Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2000-08-22
2002-04-02
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S748080, C210S198200, C210S800000, C210S804000, C209S018000, C209S131000, C209S156000, C209S422000
Reexamination Certificate
active
06365050
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to a method for performing flow field-flow fractionation (flow FFF), which is useful in the separation, isolation, and characterization of a wide range of particles and macromolecules.
BACKGROUND OF THE INVENTION
There is a tremendous need in virtually all branches of science and technology for the separation and characterization of a wide variety of analytes. Although numerous methods have been devised to accomplish such separations, including various forms of chromatography, filtration, precipitation, electrophoresis, and centrifugation, no one technique is universally applicable. One useful family of techniques is field-flow fractionation (FFF), as taught by J. Giddings in U.S. Pat. No. 3,449,938 and recently reviewed by P. Schettler,
LC
-
GC
14(10), 852 (1996), which are incorporated by reference herein. The most universally applicable of these FFF techniques is flow field-flow fractionation (flow FFF), as taught by J. Giddings in U.S. Pat. No. 4,147,621, which is incorporated by reference herein. Flow FFF devices effect the fractionation of particles by pushing an analyte contained in the “channel flow” stream (symbolized herein as ‘V
z
’) axially along the surface of a filtration membrane inside a narrow channel while simultaneously pushing a “cross flow” stream (symbolized herein as ‘V
x
’) through the channel in a direction orthogonal to the channel flow stream V
z
. Inside the channel, these two flowstreams intersect and intermingle, and the crossflow stream V
x
provides a field of hydraulic force across the planar surface of the filtration membrane which is sufficient to permit the separation of analyte samples into their constituent components based on differences in their hydrodynamic sizes or diffusion coefficients. The crossflow stream thus provides a force field which results in the retention of the analyte. Larger analytes feel or sense the force of this crossflow stream more strongly, due to their larger Stokes radii, and therefore spend their time, on average, closer to the filtration membrane. Smaller analytes feel the crossflow stream more weakly, and diffuse away from the filtration membrane to occupy a higher average position over it, where they encounter the faster flowstreams of the Poiseuille flow velocity distribution under laminar channel flow conditions and are carried along the channel faster and elute as a peak sooner than larger analytes. In this way, the analyte particles are fractionated as a function of their diffusion coefficients or apparent hydrodynamic sizes.
Many publications, such as J. Giddings et al.,
Polym. Mater. Sci. Eng
. 65, 21 (1991), P. Schettler,
LC
-
GC
14(10), 852 (1996), J. Giddings,
Sep. Sci. Technol
. 24(9&10), 755 (1989), J. Giddings et al.,
Meth. Biochem. Anal
. 26, 79 (1980), F. Yang et al.,
Anal. Chem
. 49(4), 659 (1977), and M.-K. Liu et al.,
Anal. Chem
. 63, 2115 (1991), and a number of patents, such as U.S. Pat. Nos. 4,214,981, 4,737,268, 4,830,756, 4,894,146, 5,039,426, 5,141,651, and 5,193,688, all of which are incorporated by reference herein, have disclosed applications, modifications, calibration procedures, and improvements to the flow FFF separation process, so that the set of conditions which are classically employed in flow FFF systems is well known and fully illustrated in the prior art.
One important advantage of flow FFF is the number and variety of the variables which are available in the design of channels and the operating parameters. For example, although thin, planar channels are the most common, an annular channel geometry is also known, as illustrated in
FIG. 7
of U.S. Pat. No. 4,214,981. In addition, at least three different “operating modes” are known for flow FFF, including the “normal,” “steric,” and “hyperlayer” modes. In the normal mode, the analyte exists predominantly as a “cloud” of particles hovering over the surface of the membrane, balanced between the crossflow force driving it towards the accumulation wall on the one hand and diffusion, flow-induced lift forces, and steric effects on the other hand, driving the analyte away from the accumulation wall during its travel down the channel. In this normal mode, which takes place with small particles at modest flow rates, the contribution of flow-induced lift forces is considered to be minimal, so that the primary forces acting on the particles are the hydraulic crossflow force field and the back-diffusion of the analyte away from the filtration membrane. Thus, in normal mode, smaller particles elute before larger particles. In the steric mode, which predominates for larger particles and relatively high crossflow velocities, the particles essentially reside along the surface of the filtration membrane, so that larger analyte particles protrude further into the channel and sample the faster flowstreams of the Poiseuille flow velocity distribution. Thus, in steric mode the particles are considered to essentially “roll” along the surface of the filtration membrane, so that the elution order is the reverse of that observed for normal mode, with larger particles eluting before smaller ones. In hyperlayer mode, which takes place with similar larger particles as required for the steric mode but with somewhat faster flow velocities, the flow-induced lift forces which are considered to be minimal in normal mode become significant, and lead to a fluid force lifting the particles off from the surface of the membrane and into a relatively narrow layer of fluid. As with steric mode, larger particles in hyperlayer mode elute before smaller particles.
In addition, there also exist at least two configurations of flow FFF channel. In “symmetrical” flow FFF configuration, the crossflow stream V
x
enters the channel from the crossflow inlet frit above it and passes through the channel and then through the filtration membrane, whereupon retained analyte is removed from the cross flow and the remaining V
x
stream exits through the outlet frit underneath the filtration membrane. In symmetrical flow FFF, the channel flow stream V
z
is thus physically distinct from the cross flow stream V
x
, and these two flow streams are typically “balanced” by independently adjusting the four flow streams V
z
in, V
z
out, V
x
in, and V
x
out by any convenient means until V
z
in equals V
z
out and V
x
in equals V
x
out. However, these two flow streams need not be balanced in this manner, since the only physical requirement is that the total of the sum of V
z
in and V
x
in is equal to the total of the sum of V
z
out and V
x
out. Thus, symmetrical flow FFF channels can also be operated in “unbalanced flow” mode, in which the relative velocities of the four flow flowstreams can be adjusted to obtain any desired effect. In “asymmetrical” flow FFF, the crossflow inlet frit is replaced with a non-porous solid material, so that the channel flow stream V
z
in and the crossflow stream V
x
in must be mixed and pumped into the channel together through a single channel flow inlet. Typically, the total input flux is chosen in advance, and the ratio of the fluid flow which exits from the channel flow outlet to that which exits through the filtration membrane and the crossflow outlet can be controlled by any convenient means, including pieces of constrictive tubing, pressure regulators, etc. In this way, the desired degree of retention can be achieved.
The flow FFF channel can also contain one of a number of potential channel spacer “geometries,” typically produced by cutting a portion out from the center of a thin sheet of spacer material, most often a plastic. For example, if a portion of the spacer consisting of a rectangle 2.0 cm wide by 25 cm long and bearing end-pieces consisting of isosceles triangles 2.0 cm on a side is removed, and the apparatus assembled, then the channel would exhibit a “parallel-walled” geometry. Similarly, a triangular portion of 2.0 cm breadth and 20 cm length contiguous to an isosceles triangles portion 2.0 cm on a side would yield a geometry referred to as “tapered-wall.” Although a large
Amgen Inc.
Crandall Craig A.
Levy Ron K.
Odre Steven M.
Therkorn Ernest G.
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