Field-Flow Fractionation Supporting Consumer Safety Evaluation of Silver Nanoparticle Applications in Food Packaging Polymers

Throughout the chemical industry there is a growing movement toward macromolecular research. In particular, the ongoing race for nanotechnology development has resulted in a need for analytical methods that can accurately and efficiently separate, characterize, fractionate, and undertake the speciation of nanoparticles, polymers, and proteins. Field-flow fractionation (FFF) presents a powerful method of separation and fractionation ideal for the separation of various nano- and macro-sized sample types. Depending on the FFF technique applied, separation can be undertaken on a range of sample ranges from 1 nm up to several microns. Globally, FFF finds use in a broad variety of fields of application, within environmental, food safety, biopharmaceutical, and polymer research. In particular, FFF has found widespread use within nanoparticle research.

Coupled to detection systems such as static light scattering (SLS), multiangle light scattering (MALS), dynamic light scattering (DLS), different forms of UV detectors, small-angle X-ray scattering (SAXS), or inductively coupled plasma-mass spectrometry (ICP-MS) or other detector, FFF separation technologies enable the fractionation, characterization, and speciation of large, complex polymers, biomacromolecules, and nanoparticle species. Such separations often cannot be performed using traditional chromatographic methods, predominantly because of the stationary phase in traditional chromatographic separations. Consequently, FFF online light scattering coupling presents another potential for real-time separation and analysis. This is highly recognized for any macromolecular research where the highest resolution and reproducibility are required. This article will examine the various forms and applications of FFF separation, with particular reference to the use of silver nanoparticles in food packaging.

The rise of nanotechnology

The discovery that particles on a nanoscale can display significantly different properties to their corresponding bulk material was a major turning point in materials research. Nanotechnology now ranks as one of the most studied subjects and transcends almost every branch of science.¹ Today, the interesting behavioral novelties and range of fascinating properties offered by nanoparticles bring exciting opportunities to a diverse range of industries.

There is no distinct definition as to what constitutes a nanoparticle. Generally, they are considered to be a species with at least one dimension of the order of 100 nm or less, although species much larger than 100 nm have been found to exhibit nanoparticle properties. Regardless of how they are defined, nanotechnology represents one of the most exciting, not to mention most lucrative, industries to emerge in the past couple of decades. In fact, such is the demand for nanomaterial that the conversion of clay from its natural to nanoparticulate form increases its value by over 100 times.² This explosion of interest in nanoparticle development has led to demand for a separation technique capable of accurately and sensitively analyzing these large, uniquely complex species.

Traditional separation techniques that have been in practice for generations for the separation of relatively small species are not suited to fractionation of bulky nanoparticle structures. Additionally, typical macromolecular chromatographic methods, such as size exclusion chromatography (SEC), that achieve separation via analyte and stationary phase interaction are often incapable of nanoparticle separation.

Principles of field-flow fractionation

FFF was created by American chemist Prof. J. Calvin Giddings in 1966 as a gentler, higher-resolution alternative to traditional chromatographic methods. Twice nominated for a Nobel Prize, Prof. Giddings pioneered the technique, producing the first commercially available fractometer and founding the U.S.-based FFFractionation, Inc., later to become Postnova Analytics Inc. (Salt Lake City, UT), following his death.

Figure 1 – FFF principle and the separation mechanism, first with the laminar flow that carries the sample through the separation chamber, and second, with the separation field applied perpendicular to the channel, against the sample flow.

Today, FFF’s ability to separate large macromolecular structures from solution with minimal sample preparation and without sample matrix interaction has married with the evolving nature of the chemical industry toward nanotechnology. The advantages of FFF for nanomaterial separation are given in the Appendix.

What makes FFF such an appealing technique is not only its breadth of analysis but its operational simplicity. There are two components that make up the FFF system. Firstly, the laminar flow that carries the sample through the separation chamber, and secondly, the separation field applied perpendicular to the channel, against the sample flow (Figure 1).⁴

As particles flow along the chamber, the cross-flow separation field pushes the molecules toward the bottom of the channel. As they pass by the bottom, they diffuse back into the channel against the carrier flow (Figure 2). The extent to which the molecules can diffuse back into the channel is dictated by their natural Brownian motion, a characteristic based on size that is unique to each individual species. Smaller particles have a higher Brownian motion than larger ones and are able to diffuse higher into the channel against the carrier flow.

Figure 2 – Flow field-flow fractionation (AF4) channel cross-section, where the rate of laminar flow within the channel is not uniform. It travels in a parabolic pattern with the speed of the flow, increasing toward the center of the channel and decreasing toward the sides.

The rate of laminar flow within the channel is not uniform. It travels in a parabolic pattern with the speed of the flow, increasing toward the center of the channel and decreasing toward the sides (see Figure 2). Therefore, the rate at which particles will be carried through will depend on their position within the channel. Those with a greater diffusion located in the center of the channel will be transported with a greater velocity. The larger particles in the shallow, slower-moving stream are transported with lower flow velocity and elute later than the smaller particles. This results in a gentle separation of particles based on mass with the elution order of smallest to largest.

Forms of field-flow fractionation

Around this basic principle numerous FFF techniques have been developed that are eminently suited to particular applications (Figure 3). These vary both in the nature of the separative applied field and the size of particle undergoing separation. Among them are flow FFF, centrifugal FFF, thermal FFF, and gravitational FFF, all named for the form of the separation field applied.

Figure 3 – Schematic of the different applications in which FFF technology is widely used by academia, government institutions, and industry.

Recent developments have led to the newer technique of centrifugal FFF, wherein the separation field is supplied via a centrifugal force. The channel takes the form of a ring, which spins at 4900 rpm, as illustrated in Figure 4. The flow and sample are pumped in and the mixture is centrifuged, allowing the operator to resolve the particles by size and density. The advantage of centrifugal FFF lies in the broad range of samples and high resolution that can be achieved by varying the speed and force applied.

Figure 4 – Recent developments have led to the newer technique of centrifugal FFF, in which the separation field is applied via a centrifugal force. The channel takes the form of a ring, which spins at 4900 rpm, with separation based on size and density.

In contrast to chromatographic techniques, no stationary phase interactions occur, which eliminates particle interaction and shearing.

Another advantage of the technique is that molecules can be separated by particle density, rather than just particle size. This can be particularly useful for novel products such as composite materials and coated polymer-containing nanoparticles, samples that may not vary in size but vary in density. In this way, two identically sized particles can still be separated into two peaks, provided the density is different (Figure 5).

Figure 5 – Centrifugal FFF has the advantage that molecules can be separated by particle density rather than just particle size. In this instance, two identically sized gold and silver nanoparticles can be separated into two peaks, according to differences in density in the gold and silver nanoparticles, separated with the CF2000 instrument (Postnova Analytics).

The CF2000 instrument from Postnova Analytics was developed as a modular FFF system, which can be easily interfaced with other existing detection systems such as UV, DLS, MALS, SAXS, or ICP-MS. This allows for the separation of a range of particle sizes in just a single analysis, and avoids the discrimination of smaller particles by large species, a limitation that often plagues light scattering-based techniques.

Silver nanoparticles in food packaging

One novel and surprisingly common application of nanotechnology is the use of silver nanoparticles (SNP) within food packaging polymers.⁵ Silver has been used medicinally since ancient times, and Hippocrates, often considered the father of Western medicine, believed that silver had beneficial healing and antidisease properties. The underlying effect behind this property is related to the release of silver (Ag⁰) from the SNP followed by its oxidation to antimicrobial active silver ions (Ag²⁺).⁶ These can diffuse through a bacterial cell wall, disrupting their metabolism and eventually destroying it. The benefit of this in food packaging comes from SNP’s potential to enhance the shelf-life of food.

Migration potential of nanoscale silver particles

Due to the growing use of silver as an antibacterial agent, there is an increased necessity to understand the potential effect these species will have on their environment and the consequent impact they may have on public health. To illustrate the potential of FFF, a study on the migration of silver nanoparticles in low-density polyethylene (LDPE) in food packaging⁷ was undertaken to provide some answers to the question: To what extent is the consumer exposed as a result of migration of nanoparticles in food packaging?

The study was carried out by the Fraunhofer Institute for Process Engineering and Packaging (IVV) in Freising, Germany.⁷ For the migration study, LDPE films with differing silver content were used. The migration tests were performed under varying contact conditions to simulate realistic long-term storage, using 3% acetic acid, 10% ethanol, 95% ethanol, and iso-octane. ICP-MS was interfaced with the FFF instrument to determine the content of total silver (Ag⁰ and Ag⁺) in the migration solution.⁸

Transmission electron microscopy (TEM) was utilized to view the distribution of SNP in the polymer and their particle sizes in relation to the nominal values. The separations were carried out on the Postnova Analytics AF2000MT series for AF4, equipped with a 10-kDa regenerated cellulose (RC) membrane so that nanoscale silver could not pass through the channel membrane. Equally, this separation could have been undertaken on the CF2000 to provide even higher resolution and separation of the SNP, due to the ability of the centrifugal FFF CF2000 instrument to separate particles by dynamic diffusion on the basis of both particle size and density. This allows for the separation of particles with only a 5% difference in size. In AF4 separation, it is based on a 1:1 ratio of mass to time. The addition of the third parameter of density to centrifugal fractionation produces a ratio more akin to: mass to time to the power of three. This produces significantly larger distinction between peaks and results in greatly improved resolution.

Colloidal dispersions of silver release Ag ions from the surface of silver particles, and this is utilized in drinking water sterilization. It was found that 3% acetic acid accelerates the oxidative process until complete dissolution within 24 hr at room temperature (Figure 6). In 95% ethanol and iso-octane, the silver nanoparticles remain unchanged. The Ag migration is enhanced through the penetration of small acetic acid molecules into the LDPE film. Smaller amounts of silver are found in 10% ethanol and can be explained by dissolution of Ag⁺ from the silver nanoparticles present at the surface of the film. Dissolved silver ions are released from the polymer and, for this reason, silver nanoparticles are used for the intended antimicrobial effect. Migration of silver nanoparticles themselves could not be detected.

Figure 6 – Illustrating the potential of FFF to study the migration of silver nanoparticles in LDPE from food packaging, it was found that the silver nanoparticles were stable in normal aqueous conditions, yet will solubilize into Ag⁺ ions when exposed to acidic conditions such as humic acid.

Conclusion

Since Prof. Giddings developed the first field-flow fractionation system over half a century ago, the nature of the chemical and materials industry has changed unimaginably. Today, the need for a technique that offers high-resolution separation for macromolecules has led laboratories across the world to embrace field-flow fractionation as the most effective solution to nanoparticle analysis.

References

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2. www.malvern.com/labeng/industry/nanotechnology/nanoparticle_ headlines.htm; accessed 04/02/2013.
3. Klein, T. Comparison of AF4, AUC and SEC for Monoclonal Antibody Aggregate Quantitation. Postnova application note.
4. Giddings, J.C. New separation concept based on a coupling of concentration and flow non-uniformities. Sep. Sci.  1966, 1, 123–5.
5. Varner, K.E.; El-Badawy, A. et al. U.S. EPA Report, 2010 (EPA/600/R-10/084); 155–9.
6. Lee, Y.-J.; Kim, J. et al. Envir. Toxicol. Chem. 2012, 31, 155–9.
7. Bott, J.; Störmer, A. et al. Migration potential of nanoscale silver particles in food contact polyolefins. Poster presented at the 5th International Symposium on Food Packaging, Berlin, Nov 14–16, 2012.
8. Bolea, E.; Jiménez, J. et al. Anal. Bioanal. Chem. 2011, 401, 2723–32.

Dipl. Ing. Johannes Bott, Ph.D., is a Student, Department of Product Safety and Analysis, Fraunhofer Institute for Process Engineering and Packaging (IVV), Freising, Germany. Dr. Soheyl Tadjiki is Science Director, Postnova Analytics, Inc., 230 S. 500 E #120, Salt Lake City, UT 84102, U.S.A.; tel.: 801-521-2004; e-mail: [email protected].