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 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