Two-Phase Electrophoresis

William M. Clark, Chemical Engineering Department, Worcester Polytechnic Institute
100 Institute Road, Worcester, MA 01609-2280

Two-phase electrophoresis is a hybrid separation method where an electric field is applied perpendicular to a liquid-liquid phase interface as shown in Figure 1. The distribution of charged species between the phases is governed by phase partitioning and electrophoretic mobility. The method has also been called electropartitioning [1] and electroextraction [2] but two-phase electrophoresis [3] describes the process better because it is more akin to electrophoresis than liquid-liquid extraction. As shown in Figure 1, the original motivation for introducing the phase interface was to control convective mixing due to ohmic heating during electrophoresis [1, 2]. Mixing might still occur within a phase, but not between the phases. Recently, it has been shown that the phase interface can also play an active role in the separation process [4].

The first requirement to achieve the objective shown in Figure 1 is to have two stable liquid phases that are both electrically conductive. This requirement has been met by using aqueous two-phase polymer solutions or by using an aqueous solution together with an organic solvent, like n-butanol, that is partially miscible with water and dissolves enough water to make the organic phase conductive. For example, it has been shown that organic acids and dyes can be transferred from an aqueous phase to an n-butanol-rich phase using an electric field [2, 5]. Two-phase electrophoretic transfer of carboxylic acids from an aqueous phase to an organic phase containing trioctylamine in n-hexanol and transfer of the acids back from this organic phase to the aqueous phase has also been accomplished [6]. These studies utilized simple U-tube electrophoresis devices for batch separation and countercurrent flow equipment for continuous processing. Efficient recovery of citric acid from water was demonstrated in a 10-stage apparatus that used co-current flow of the phases on each stage but provided an overall countercurrent flow [2]. Platinum electrodes were immersed directly in the phases in this apparatus as well as in other flow equipment that facilitated excellent removal of dyes from water using a single stage with the phases flowing countercurrent to each other [5].

Aqueous two-phase polymer solutions can be formed when two polymers like polyethylene glycol (PEG) and dextran are dissolved in water. For example, when 3.8 g of PEG (average molecular weight 8000) and 5.5 g of dextran (average molecular weight 500,000) are dissolved in 90 g of water, two phases (a PEG-rich phase and a dextran-rich phase) are formed with nearly equal volume and each containing about 90 weight % water [7]. These two-phase systems have been utilized extensively for many years to make biocompatible extractive separations [7]. Buffered aqueous two-phase systems of this type have been employed for two-phase electrophoretic separation of proteins [1, 3, 8-11], amino acids [12, 13], and DNA [4, 14, 15] in both batch and flow devices. In these studies, membranes or polymer gels have usually been employed to isolate the phase systems from the electrodes to prevent electrolysis gases from disrupting the phase boundary. Batch studies have been made in U-tube and single cylinder electrophoresis devices. For flow systems, pumps have been used to establish two (PEG-rich/dextran-rich) or three (PEG-rich/dextran-rich/PEG-rich) layers in co-current laminar flow.

Flow (and stopped-flow) studies have also been conducted in microfluidic devices that have proven particularly useful for detailed observation of solute interaction with the phase interface [4, 10, 11, 14, 15]. Earlier investigations with proteins had indicated that the phase interface might provide a resistance to solute transport in some cases, but the cause and magnitude of this resistance was unclear [8, 16-18]. Recent studies showed that DNA molecules initially dissolved in a dextran-rich phase can be made to accumulate at the interface using a weak electric field. Subsequent increase of the electric field was found to provide a size-selective detachment of the DNA from the interface [4, 14, 15]. The interfacial resistance is not fully understood but has been attributed to a free energy minimum caused by an electrical potential difference and interfacial tension at the interface [4, 14, 15]. A simple mathematical model based on only electrokinetic flow in the phases and equilibrium at the interface can describe the observed behavior, at least qualitatively [18].

Applications of two-phase electrophoresis to date have been largely academic demonstrations of improvement over liquid-liquid extraction (with no applied field) and electrophoresis in free solution, but there should be commercial applications in the future. The efficient recovery of small molecules using water-alcohol phase systems described above is attractive for treating dilute solutions like fermentation broth and wastewater. A novel application of electrophoresis for downstream processing has also been developed where protein products from E. coli fermentation can be recovered directly from lysed cells when positively charged proteins are directed into the PEG-rich phase leaving negatively charged cell debris and DNA in the dextran-rich phase [9]. The recent observation of size-dependent DNA release from the interface holds promise for concentration, analysis, and selective recovery of large molecules and small particles [15]. A computational model has been developed that should prove useful for optimizing both large scale and microfluidic separations [18]. Two recent reviews provide more details of the development, application, and promise of the technique [19, 20].

Figure 1: Two-phase electrophoresis concept.
  1. Clark, W. M., Electrophoresis-enhanced extractive separation, CHEMTECH, 1992, 22: 425-429.
  2. Stichlmair, J.; Schmidt, J.; Proplesch, R., Electroextraction: a novel separation technique, Chem. Eng. Sci., 1992, 47(12): 3015-3022.
  3. Marando, M. A.; Clark, W. M., Two-phase electrophoresis of proteins, Sep. Sci. Technol., 1993, 28(8): 1561-1577.
  4. Hahn, T.; Munchow, G.; Hardt, S., Electrophoretic transport of biomolecules across liquid-liquid interfaces, J. Phys.: Condens. Matter, 2011, 23, 184107+08.
  5. Luo, G. S.; Jiang, W. B.; Lu, Y. C.; Zhou, S. L.; Dai, Y. Y., Two-phase electrophoresis separation of dyestuffs from dilute solution, Chem. Eng. J., 1999, 73: 137-141.
  6. Pan, S.; Luo, G. S.; Liu, J. G.; Wang, J. D.; Back-Extraction of Carboxylic Acids by Two-Phase Electrophoresis, Sep. Sci. Technol.; 2003, 38(15), 3731-3746.
  7. Albertsson, P.-A., Partition of Cell Particles and Macromolecules, 3rd ed., Wiley, New York, 1985.
  8. Theos, C. W.; Clark, W. M., Electroextraction: two-phase electrophoresis, Appl. Biochem. Biotech., 1995, 54, 143-157.
  9. Oehler, R. D. and Clark, W. M.; β-Lactamase Recovery from E. coli Cell Lysate via Two-Phase Electrophoresis, Biotechnol. Prog. 1996, 12, 873-876.
  10. Munchow, G.; Hardt, S.; Kutter, J. P.; Drese, K. S., Protein transport and concentration by electrophoresis in two-phase microflows, JALA, 2006, 11: 368-73.
  11. Munchow, G.; Hardt, S.; Kutter, J. P.; Drese, K. S. Electrophoretic partitioning of proteins in two-phase microflows, Lab Chip, 2007, 7, 98-102.
  12. Zhai, S. L.; Luo, G. S.; Liu, J. G., Selective recovery of amino acids by aqueous two-phase electrophoresis, Chem. Eng. J., 2001, 83 (1): 55-59.
  13. Chen, C.-Y.; Fang, W.-F.; Chen, C.; Yang, J.-T.; Lyu, P.-C., Separation of Amino Acids by Aqueous Two-Phase Electrophoresis on the Micro-Pillar Chips, Proceedings IEEE Int. Conf. on Nano/Micro Engineered and Molecular Systems, 2006, 513-518.
  14. Hahn, T.; Hardt, S., Size-dependent detachment of DNA molecules from liquid-liquid interfaces, Soft Matter, 2011, 7, 6320-6326.
  15. Hahn, T.; Hardt, S., Concentration and Size Separation of DNA Samples at Liquid-Liquid Interfaces, Anal. Chem., 2011, 83, 5476-5479.
  16. Levine, M. L.; Bier, M., Electrophoretic transport of solutes in aqueous two-phase systems, Electrophoresis, 1990, 11: 605-611.
  17. Levine, M. L.; Cabezas, H.; Bier, M., Transport of solutes across phase interfaces by electrophoresis. mathematical modeling, J. Chrom., 1992, 607: 113-118.
  18. Clark, W. M.; Lindblad, M. A., Numerical Analysis of Two-Phase Electrophoresis, Sep. Sci. Technol., 2011, 46, 1546-1554.
  19. Kuban, P.; Slampova, A.; Bocek, P., Electric field-enhanced transport across phase boundaries and membranes and its potential use in sample pretreatment for bioanalysis, Electrophoresis, 2010, 31, 768-785.
  20. Hardt, S.; Hahn, T; Microfluidics with aqueous two-phase systems, Lab Chip, 2012, 12, 434-442.

William Clark
Worcester Polytechnic Institute