Milli‐free flow electrophoresis: I. Fast prototyping of mFFE devices

Abstract

We coin a term of milli-free flow electrophoresis (mFFE) to describe mid-scale FFE with flow rates intermediate to macro-FFE and micro-FFE (μFFE). Introduced decades ago, mFFE did not find practical applications. We revive mFFE, as we view it as a viable purification complement to continuous synthesis in capillary reactors with product flow rates of ∼5 to 2000 μL/min, too small for macro-FFE but too large for μFFE. The development of the tandem of continuous synthesis/purification will require the production and evaluation of a large number of prototypes of mFFE devices. As the first step, we developed a fast (<24 h) and economical (∼$10) method for prototyping mFFE devices using a robotic milling machine. mFFE prototypes are constructed from two machined matching poly(methyl methacrylate) (PMMA) substrates, which are bonded in 10 min using dichloromethane to provide a strong and irreversible seal. Using the developed prototyping technology, we designed and evaluated 25 prototypes of mFFE devices. By optimizing the feed rates and rotational speeds of the drills, the depth of the electrode channels, the dimensions of the entrance and exit reservoirs, the sample flow rate, and the diameter and position of the sample input, we were able to achieve indefinitely long operation of the device with cycles of alternating 15-min electrophoresis and 0.5-min regeneration (bubble removal). The test analytes, rhodamine B and fluorescein, were baseline resolved by mFFE for flow rates ranging from 10 to 600 μL/min. These results prove that our prototyping approach is suitable for the challenging task of multi-parameter optimization of mFFE devices.

Quantitative investigation of resolution increase of free‐flow electrophoresis via simple interval sample injection and separation

“…As an important preparative purification technique [1][2][3][4][5], freeflow electrophoresis (FFE) has been used for separation of small ions [6,7], DNA [8,9], peptides [10,11], proteins [12][13][14], and membrane vesicles [15,16] as well as cells [17][18][19][20]. Due to its complete liquid-phase electrophoresis, FFE has numerous advantages, such as being with quite gentle and controllable experiment conditions, relatively high-yield preparation, well protection of sample activity, and wholly continuous sepa-ration mode in contrast to a batch one in chromatography [21][22][23][24].…”

Advances in steady-state continuous-flow purification by small-scale free-flow electrophoresis

“…FFE devices can be classified into four categories based on their scale -macroscale [36,37], midscale [38], milliscale (mFFE) [39][40][41], and microscale (μFFE) [42,43]. The scale categories are mainly defined by the volumes of the separation channels but also by acceptable ranges of flow rates and electric field strengths ( Table 1).…”

“…In general, however, µFFE flow rates are too small for high-throughput preparative technology and do not reflect flow rates that are typically used in continuous-flow micro-reactors. The flow rates used in larger-scale FFE are compatible with the flow rates in most continuous-flow micro-reactors, making these purification scales better suited to CFC design [39].…”

Recent developments in capillary and microchip electroseparations of peptides (2011–2013)

“…Various FFE modes, particularly the FF‐ZE and FF‐IEF have been used for prefractionation of complex peptide/protein mixtures in proteomic studies of cells and body fluids . FFE systems have been designed also in the midscale , milliscale , or microchip formats . However, due to the limited preparative capacity of the latter devices, they are more suitable for continuous monitoring of selected analytes than for really preparative purposes.…”