Vol. 21, No. 1 Printed in U.S.A.
JOURNAL OF VIROLOGY, Jan. 1977, p. 131-138 Copyright © 1977 American Society for Microbiology
Flow Field-Flow Fractionation: New Method for Separating, Purifying, and Characterizing the Diffusivity of Viruses J. CALVIN GIDDINGS,* FRANK J. YANG, AND MARCUS N. MYERS Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
Received for publication 6 July 1976
The nature and theory of flow field-flow fractionation is described, and its potential applicability to virus-like particles is discussed. Different virus types are shown to be retained at different levels. Retention can be controlled by variation of the experimental parameters, in good agreement with theory. However, a mild adsorption effect is indicated and requires the development of alternate strategies for measuring diffusion coefficients. For Q(3, our value agrees well within 10% of literature values; the values obtained for other viruses, using Q/3 as an internal standard, are untested. Finally, it is demonstrated that flow field-flow fractionation can cleanly fractionate two viruses from one another and from an albumin impurity, that samples as large as several milligrams in size can be analyzed, and that the method has potential utility in the quantitative and qualitative analysis of virus systems.
Field-flow fractionation (FFF) is a relatively new tool, developed primarily for the separation and characterization of macromolecdles and particles (4, 5, 10). The technique has demonstrated a capability of dealing with an enormous mass range. Various subtechniques have been used to handle components varying from a molecular weight of 600 up to particles of 1 ,um in diameter. This represents a mass range of approximately 109. FFF takes advantage of the nature of viscous flow in narrow channels. Under laminar flow conditions, the velocity of flow approaches zero as one approaches the wall of the channel. Any solute or particle confined in the quiescent region near the wall will have its motion retarded relative to solutes distributed over the total flow cross section. FFF employs an external field to partition the desired solutes into the quiescent wall regions of a narrow column. The field is applied along an axis perpendicular to the flow axis. As the strength of the field is increased, the solute is driven further and further toward the wall and its downstream motion is increasingly retarded. Different solutes will be retarded differentially because they will interact to a different degree with the field and/or they will exhibit a different level of diffusivity that will selectively oppose the induced drift toward the channel wall. The subtechniques of FFF are characterized according to the kind of field employed. Many possibilities exist, but we have so far utilized thermal gradients (giving us thermal FFF) (1,
6, 14, 19), electrical fields (7), sedimentation fields (SFFF) (7, 8, 20), and flow gradients (9) (Giddings, Yang, and Myers, Science, in press). A combination of these techniques has given evidence of being applicable to an extraordinary range of solute mass and properties and of solvent types. The rate of migration of solutes in FFF depends on the magnitude and type of field, the channel dimensions, the solute-field interaction, and the solute-solvent diffusion coefficient. Inasmuch as the channel is designed to have a simple and tractable geometry with perfectly understood flow characteristics, the retention of components can be, in the ideal case, mathematically described in terms of these parameters. Therefore, measurements of retention can be inverted to yield some combination of parameters related to field-solute coupling and diffusivity. It has been shown, for example, that sedimentation FFF can be used to yield the effective mass and, therefore, ultimately the molecular weight of viruses (8). In flow FFF, the "field" or influence selected to drive solute toward a channel wall is simply the superposition of lateral cross-flow from one side of the channel to the other. This lateral or secondary flow is superimposed upon the primary axial flow which carries solute from one end of the column to the other (see Fig. 1). The maintenance of lateral flow is assured by constructing the column such that it consists of the space between two flat, semipermeable membranes. The lateral flow proceeds continuously throughout the experiment, passing crosswise 131
132
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JOURNAL OF VIROLOGY, Jan. 1977, p. 131-138 Copyright © 1977 American Society for Microbiology
Flow Field-Flow Fractionation: New Method for Separating, Purifying, and Characterizing the Diffusivity of Viruses J. CALVIN GIDDINGS,* FRANK J. YANG, AND MARCUS N. MYERS Department of Chemistry, University of Utah, Salt Lake City, Utah 84112
Received for publication 6 July 1976
The nature and theory of flow field-flow fractionation is described, and its potential applicability to virus-like particles is discussed. Different virus types are shown to be retained at different levels. Retention can be controlled by variation of the experimental parameters, in good agreement with theory. However, a mild adsorption effect is indicated and requires the development of alternate strategies for measuring diffusion coefficients. For Q(3, our value agrees well within 10% of literature values; the values obtained for other viruses, using Q/3 as an internal standard, are untested. Finally, it is demonstrated that flow field-flow fractionation can cleanly fractionate two viruses from one another and from an albumin impurity, that samples as large as several milligrams in size can be analyzed, and that the method has potential utility in the quantitative and qualitative analysis of virus systems.
Field-flow fractionation (FFF) is a relatively new tool, developed primarily for the separation and characterization of macromolecdles and particles (4, 5, 10). The technique has demonstrated a capability of dealing with an enormous mass range. Various subtechniques have been used to handle components varying from a molecular weight of 600 up to particles of 1 ,um in diameter. This represents a mass range of approximately 109. FFF takes advantage of the nature of viscous flow in narrow channels. Under laminar flow conditions, the velocity of flow approaches zero as one approaches the wall of the channel. Any solute or particle confined in the quiescent region near the wall will have its motion retarded relative to solutes distributed over the total flow cross section. FFF employs an external field to partition the desired solutes into the quiescent wall regions of a narrow column. The field is applied along an axis perpendicular to the flow axis. As the strength of the field is increased, the solute is driven further and further toward the wall and its downstream motion is increasingly retarded. Different solutes will be retarded differentially because they will interact to a different degree with the field and/or they will exhibit a different level of diffusivity that will selectively oppose the induced drift toward the channel wall. The subtechniques of FFF are characterized according to the kind of field employed. Many possibilities exist, but we have so far utilized thermal gradients (giving us thermal FFF) (1,
6, 14, 19), electrical fields (7), sedimentation fields (SFFF) (7, 8, 20), and flow gradients (9) (Giddings, Yang, and Myers, Science, in press). A combination of these techniques has given evidence of being applicable to an extraordinary range of solute mass and properties and of solvent types. The rate of migration of solutes in FFF depends on the magnitude and type of field, the channel dimensions, the solute-field interaction, and the solute-solvent diffusion coefficient. Inasmuch as the channel is designed to have a simple and tractable geometry with perfectly understood flow characteristics, the retention of components can be, in the ideal case, mathematically described in terms of these parameters. Therefore, measurements of retention can be inverted to yield some combination of parameters related to field-solute coupling and diffusivity. It has been shown, for example, that sedimentation FFF can be used to yield the effective mass and, therefore, ultimately the molecular weight of viruses (8). In flow FFF, the "field" or influence selected to drive solute toward a channel wall is simply the superposition of lateral cross-flow from one side of the channel to the other. This lateral or secondary flow is superimposed upon the primary axial flow which carries solute from one end of the column to the other (see Fig. 1). The maintenance of lateral flow is assured by constructing the column such that it consists of the space between two flat, semipermeable membranes. The lateral flow proceeds continuously throughout the experiment, passing crosswise 131
132
GIDDINGS, YANG, AND MYERS CROSS
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PLNII
PUNIP
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DEI ECTOR
Upper
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