles, in naturally occurring colloids in the environment, or sometimes both in the case of engineered titanium dioxide released from consumer products to the environment.

Where biopharmaceuticals and nanotechnology come together is the rapidly growing multidisciplinary field of nanomedicine, which uses nanoparticles or vesicles to create a new generation of targeted treatments for a variety of diseases. Since FFF was already widely used for the analysis of nanoparticles, it was well-positioned to become a leading technique for the characterization of nanomedicines such as liposomes and polymer particles which can be loaded with drug molecules.

For ultra-high molecular weight polymers, there can often be a mixture of dissolved polymer and undissolved particle or gel content present. Using SEC for separation is a poor choice in this case, as the particulate content will be filtered out by the column, resulting in an incomplete picture of your sample’s distribution. Again, the open channel architecture of FFF makes it a great technique here – we can use AF4 or TF3 to separate dissolved polymer and gels in a single run, in either aqueous or organic solvents.

How does Postnova’s FFF platform differ from other offerings on the market in the past or present?

We are the only instrument vendor which specializes in FFF. Postnova offers a complete suite of FFF sub-techniques as I mentioned earlier: AF4, EAF4, CF3, and TF3, with the last three currently unique to Postnova. The way we differentiate our AF4 (current model AF2000) system is that it’s not an add-on module to an HPLC system. The two techniques may share some pieces of hardware but the analytical requirements are quite different.  For example, we use three separate pumps to control the three constantly changing flows required for AF4 during sample injection, focusing, and elution. This provides more precise control over flow rates and a wider range of possible flows. Simpler, HPLC add-on systems, use a crude splitting device for the flows but cannot achieve the same flow range, reproducibility, or precision required to cover all applications. When we free the AF4 from being simply an add-on module, we are able to control the parameters which are important to FFF. Also, by supplying the complete system from a single vendor, we can provide easier installation, better training, support, and maintenance, and only require the user to learn and use one software. Important little details like AF4 membrane quality matter too: we offer the broadest range of membrane materials and pore sizes on the market, and perform rigorous QC on them before shipping to customers. We also offer more options for channel configurations than ever before, enabling a wider variety of methods: in addition to our standard analytical scale channel, we offer a microchannel for faster separation, a frit-inlet channel for aggregation-prone samples, disposable hollow fiber channel for hazardous samples, and a semi-preparative scale channel.

What are some applications for these unique systems such as Centrifugal and Thermal FFF?

Centrifugal FFF separates by mass and is often used for separating nanoparticles with relatively high density. It can also be used to measure the amount of drug mass loaded into a drug delivery particle such as a liposome, or to measure the density of materials such as silver nanoparticles. It has also frequently been used in separation of environmental colloids or clays, often coupled to ICP-MS to enable additional elemental characterization.

Thermal FFF separates by thermal diffusion, which is influenced by the chemical composition of the analyte. Therefore, it is possible to separate analytes, e.g. polymers, with the same size or molecular weight but different composition to elucidate structural differences like branching or core-shell information. It is also finding a novel application in biopharmaceutical analysis, for example, separating liposomes from nucleic acids in solution.

The Electrical-Asymmetrical Flow FFF is your most recent addition to the FFF family. Can you provide some background on how this works and some key application areas for this?

Gladly, the Postnova EAF2000 has been an established member of our FFF family, as you called it, for about three years now. We combine the use of two fields: the flow field separates particles by size, and the electrical field separates by charge. By performing multiple runs with the same flow field conditions but varied electrical field conditions, we are able to determine particle electrophoretic mobility. Electrodes on the top and bottom of the channel generate the electrical field. The electrode material can be either titanium or platinum, and the field’s polarity can be switched. You can add the electrical field capability to an existing Postnova system easily by adding the electrical field generating module and the proper channel with electrodes.

As far as EAF4 applications go, any sample we can separate by size can have its electrophoretic mobility measured. One of the coolest applications is for samples with a multi-modal size distribution: different particle sizes may have different surface charges, which would not be detected by ensemble electrophoretic mobility or zeta potential measurements. This includes biomolecules and their aggregates like viruses and antibodies, but also drug delivery formulations like liposomes and polydisperse nanoparticle or environmental colloid samples, just to name a few important application areas.

You said that Postnova specializes in FFF. Besides having a diverse platform of FFF instruments, what does that mean?

All of our employees focus on FFF in one way or another, from marketing to sales, from R&D to the demo lab and from manufacturing to administration, all culminating in an installation at the customer site and ongoing support of the instrument. In addition, we employ an active FFF research group that engages with the worldwide scientific community through collaborative research projects, joint publications, and adding our expertise to standardization activities. Our application labs in the USA and Europe have staff with decades of experience supporting existing customers, and state-of-the-art instrumentation allows us to provide exciting demo results quickly. In other words, it means you can bring your difficult particle and macromolecule separation problems to us and we will be happy to discuss them and collaborate with you. These collaborations are what drive the science of FFF forward!   

About Dr. Soheyl Tadjiki

Dr. Soheyl Tadjiki received his B.Sc. in Chemistry from Middle Eastern Technical University in Ankara, Turkey, and his M.Sc. in Analytical Radio-Chemistry from Bilkent University, Ankara, Turkey. Dr. Tadjiki earned his Ph.D. in Chemistry from Monash University, Melbourne, Australia under the supervision of Dr. Ron Beckett and Dr. David Chittleborough. His postdoctoral studies were at Water Studies Centre, Melbourne, Australia, and UFZ, Magdeburg, Germany. He joined Postnova Analytics in 2001, and since 2016 he is Managing Director of Postnova Analytics USA. He has published 14 papers and presented over 50 oral and poster presentations.