Free Flow Electrophoresis (FFE)

(Contributor: Johann Bauer, Max-Planck Institute Martinsried, Germany)

Introduction

Free Flow Electrophoresis (FFE) is an electrophoresis procedure working continuously in the absence of a stationary phase (or solid support material such as a gel). It separates preparatively charged particles ranging in size from molecular to cellular dimensions according to their electrophoretic mobilities (EPMs) or isoelectric points (pIs). Samples are injected continuously into a thin buffer film, which may be segmented or uniform, flowing through a chamber formed by two narrowly spaced glass plates. Perpendicularly to the electrolyte and sample flow, current may be applied while the fluid is flowing (continuous FFE) or while the fluid flow is transiently stopped (interval FFE). In any case the applied electric field leads to movement of charged sample components towards the respective counterelectrode according to their electrophoretic mobilities or isoelectric points. The sample and the electrolyte used for a separation enter the separation chamber at one end and the electrolyte containing different sample components as separated bands is fractionated at the other side.

Applicability

  1. Conventional machines may be operated with a low ionic strength uniform electrolyte in the zone electrophoretic mode only (see middle scheme). Peptides, proteins, DNA, viruses, organelles, bacteria or cells can be separated at resolutions of 3-5% of their electrophoretic mobilities and a throughput of up to 50 mg protein or 20 million cells per hour may be achieved.
  2. Highly developed modern machines may be operated continuously or at intervals with segmented electrolyte (see both edge schemes) in various modes and with buffers containing up to 60 mM ions. Typical separation capabilities are shown in this Table:

    FFE-mode samples for separation resolution* throughput*
    zone electrophoresis (ZE) cells, bacteria, proteins, peptides, (DNA), viruses, membranes, organelles, enantiomers 3% EPM 40 Mio/h**
    50 mg/h***
    isoelectric focusing (IEF) proteins, peptides, membranes, organelles, viruses 0.03 pI 500 mg/h***
    isotachophoresis (ITP) proteins, peptides, membranes, organelles, (bacteria), viruses, enantiomers 1% EPM 150 mg/h***
    field step electrophoresis (FSE) preconditioning of any sample 30% EPM 5 g/h***
    * attainable at optimal conditions       ** eucaryotic cells     *** proteins or peptides

Literature References

  1. Bauer J. (ed.), Carrier Free Electrophoresis, Electrophoresis 19 (7): pp.1057-1233 (1998)
  2. Hymer W., Kirshnan K., Kraemer W., Welsch J., Lanham W., Mammalian pituitary growth hormone: Applications of free flow electrophoresis, Electrophoresis 21 (2): pp.311-317 (2000)
  3. Weber G., Grimm D., Bauer J., Application of binary buffer systems to free flow cell electrophoresis, Electrophoresis 21 (2): pp.325-328 (2000)
  4. Bauer J. (ed.), Cell Electrophoresis, CRC-Press, Boca Raton, 1994
  5. Bauer J., Hymer WC., Morrison DR., Kobayashi H., Seaman GVF., Weber G., Electrophoresis in Space, (Bonting SL, ed.) Advances in space biology and medicine, 7: pp.163-212 (1999).
Further relevant literature may be found at http://members.aol.com/ffeweber/lit.htm.