Free Flow Electrophoresis

Most electrophoretic methods have been tried in a free-flow format, including isoelectric focusing, native zone electrophoresis, and isotachophoresis. Most free-flow electrophoresis equipment has very low (ca 1 g/(L-h)) capacity, and resolution is reduced by heating and electroosmotic considerations. 

Preparative continuous free flow electrophoresis was first reported in 1958 [15]. As in the case of classical gel electrophoresis, most of the work done in this area has been primarily in the purification of biopolymers. Continuous free flow electrophoresis for the separation of small molecules has remained relatively unexplored [16], although this is beginning to change. 

In preparative continuous free flow electrophoresis, continuous buffer and sample feed are introduced at one end of a thin, rectangular electrophoresis chamber. A schematic is presented in Fig. 11-5. The sample stream is usually introduced through a single port while buffer is introduced through several ports, essentially producing a buffer curtain . Because the buffer streams are introduced independently, it is fairly easy to establish a variety of gradients (e.g., pH, density, ionic strength) across the buffer curtain . 

Fig. 11-5. Schematic of continuous free flow electrophoresis apparatus.

Continuous free flow electrophoresis has been used for the separation of biopolymers (e.g. ovalbumin and lysozyme) [20] as well as smaller inorganic species (e.g. [Co sepulchrate)] and [Co (CN)g] ) [21]. Sample processing rates of 15 mg h were reported for a mixture of Amaranth (MW 804) and Patent Blue VF (MW 1159) [22]. 

As in CE, changing system variables (e.g., pH, ionic strength, additive concentration) is very easy in any of the continuous free flow electrophoresis systems reported here because all the interactions take place in free solution. Indeed, changing system variables may be easier in continuous free flow electrophoresis systems than in a CE system because there are essentially no wall effects. Of course, changing system variables in the continuous free flow electrophoresis apparatus may also be easier... 

The author would like to acknowledge R S Technologies, Inc. (Wakefield, RI, USA) for the loan of the continuous free flow electrophoresis system, and Cerestar, Inc. for the donation of the sulfated cyclodextrin. The author would also like to thank Drs. Chris Welch and Prabha Painuly for helpful discussions. 

Douglas et al. [98] have measured protein (serum albumin, ovalbumin, and hemoglobin) mobilities over a range of pH values using a free-flow electrophoresis apparatus and a particle electrophoresis apparatus. They found good agreement between the two measurements however, they also found some differences between their measurements and those reported in the older literature. They attributed the differences to the use of moving-boundary electrophoresis methods in the early experimental work and to differences in... 

Kuhn, R. and Wagner, H., Application of free flow electrophoresis to the preparative purification of basic proteins from an E. coli cell extract,... 

Fig. 11-6. Histograms showing the distribution of piperoxan enantiomers in the absence (a) and presence (b) of sulfated cyclodextrin in continuous free flow electrophoresis.

Hoffmann, R, Ji, H., Moritz, R. L., Connolly, L. M., Frecklington, D. F., Layton, M. J., Eddes, J. S., Simpson, R. J. (2001). Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215 a non two-dimensional gel electrophoresis-based proteome analysis strategy. Proteomics 1(7), 807. 

Free-flow electrophoresis, 9 752 Free lime, 75 27 hydration, 5 478... 

Sengelpv, H., Nielson, M. H., Borregaard, N. (1992). Separation of human neutrophil plasma membrane from vesicles containing alkaline phosphatase and NADPH oxidase activity by free flow electrophoresis. J. Biol. Chem. 267,14912-17. 

Free-Flow Electrophoresis. Free-flow electrophoresis is the most common technique for scaling up electrophoresis for commercial application, in this technique, sample compounds are injected into a curtain of buffer which flows between two flat plates, with electrodes parallel to the flow at each end. The electric field is then applied perpendicularly to the flow direction, so that as compounds flow down between the electrodes they separate horizontally and exit the flow field at different locations. 

Schmitt-Kopplin, P., and Kettrup, A. (2003). Capillary electrophoresis-electrospray ionization-mass spectrometry for the characterization of natural organic matter An evaluation with free flow electrophoresis-off-line flow injection electrospray ionization mass spectrometry. Electrophoresis 24, 3057-3066. 

Raymond, D.E., Manz, A., Widmer, H.M., Continuous sample pretreatment using a free-flow electrophoresis device integrated onto a silicon chip. Anal. Chem. 1994, 66, 2858-2865. 

Heidrich HG, Dew M (1977) Homogeneous cell populations from rabbit kidney cortex. Proximal, distal tubule, and renin-active cells isolated by free-flow electrophoresis. J Cell Biol 74 780-788... 

To remove carrier ampholytes when electrospray ionisation mass spectrometry is used on-line with capillary isoelectric focusing, a specially designed free-flow electrophoresis device can be coupled to the CIEF system [67],... 

Moritz, R.L., Clippingdale, A.B., Kapp, E.A., Eddes, J.S., Ji, H., Gilbert, S., Connolly, L.M. and Simpson, R.J. (2005) Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis of human plasma depleted of multi high-abundance proteins. Proteomics 5, 3402-3413. 

The technique of choice in the detection and isolation of protein complexes is immunoprecipitation [15], whereas in some cases centrifugation or free-flow electrophoresis can be applied for the purification of cellular organelles. Immunoprecipitation permits the purification of specific proteins against which an antibody has been raised. Affinity-based methods have been developed for a variety of organelles. When apphed to the isolation of protein complexes, the antibody should target one of the proteins in the complex. The antibody-protein complex precipitates from solution. Alternatively, inunobilization of the primary antibody to agarose (beads) can be applied. Mrrlti-step procedttres have been developed as well. 

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