The class of particle separation techniques known as field flow fractionation.
or FFF, has become increasingly popular in recent years. This is evident from an
examination of the extensive and detailed bibliography to be found on the World
Wide Web at http://www.rohmhaas.com/fff/fff.html. These FFF techniques consist
of constraining a sample bearing fluid to flow within a long thin channel by means
of an applied pressure gradient along its long dimension. The channel is often
comprised of upper and lower flat plates separated by means of a spacing element,
of thickness much smaller than the channel width, which also seals the channel
and defines its horizontal dimension. In response to the pressure gradient, the
fluid moves and its velocity assumes the well known quadratic Pouiselle profile.
The fluid touching the plates and spacer is stationary and its velocity reaches
a maximum in the center of the channel. A field is then applied perpendicular to
the direction of flow. The resulting force on the particles causes them to migrate
towards one or both of the plate surfaces, depending on the sign of the force.
The magnitude and size dependence of the force depends on the nature of the applied
field, but in all of these techniques, the particle's concentration profile will
be due to a balance between the applied force which tends to concentrate the particles
near the surface, and effects of diffusion which tend to reduce the concentration.
The assumption here is that the local concentration is small enough that the particles
do not interact. If the field is too large, the particles will be forced onto the
surface. This is known as steric mode separation and will not be discussed further
in this specification.
For any specific particle population, the mean distance from the plate
surface can vary quite dramatically, depending on the particle size, shape, and
the magnitude of the field coupling. Since there is a strong velocity shear near
the surface of the plates, the particles with larger mean displacements will be
in the faster moving part of the fluid stream and will be transported more rapidly
along the channel. Therefore they will elute earlier than those closer to the surface.
Many different applied fields have been used, each of which separates
the particles by a different mechanism. The most frequently used fields have been
i) gravitational which is usually referred to as sedimentation, produced by rotating
the channel in a centrifuge; ii) therrnal. produced by a temperature difference
between two isothermal plates; iii) hydrodynamic generally called flow, produced
by flow through a semipermeable membrane; and iv) electrical, produced by conducting
plates which act as electrodes. It is this latter FFF process to which this specification
is primarily directed.
There has long been a need to find better electrode means for electrical
FFF separation devices. The traditional electrical FFF channel is comprised of
two solid conducting bars of metal or graphite, which are separated by a thin spacer
which has a channel cut from it. Typically, the spacer is made of made from MYLAR®
or similar material which is slightly deformable under an applied clamping pressure.
The spacer thus serves to define the lateral extent of the cell, provides the fluid
seal, and electrically insulates the plates from each other. The fluid is introduced
into the cell through holes at the ends of the conducting plates. Since the separation
efficiency depends on the length of the channel and the strength of the field,
there is a strong incentive to make the cells long and to make the spacing between
the electrodes as small as possible. Moreover, as the field strength is increased,
the particle mean distance from the electrode surface decreases. It it not uncommon
for this distance to be a few micrometers. There is, therefore, a strong incentive
to make the electrode surfaces optically smooth. In addition to minimizing surface
roughness, there is a need to maintain the plates spacing over the entire length
of the channel since the homogeneity of the field is essential in achieving consistent
and reproducible separations while, at the same time, minimizing zone broadening
and sample remixing. Finally there is a need to improve the hardness and stability
of the plates. As with all FFF devices, it has always been useful to visualize
the separation process, but since the plates are generally opaque, this feature
is rarely achieved
Many of the materials used in extant electrical FFF separators are
chemically or mechanically unstable. For example, graphite electrodes, no matter
how well prepared, tend to shed particles and deteriorate with time. Electrodes
made of titanium often oxidize under exposure to some of the mobile phases frequently
used in the device. Platinum coated electrodes, traditionally made from a very
thin plating onto steel plates, cannot withstand the typical electrical fields produced
and tend to fracture exposing the reactive steel supporting structures. Plates
made entirely of platinum would be ideal but because of their prohibitive cost
are never used.
A new concept and design for the electrical FFF structure has been
developed and is described herein. The new plate structure incorporates all of
the most desirable features for electrical FFF electrodes and provide means for
visualizing the flow as well.
Brief description of the invention.
The new design disclosed for an electrical FFF device uses coated
glass plates as the electrodes. Glass is an ideal substrate since it is hard and
may be readily fabricated in an extremely flat and smooth form. However, it is
not conducting. Metal films evaporated on glass surfaces have been used with limited
success, but they are not transparent and ions generated at the electrode surfaces
tend to oxidize the films and cause them to separate from the glass. A superior
conducting surface that would permit the use of a glass substrate is disclosed
which consists of a conducting oxide such as indium-tin-oxide or tin-oxide. The
incorporation of electrodes of this composition allows the invention to achieve
all of the seven most desirable features for such an electrical FFF channel, viz.
1) hardness, 2) flatness, 3) smoothness, 4) chemical inertness, 5) durability (does
not generate particles), 6) high conductivity, and 7) transparency. Since the surface
is optically smooth, the clamping pressures required to seal the cell are much
smaller than required with other electrodes.
Brief description of the figures
Figure 1 illustrates an exploded view of the major elements of an
electrical FFF system.
Figure 2 illustrates a preferred means for reducing the resistance
of the coated glass electrode.
Detailed description of the invention
Figure 1 shows an exploded view of the arrangement of the elements
of an electrical FFF channel. The upper and lower electrodes 1 are separated by
a spacer 2 which is generally cut from a sheet of MYLAR® or similar insulating
material. The spacer is cut and shaped to provide conductive means to guide the
9 particle bearing fluid from the entrance inlet 3 through the channel and into
the outlet 4 and incorporates V-shaped ends 5 to minimize regions of stagnation.
The spacer is maintained between the plates by clamping between two compression
plates 6 with nut and bolt fittings 7. An additional port 8, though not generally
required, may be added to provide means for the injection of the particle bearing
sample. Such samples are usually introduced via the inlet port 3. Once the sample
has been introduced thus, the flow is usually stopped briefly to permit relaxation
of the sample within the applied electrical field. With the added injection port
8, the sample may be introduced for subsequent relaxation without need to have
started the channel flow to transfer the sample into the fractionation channel itself.
In the preferred embodiment of the electrical FFF device, the spacer may serve
also as a liquid seal preventing thereby the fluid therein from entering other
parts of the device. One or both of the clamping plates 6 may have observation
ports or slots 9 cut therein to permit direct observation of the flow cell during
operation or for alignment purposes.
In the preferred embodiment of this invention, the electrodes 1 are
transparent and prepared by chemically bonding a conductive metallic oxide coating.
Such electrodes may be prepared from tin oxide coated plates such as used for liquid
crystal displays. Glass surfaces prepared in this manner are available, for example,
from Owens Corning Glass and are commonly used for the plates of liquid crystal
displays or as transparent resistive heaters. To the best of the inventor's knowledge,
they have never been used as electrodes for conducting fluids such as is
the case for electrical FFF. Since the substrate is glass, it is easy to make large,
flat, and smooth surfaces and its transparency allows one to visualize the separation
process by means of dye injections. An added benefit of transparent glass plates
is that by illuminating the sample cell comprised thereof with a beam of coherent
light, a series of interferometric fringes formed by reflections from the top and
bottom plates, can be observed. These fringes directly measure the uniformity of
the plate spacing. A uniform plate spacing is crucial to insure that the electrical
and flow fields do not vary along the length of the channel. Until the advent of
the current invention. the degree of uniformity achievable was inferred only by
reliance on the uniformity and the mechanical stability of the spacer between the
two plates and the assumed ability to clamp the plates uniformly. It has heretofore
been impossible to measure accurately the cell spacing uniformity in situ
during operation. In order that the interferometric examination of the sample cell
be possible, the compression plates 6 need be made of transparent materials such
as poly methyl methacrylate. Alternatively, they may be made of similarly rigid
opaque material into which have been cut observation ports 9 which permit direct
observation of the glass surfaces.
Another embodiment of the invention consists of the incorporation
of plates made of doped silicon. Such materials are commonly made to produce semiconductor
devices and are available in sections large enough to serve as electrodes. Because
silicon is transparent to many infrared wavelengths, such plates may be adjusted
also by interferometric examination.The standard processes of doping generally
result in the silicon plates being transformed into conducting plates.
One drawback to the tin-oxide plated glass electrodes is that the
conductivity of each electrode is much poorer those of metal or graphite. Similar
comments refer to plates made of doped silicon. This can cause a substantial voltage
drop along the electrode. For example, the resistance of a 4 cm by 40 cm plate
of tin-oxide coated glass is approximately 100 ohms depending on the thickness
of the coating. One can reduce this-resistance significantly by adhering, plating,
or otherwise affixing a highly conducting strip 10, as shown in Fig. 2, along one
or more edges of the electrode outside the fluid bearing region of the cell. We
have found that the plate resistance can be reduced to about 1 ohm by coating the
edge with a conductive silver paint such as manufactured by GC Electronics.
Although the preferred embodiments of this invention use metal oxides
such as tin-oxide and indium-tin oxide to achieve a transparent conducting surface
on flat glass plates, there are certainly other materials that may be used to equal
effect including conducting polymers and similar non-metallic materials. One advantage
of a tin-oxide coating is that its mechanical hardness prevents the surface from
being damaged during cleaning.
Throughout this specification, the electrical FFF separations have
referred to the separation of particles. Such designation refers equally to macromolecules
including, but not limited to, proteins. DNA, oligomers, dendrimers, polymers,
gels, as well as a variety particle types such as micelles, liposomes, emulsions,
bacteria, viruses, algae, protozoa, and various molecular aggregates such as gels.
In addition, all other coatings that provide similar properties to the preferred
embodiment of tin or indium-tin oxides are included as but simple variations of
As will be evident to those skilled in the arts of electrical field
flow fractionation and chromatography, there are many obvious variations of the
channel I have invented and described that do not depart from the fundamental elements
that I have listed for its practice; all such variations are but obvious implementations
of my invention described hereinbefore and are included by reference to my claims,