A method for separating according to mass a mixture of macromolecules or small particles suspended in a fluid. II. Experiments in a gravitational field.

We have described a method for separating according to mass a mixture of macromolecules or small particles.' The particles are suspended in a fluid and exposed to a uniform acceleration. The separation occurs as a consequence of the motion of the fluid. If the particles are heavy, the method works in the earth's gravitational field; if they are light, centrifugal fields are required. We will describe here experiments designed to test the method using heavy particles and a gravitational field. A third paper will discuss experiments done in a centrifugal field.2 The apparatus used in the gravity experiments, a long rectangular trough, is shown in schematic side view in Figure 1. A glass plate on the bottom of the trough is covered with a thin layer of fluid. The particles to be separated are added as a spot or a narrow band near one end of the trough. The gravitational field concentrates the particles near the surface of the glass in exponential Boltzmann distributions. The height of a distribution depends on what we have called the effective mass of the particle, the mass of the particle less the mass of the fluid it displaces. The lighter the particle, the higher its distribution. The fluid is made to flow slowly down the length of the trough. In this laminar flow the velocity of the fluid increases with height. The higher the particle, the more rapidly it moves down the trough. Light particles are on the average higher in the fluid than heavy particles; therefore, they travel more rapidly. At any moment, however, the particles in a given distribution have various heights and therefore various velocities. Brownian motion of an individual particle causes it to sample all these heights and velocities. Consequently, the average velocity of one particle is nearly the same as that of another particle of the same kind, and particles of the same kind move as a group. The method is capable of high resolution only if enough time is allowed for this sampling to occur. Materials and Methods.-Particles: We used "monodisperse" polystyrene latex spheres of diameter 0.796 At (run no. LS-449-E) and 1.305 ,u (run no. LS-464-E) obtained from the Dow Chemical Company Bioproducts Department through the courtesy of L. S. Lippie. The diameters had been measured at Dow in an electron microscope by the method of Bradford and Vanderhoff.3 The particles came in an aqueous suspension (10% solids) containing an alkyl benzenesulfonate emulsifier. We diluted the 1.3-,u suspension about 1: 20 and the 0.8-,4 suspension about 1: 6 with the fluid to be used in the experiment and allowed the mixtures to settle at least six hours in columns about 1.5 cm high. The suspensions used in the experiments were withdrawn from the middle of these columns. We measured the density of this material by observing whether the particles would float or sink when centrifuged in sucrose solutions of graded concentrations, obtaining a value 1.051 ±4 0.001 gm/cm3 (250C).