Exploring Free Flow Electrophoresis: A Comprehensive Review

In FFE, pressure drives a sample stream through a planar separation channel (Fig. 1b). An electric field is applied perpendicularly to the flow direction to deflect analytes into distinct streams^.1 The separation chamber is a flat compartment between two parallel plates separated by a spacer through which a laminar hydrodynamic flow is generated. Electrophoretic separation is feasible with separation times in the 1-2 min range. These features make FFE suitable as an interfacing separation technique for compounds possessing different electrophoretic mobilities.³

The mechanics of FFE

In FFE, the electric field is set perpendicular to a hydrodynamically pumped liquid system consisting of a sample fed into a carrier electrolyte (also named carrier buffer or separation buffer) flowing through a channel in the laminar regime. Each charged analyte molecule or particle i is deflected from the flow direction by the interplay of the drag velocity νhd and their specific electrophoretic velocity νi,ep. The magnitude of the individual νi,ep is a function of the type of analyte, the local buffer environment, and electric field strength. In general terms and for charged species, the resulting analyte velocity vector, the sum of both velocities mentioned above, will result in diagonally flowing analytes and, therefore, in a two-dimensional separation with subsequent fractionation at the outlets. FFE can be applied to all separation modes known from capillary electrophoresis. The difference between the separation modes lies in the number and the composition of the separation buffer and gradients generated or externally applied. The simplest separation mode is zone electrophoresis (ZE), in which only one separation buffer is used. The properties of the separation buffer, e.g. concentration, conductivity, and pH, are comparable to those of the sample. Because of its simplicity, this separation mode is often used for inaugural proof-of-concept experiments demonstrating the capability and performance of conceptually new devices.⁴

The stable buffer flow in FFE is achieved by inducing gravity-induced pressure and using a gas cushion injector to reduce pulse flow, ensuring a stable flow in the FFE chamber.⁵ 

Particles are separated in FFE based on their charge, size, and electric field strength. The overall charge of bio-particles, including cells, organelles, and macromolecules, determines their separation in FFE.⁶ Additionally, introducing organic solvent into the electrolyte system has been shown to increase the solute solubility and throughput of the sample, enhancing the separation of weak polarity solutes.⁷    

Applications 

FFE is a continuous and relatively fast electrophoretic separation technique used in various regular and miniaturized analytical systems.³ Free-flow electrophoresis (FFE) has broad applications in biochemistry, molecular biology, diagnostics, and therapeutic manufacturing, particularly for separating proteins, enzymes, membrane particles, organelles, and cells.2,8,9 This technique has been instrumental in various biomedical applications, including proteomic sample preparation, continuous particle separation, biochemical detection, and the enrichment of viable bacteria.3,10-12 

Biochemical Detection

Mazereeuw et al., presented a study on developing a free flow electrophoresis (FFE) device for continuous electrophoretic separation of charged compounds, which was then integrated into a continuous flow biochemical detection (BCD) system. In the study, the continuous separation capabilities of FFE make it suitable for online implementation in chromatographic or flow injection analysis systems, where the additional separation of charged compounds is required. The authors demonstrate the application of this system in a heterogeneous biochemical flow assay for the determination of biotin, where an analyte zone reacts with an excess of an affinity protein, followed by the reaction of the free binding sites of the affinity protein with an excess of fluorescein-labeled ligand. The FFE device separates the free and affinity protein-bound labels before fluorescence detection of the separated fractions. The study specifically focuses on separating biotin and streptavidin as model ligands and affinity proteins, respectively, due to their different electrophoretic properties.³ 

The authors successfully perform quantitative analysis after completely separating the fluorescent affinity complex and labeled biotin within 2 minutes using the FFE device. The separation of free FB and streptavidin-bound FB was demonstrated by mixing FB and streptavidin at a molar ratio of streptavidin binding sites to biotin of 1:8. The concentration of FB was 6 µmol/L. The free FB and streptavidin-FB mixture was continuously infused into the device with a flow of 1 µL/min. The compound lines are visualized by illuminating the entire separation compartment. A drawing of the separated compound lines is shown in Fig 3. Since the device is optically transparent, the separated zones can be detected in the separation compartment using laser-induced fluorescence. The applicability of the BCD-FFE system in combination with an HPLC separation is demonstrated in the bioanalysis of biotin in human urine at the micromole per liter level.³

Microfluidic free-flow electrophoresis proteomics

Microfluidic free-flow electrophoresis is the most promising technique for proteomics studies involving large-scale purification and analysis of proteins. This technique can perform real-time separation of proteins with electrophoretic mobility or isoelectric point in a small device where a continuous flow of carrier buffer is driven, and an external electric field is applied perpendicular to the buffer flow. The capability of μFFE has offered significant benefits for separating, isolating, and characterizing target proteins in biological systems. High-resolution separation of the entire protein sample can be achieved quickly. A small volume of protein sample is consumed in the separation process. Excellent coupling with mass spectrometry is ensured for protein analysis. These benefits have found extensive applications of μFFE in the proteomics fields that require prefractionation, enrichment, and higher-level purification of proteins while preserving their integrity.¹³

Enrichment of viable bacteria in a micro-volume by free-flow electrophoresis¹²

Macro- to micro-volume concentration of viable bacteria is performed in a microfluidic chip. The enrichment principle is based on free-flow electrophoresis and is demonstrated for Gram-positive bacteria.

Podszun et.al., demonstrated the highly efficient enrichment of bacteria in a continuous free-flow electrophoresis chip. Enrichment efficiencies up to 80% and concentration factors of over 13 from 100 ml and 25 from 200 ml samples were obtained. The results and the applied enrichment principle showed that feeding more sample volume can quickly achieve higher enrichment factors. Moreover, the applicability for analytically relevant concentrations of as little as 266 cells per ml was shown. Using free-flow electrophoresis, the researchers demonstrated the feasibility of efficiently capturing target species in a micro-volume. They introduced several novel handling techniques that make the system easy to operate, highly reproducible, and allow quantification of bacteria trapping and viability.¹²

Advantages of FFE

The advantages of FFE include its precision, ability to process large volumes, and gentle conditions that preserve biomolecular integrity.14 FFE offers high precision in separating biomolecules, cells, and nanoparticles based on their charge differences, enabling the continuous and efficient separation of analytes with minimal band broadening.1,14Moreover, the technique’s capability to process large volumes of samples makes it suitable for preparative applications in therapeutic manufacturing and proteomic sample preparation.10,14 Importantly, FFE operates under gentle conditions, preserving the integrity of biomolecules during separation, which is crucial for maintaining the functionality of separated analytes.14

Integration of FFE with a pressure flow controller 

There are significant enhancements to free-flow electrophoresis (FFE) processes, particularly in improving precision, efficiency, and scalability in FFE applications. Pumps and controllers provide precise and stable flow control, enabling accurate manipulation of sample and buffer flows in FFE systems. This precision is crucial for achieving consistent and reliable separations in FFE. 

A close example of integrating microfluidics solutions in FFE was in a pressure-controlled microfluidic system proposed to automate single-molecule sample preparation.

Conclusion

The potential for integrating FFE techniques with cutting-edge products has been strongly reinforced by the swift evolution of micro-free-flow electrophoresis (μFFE) techniques. This progress has opened avenues for the ongoing separation of biomolecules, showcasing the promise of advancements in both research and analytical capabilities. Through the utilization of these techniques, researchers and professionals stand to elevate their decision-making processes, enhance project performance, and attain breakthroughs in product development.

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