Utilizing Field-flow Fractionation Techniques in Molecular Biology and Biotechnology

Johanna Tim^*
*Correspondence: Johanna Tim, Department of Biochemistry and Molecular Cell Biology, Radboud University Medical Centre, 6525 GA Nijmegen, The Netherlands, Email:

Introduction

In the realm of molecular biology and biotechnology, the quest for understanding the intricate world of macromolecules and nanoparticles has propelled the development of innovative analytical techniques. Among these, Field-Flow Fractionation (FFF) has emerged as a powerful tool for the separation, characterization and analysis of a diverse range of particles, including proteins, nucleic acids, nanoparticles and biological cells. The versatility and precision of FFF make it invaluable for researchers aiming to unravel the complexities of molecular interactions and dynamics. Field-flow fractionation encompasses a family of techniques that separate particles based on their size, shape and other properties by applying an external field. This external field, such as a gravitational, electric, magnetic, or thermal field, influences the movement of particles within a flow channel, allowing for high-resolution separation. Unlike conventional chromatographic methods that rely on stationary phases and interactions between the analytes and the stationary phase, FFF techniques utilize a continuous flow field, which offers unique advantages in terms of resolution, efficiency and the ability to analyze particles in their native state [1].

Description

Field-Flow Fractionation (FFF) techniques can be classified into several types based on the nature of the applied field. Each type of FFF technique has specific advantages and applications, making it suitable for different analytical needs. Centrifugal Field-Flow Fractionation (CFFF) utilizes a centrifugal force to separate particles based on their size and density. In this technique, a centrifugal field is applied perpendicular to the flow direction in a thin channel. Larger particles experience a greater centrifugal force, causing them to migrate towards the channel wall, while smaller particles remain closer to the center. The separation occurs as particles exit the channel at different rates, allowing for their characterization based on their size and density [2]. CFFF is particularly useful for analyzing macromolecules, such as proteins and polysaccharides, as well as particles with varying densities. Electrical Field-Flow Fractionation (ElFFF) applies an electrical field to separate particles based on their charge and size. In this technique, particles with different electrophoretic mobilities are influenced by the electric field, causing them to migrate at different velocities. The separation is achieved as particles travel through a channel with a continuous flow of buffer solution. ElFFF is commonly used for analyzing charged particles, including nucleic acids, proteins and synthetic polymers. It provides high resolution and sensitivity, making it suitable for detailed characterization of complex biomolecules. Magnetic Field-Flow Fractionation (MFFF) employs a magnetic field to separate particles based on their magnetic properties [3].

Particles with different magnetic susceptibilities experience varying forces in the magnetic field, causing them to move differently within the flow channel. MFFF is particularly useful for analyzing magnetic nanoparticles and particles with inherent magnetic properties. It provides unique insights into the behavior and distribution of magnetic particles in complex biological systems. Thermal Field-Flow Fractionation (ThFFF) uses a thermal gradient to separate particles based on their thermal diffusivity. In this technique, a temperature gradient is applied across the flow channel, causing particles to migrate according to their thermal properties. ThFFF is valuable for analyzing particles with different thermal behaviors, such as proteins and polymers with varying thermal stabilities. It offers high resolution and is capable of distinguishing between particles with subtle differences in thermal properties. Field-flow fractionation techniques have revolutionized molecular biology by providing detailed insights into the size, shape and interactions of biomolecules. FFF techniques are widely used for the separation and characterization of proteins. For example, CFFF allows researchers to analyze proteins based on their size and density, providing information on protein aggregates, conformations and post-translational modifications. ElFFF enhances the understanding of protein charge properties, while ThFFF offers insights into protein thermal stability. By combining FFF with other analytical methods, such as mass spectrometry, researchers can gain a comprehensive understanding of protein structures and functions. ElFFF is particularly effective for the separation and analysis of nucleic acids, including DNA and RNA. By leveraging the electrophoretic mobility of nucleic acid fragments, ElFFF enables high-resolution separation of different sizes and conformations of nucleic acids [4].

This technique is valuable for studying gene expression, DNA fragmentation and RNA modifications. Additionally, FFF techniques can be employed to analyze nucleic acid interactions with proteins and other biomolecules, providing insights into cellular processes and genetic regulation. The rise of nanotechnology has led to the development of various nanoparticles with diverse applications in medicine, electronics and materials science. FFF techniques, particularly MFFF, are essential for characterizing magnetic nanoparticles and other nanomaterials. By separating nanoparticles based on their size, shape and magnetic properties, FFF provides crucial information for designing and optimizing nanoparticle-based technologies. For instance, MFFF can be used to analyze the distribution and behavior of magnetic nanoparticles in drug delivery systems or imaging agents. The analysis of biological cells, including their size, shape and internal properties, is another important application of FFF techniques. For example, ThFFF can be used to study the thermal properties of cells and their response to temperature changes. Additionally, CFFF can separate cells based on their size and density, providing insights into cell populations and heterogeneity. FFF techniques enable researchers to investigate cellular processes, such as cell division, differentiation and response to environmental stimuli [5].

Conclusion

Field-flow fractionation techniques have significantly advanced the field of molecular biology and biotechnology by offering unique and powerful methods for the separation, characterization and analysis of a wide range of particles. From proteins and nucleic acids to nanoparticles and cells, FFF techniques provide high resolution and detailed insights into the properties and behaviors of these biomolecules. The versatility of FFF techniques allows researchers to address complex questions in molecular biology, such as protein structure and function, gene expression and cellular dynamics. By applying different types of field-flow fractionation, researchers can tailor their analysis to specific experimental needs, leading to a deeper understanding of biological processes and the development of novel technologies. As the field of molecular biology continues to evolve, FFF techniques will remain a crucial tool for advancing our knowledge of macromolecules and nanoparticles. The integration of FFF with other analytical methods, such as mass spectrometry and imaging techniques, will further enhance its capabilities and applications. Ultimately, the continued development and refinement of field-flow fractionation techniques will contribute to breakthroughs in molecular biology, biotechnology and related fields, paving the way for new discoveries and innovations.

Acknowledgement

None.

Conflict of Interest

None.

References

1. Marassi, Valentina, Marco Macis, Stefano Giordani and Lucia Ferrazzano, et al. "Application of af4-multidetection to liraglutide in its formulation: Preserving and representing native aggregation." Molecules 27 (2022): 5485.

Google Scholar, Crossref, Indexed at

2. Yang, Jun, Ying Huang, Xiao-Bo Wang and Frederick F. Becker, et al. "Cell separation on microfabricated electrodes using dielectrophoretic/gravitational field-flow fractionation." Anal Chem 71 (1999): 911-918.

Google Scholar, Crossref, Indexed at

3. Yohannes, Gebrenegus, M. Jussila, Kari Hartonen and M-L. Riekkola. "Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles." J Chromatogr A 1218 (2011): 4104-4116.

Google Scholar, Crossref, Indexed at

4. Reschiglian, Pierluigi andrea Zattoni, Barbara Roda and Elisa Michelini, et al. "Field-flow fractionation and biotechnology." Trends Biotechnol 23 (2005): 475-483.

Google Scholar, Crossref, Indexed at

5. Fuentes, Catalina, Jaeyeong Choi, Claudia Zielke and J. Mauricio Peñarrieta, et al. "Comparison between conventional and frit-inlet channels in separation of biopolymers by asymmetric flow field-flow fractionation." Anlst 144 (2019): 4559-4568.

Google Scholar, Crossref, Indexed at