Helical path electrophoresis in vertical fluid sheaths.

The general principle underlying the method described below is that of deviation electrophoresis. The mixture of ions to be spearated from each other enters an electric field in form of a thin streak carried by a buffer flow transversely to the electric field. The streak is affected by the electric field similarly to a zone in zone electrophoresis. The different ions are moved by the electric field transversely to the direction of flow of the surrounding buffer solution with velocities determined by their ionic mobilities. As a result, the streaks are deviated from the course they would have followed in the absence of the electric field. Since the slopes of the deviated streaks depend on the ratio of the electrophoretic velocities of the ions to the buffer flow velocity perpendicular to the electric field, one obtains a series of streaks deviated from the course of the reference streak corresponding to an ion of zero charge by angles depending on the ionic mobilities, the electric field strength, and the rate of buffer flow. This is the well-known arrangement used in filter paper curtain electrophoresis.1 The resolving power of filter paper electrophoresis as applied to macromolecular ions is greatly impaired by interaction between the ions and the filter paper. As a rule, suspensions of particles cannot be separated by this method due to adsorption by the curtain matrix. To eliminate the troublesome adsorption, Barrolier et al.2 replaced the filter paper curtain by a nonvertical thin sheath of buffer solution about 0.5 mm in thickness. This arrangement has, however, the disadvantage that small particles tend to precipitate on the bottom plate of the inclined sandwich containing the buffer sheath between two glass plates measuring about 50 X 50 cm. This drawback was eliminated by Hanning by mounting the fluid curtain in the vertical plane.' His plates measured 50 X 50 cm and were 0.5 mm apart. This orientation of the fluid curtain eliminated the problem of sedimentation of the particles on the bottom plate and made separation of cells and subcellular particles possible. The small distance between the plates and an elaborate thermoelectric cooling system serve in this apparatus to suppress thermal convection. Two different approaches were used by the author to suppress thermal convection and particle sedimentation. In one of them, the liquid sheath follows a serpentine path.4' 5 Figure la shows how particles issuing from injector IN and carried by the buffer flow following the serpentine path in the direction of the arrow are falling toward the bottom wall of the first horizontal leg of the serpentine path. However, as the particles enter the upper horizontal leg, the wall which previously was the bottom wall is now the top wall and the particles begin to recede from it due to sedimentation. Thus, the particles oscillate between the walls without depositing on them, provided the speed of the buffer flow exceeds a certain minimum value which depends upon the rate of particle sedimentation.