Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~2962S7
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This invention relates to a novel molecular
separation column, e.g. chromatography column, and
more particularly to a novel column using a solid
stationary phase.
Chromatography is a general term applied to
a wide variety of separation techniques based upon the
sample interchange between a moving phase, which can
be a gas or liquid, and a solid stationary phase.
When ~as is the moving phase (or "mobile phase" as
referred to in chromatographic terminology), the
technique is termed gas chromatography and when liquid
is the mobile phase, the technique is termed liquid
chromatography.
Separations can be classified into either
analytical or preparative depending on the objective.
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In analytical separations, the objective is high reso-
lution separation, identification and quantification
of the various components of a sample mixture. In
preparative chromatography, on the other hand, the
objective is the isolation of large pure quantities of
the desired constituents in the sample.
The collection of liquid chromatographic column
techniques can be classified in several ways. The
most fundamental is based on naming the types of
phases used. Liquid absorption chromatography is used
extensively for organic and biochemical analysis. Ion
exchange chromatography is a special field of liquid-
solid chromatography and is specifically applicable to
ionic species. Affinity chromatography is based on
the attraction (affinity~ of a ligand bondea to the
solid stationary phase for a giveD component of the
sample. Liquid-liquid or partition chromatography
involves the use of a thin layer of liquid held in
place on the surface of a porous inert solid as the
stationary phase.
In the chromatographic process, it is customary to
pass a mixture of the components to be resolved in a
carrier fluid through a chromatographic apparatus or a
separative zone. The separative or resolving zone,
i.e. the stationary phase, generally consists of a
material referred to as a chromatographic media, which
has an active chromatographic sorptive function for
separating or isolating the components in the carrier
fluid. The separative zone usually takes the form of
a column through which the carrier flu-id passes.
A major problem in the art of column chromatogra-
phy is to obtain unlform fluid flow across the column.
~6~i7
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.
It has been recognized that the solution to this prob-
lem resides in an ability to obtain uniform packing,
distribution and density of the chromatographic media
within a column. To a large degree, the packing
problem is surmounted in the laboratory chromatography
columns by using columns having a small internal diam-
eter, generally on the order of 1/8 inch to 1-1/2
inches. In such columns, an uneven chromatographic
fluid flow resulting from nonuniform packing of the
chromatographic media is quickly relaxed across the
column diameter and does not significantly affect
analytical results.
To provide an economically feasible preparative
chromatography column, the column diameter must be
larger than one inch and preferably on the order of
one foot or more. Attempts to scale analytical
chromatography columns to a size feasible for prepara-
tive and/or production chromatography have met with
substantial losses in column efficiency. It has been
found that as the column diameter or cross-sectional
area is increased, the separation or resolving power
of the chromatography column decreases. The resolu-
tion losses can be attributed primarily to a lack of
effective fluid flow distribution in the column.
Various internal column devices have been proposed
to overcome the difficulties of producing large diame-
ter preparative and production chromatography columns.
Other approaches have been to provide homogenous dis-
tribution of chromatographic media and maintenance of
uniform media density across the column or to develop
novel type media and/or packing.
--4--
Of recent date, ~he assignee herein has developed
unique chromatographic media, comprisin~ in its physi-
cal form a homogeneous fibrous matrix, preferably in
sheet form. 5uch chromatographic media are described
in the following U.S. patents and patent applications:
U~S. Patent No. 4,384,957 to Crowder, III, et
al.t
U.S. Patent No. 4f512,897 to Crowder, III, et
al.;
U.S. Patent No. 4,404,285, entitled "Process
For Preparing Zero Standard Serum" to Hou;
U.S. Patent No 4,488,969, entitled "Fibrous
edia Contalnlng Millimicron Sized Particles"
to Hou;
U.S. Patent No. 4,559,145~
entitled "Process for Preparing a Zero Stan-
dard Serum" to Hou et al.;
U ~ Patent No. 4,578,150,
entitled l'Fibrous Media Containing Millimi-
cron Sized Particles" to Hou;
U.S. Patent No. 4l663,163,
1984 entitled 'rModl~ied Polysaccharide
~` Supports" to Hou et al.;
U.S. Patent NG. 4,687,820,
1984 entitled "Modified Polypeptide Supports"
to Hou et al.;
__ _
U.S. Patent no. 4,724,207,
1984 entitled "~odified Siliceous Supports"
to Hou et al.;
~U.5. Patent No. 4,639,513,
1984 entitled "Intravenous Injectable Immuno-
globulin (IgG) and Method ~or Producing Same"
to Hou et al. and
~2~2~i7
U.S. Patent No. 4,606,824,
entitled "Modi~ied Cellulose Separation
Matrix" to Chu et al.
Crowder, XII et al., in each patent, describes a
chromatography column having a substantially homogene-
ous stationary phase which~ comprises a porous matrix
of iber having par~iculate immobilized therein. At
least one of the fiber or particulate is effective for
chromatographic separations. Preferably, the station-
ary phase comprises a plurality of sheets in disc form
stacked inside a column. The edges of the discs coop-
erate with the interior wall o~ the column to form a
substantially fluid tight seal therewith, thus pre-
venting any appreciable skewing or by-pass of fluid
around the edges of the elements. In its preferred
~orm, the fluid tight seal is produced by the hydro-
philic swelling o~ the stationary phase.
( E~ou ~U.S. Patent No~ 4,404/285 and No. 4,559,145
~ describes a method for removing thyroid or
steroid hormones from a serum by using a composite
sheet, comprising a matrix of self-bonding fibers
having dispersed therein carbon particles. The sheets
are used preferably in the chromatographic column
described in Crowder, III et al. and are also hydro-
philic swellable discs or pads
Hou ~U.S. Patent No. ~ 88,969 and U.S. Patent No.
4,578,150 describes a self-supporting fibrous matrix
having immobiIized therein at least about 5~ by weight
:
~r9ç~
of micro particulate (average diameter less than 1
micron), preferably fumed silica or alumina. The
media i9 also pre~rably used in the chr~matographic
c~lumns disclosed in Crowder~ III et al. and the sol-d
stationary phase is also hydrophilic swellable.
Hou et al. U.S. Patent 4,663,163 describe a polysaccharide
material which comprises a polysacchaxlde covalently
bonded ~o a synthetic polymer. The synthetic polymer
is made from a polymerizable compound which is capable
of being covalently coupled directly or indirectly to
the polysaccharide and one or more polymerizable
compounds. The polymerizable compound contains an
ionizable chemical group, a chemical group capable of
transformation to an ionizable chemical group or a
chemical group capable o~ causing the covalent
coupling of the compound to an affinity ligand or
biologically active molecule. The media is capable of
acting as a chromatographic support ~or ion exchange
chromatogxaphy, for affinity chromatography or as
reagents for biochemical reactors. Pre~erably sheets
of this material are loaded into an appropriately
sized cylindrical column to form the desired station-
ary phase in a manner similar to Crowder, III et al.
The preferred solid stationary phase is also hydro-
philic swellable.
All of these media in their preferred embodiment
are fibrous matrices which are hydrophilic swellable,
i.e. they tend to swell upon contact with aqueous
systems. In a stacked disc type chromatographic
column such swelling is useful in assistin~ producing
a fluid tight seal with the interior wall of the
column to form a water swellable fit therewith. Such a
. ,,~
257
seal prevents skewing or bypass of the f1uid around
th~? edaes of the elements.
In Hou et al., U.S. Patent No. 4,663,163, it is indic~ted that the
media could be used in a "jelly roll" type column,
i.e. a sheet of media spirally wound around a forami-
nous core to form a cylinder having a plurality of
layers around the axis thereof. It was subsequently
found that the radial flow of a sample through such a
"jelly roll" type solid phase was not evenly distri-
buted, and there was substantial bypass of the fluid
around certain areas of the media. It is believed
that this is due to the swelling and resulting com-
pression o~ the chromatographic media upon contact
with the fluid flowing therethrough thus produciny an
irregular homogeneity in the solid stationary phase
leading to an irregular hydrodynamic profile through
the column and consequently to the establishment of
preferential hydrodynamic routes which rapidly
diminish the efficacy and selectivity of the chromato-
graphic column.
~ lou et al., U.S. Patent 4,687,820, describes a modified polypep-
tide material which comprises a polypeptide covalently
bounded to a synthetic polymer, which synthetic poly-
mer is made from a polymerizable compound as described
in Hou et al., U.S. Patent 4,663,163. The material is capable of
acting as chromatographic support for ion exchange
chromatography, affinity chromatography and reverse
phase chromatography or as reagents for biochemical
reactors. The materials are disclosed as suitable, in
sheet form, as the stationary phase for loading into
chromatographic columns.
Hou et al., U.S. Patent 4,724,207~ describes a modlfied sili-
ceous material which comprises a siliceous material
covalently bound ~o a synthetic polymer, the synthetic
/~ '`
12~25~
--8--
polyrner sim~lar -to that described in Hou e~ al., U.S. P~tent4,663,163
and Hou et al., US Patent 4,687,820. The material is
described as suitable for chxom~tographic separation
media, the separ~tion snedia comprising the stationary
phase for chromatographic columns.
Hou et al., U.S. Paten-t 4,639,513 describ~s a host
of additional chromatographic media, many of which include
the media disclosed in Hou e~ al., U.S. Pat~nt Nos. 4,663,163,
4,687,820 and 4,724,207. Additionally, this application describes
further embodiments directed to specific affinity
media, ion exchange media, and reverse phase media,
. .
all suitable for use in chromatographic separations in
general and in the preparation of intravenous inject-
able IgG specifically.
Chu et al., U.S. Patent 4,60G,824 describes rnodified cellulosic
materials which are essentially ~ree of LAL reactive
extractables.
Of additional relevance to this invention are the
following references:
Wang et al., Biotechno~y and_ Bioenqineerinq XV,
page 93 ~1973), describes the preparation of a
"Bio-Catalytic Module" wherein cc llagen-enzyme mem-
branes are layered on a supporting material, such as
cellulose acetate membrane, and coiled around a cen-
tral rod. Glass rods are used as spacers, which are
so arranged that the distance between them is small
enough to prevent the adjacent layers from contacting
each other. After coiling the complex membrane upon
the spacers, the cartridge is then fitted into a
plastic shell to form a flow-through reactor configu-
ration. The flow through the column is axial, i.e.
the sample flowing through the column contacts the
membrane in a crsss-flow manner.
~'~9~2~i~
g
Wang et al. (page 583) also recognizes that the
flow of sample through ~uch a device is mainly paral-
lel to the membrane surface and that some of the
enzyme molecules located within the matrix may not be
readily accessible. In order to improve the contact
efficiency, Wang et al. suggests that the sample flow
through the permeable membrane under hydraulic pres-
sure. In this configuration of the reactor, a filter
fabric serves as a backing material which separates
successive layers of invertase-collagen membrane, thus
preventing overlapping of the membrane layers. A per-
forated stainless steel tube is used as a central core
element which is also used for feeding the sample. A
uniform radial distribution of the substrate is
achieved by meterinq ~low through a n~nber of holes
drilled ninety degrees ~90) apart radially along the
stainless tube. A spiral reactor configuration is
formed by coiling alternate layers of the membrane and
backing around the steel tube. The spiral cartridge
is ~itted into a plexiglass outer shell. The plastic
housing is affixed to two threaded aluminum end
plates. The sample is fed ~rom the central tube while
the reaction product is collected through a central
poxt located on the periphery of the reactor shell.
U.S. Patent 3,664,095 to Asker describes a packing
material which may be spirally wound around a central
axis for fluid treatment such as drying, heat ex-
change, ion exchange, molecular sieve separations and
the like. Flow is axial through the apparatus, i.e.
parallel to the surfac~e of the packing material.
U.S. Patent No. 3,855,681 to Huber describes a
preparative and production chromatography column which
* Registered ~ademark
1~96257
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includes a relatively inert inner core onto which is
wound in a spiral pattern a xelatively inert sheet of
material, such as synthetic polymeric film. Prior to
winding, the film is coated with a chromatographic
media. A thicXness dimension of the chromatographic
media is arranged substantially perpendicular to the
primary direction of fluid flow through the column,
i.e. flow is axial thereof and thus parallel to the
surfaces of the chromatographic media.
V.S. Patent 4,~42,461 to Bartoli et al. describes
a reactor for effecting enzymic reactions in which the
flow of the solution to be treated through the
catalytic bed takes place radially. It is preferred
to have the catalytic bed in the form of coils of
enzyme-occluding fibers. The catalytic bed is formed
by winding fibers on which the enzymes are supported,
so as to form coils with filaments or groups of fila-
ments arranged helically. The fibers inserted in the
reactor can also support, instead of enzymes, chela-
tion agents, antibodies, or similar products which are
immobilized, like the enzymes, by physical bonds, ion
exchange, absorption or occlusion in the filamentary
polymeric structures.
U.S. Patent 4,259,186 to Boeing et al. 51981)
describes an elongated gel filtration column having an
outer wall and at least one gel chamber defined
therein and adapted to be filled with a filtex gel.
The gel chamber is sub-divided by a plurality of
interior partition walls arranged in parallel to the
column wall. The partition walls are of a length
shorter than the length sf the gel chamber~
~2g6~S~
U.S. Patent 4,299,702 to Bairingi et al. (1981)
describes a liquid separation apparatus of the spiral
type employing semi-permeable membrane sheets, between
which a sp~cing layer is located, and utilizing the
principal of reverse osmosis or ultrafiltering for
separating a desired liquid component, i.e. a solvent
or a solute, from a pressurized feed solution. In
this type of apparatus, the feed flows substantially
spirally through the apparatus, i.e. parallel to the
membrane. See also U.S. Patent 4,301,013 to Setti et
al. (1981).
- None of these references describe the problems
associated with the use of a swellable fibrous matrix
chromatographic media in sheet form, particularly uti-
lized in a "jelly roll" type column nor the solution
to such problems. Further, none of the references
address the problems of multiple layers of swellable
chromatographic media.
It is an object of the invention to provide an
efficient preparative or production chromatography
column using a solid stationary phase which is in
cartridge form, which may be disposable.
Another object of this invention is to provide a
solid stationary phase of a chromatography column
which can be made in cartridge form, which may be
disposable.
A further object of this invention is to provide a
chromatography column which has a solid stationary
phase which provides even distribution o~ a sample
flowing through the stationary phase.
62S7
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Still a further object of this invention is to
provide a chromatographic column which accommodates a
swellable fibrous matrix in sheet form as the solid
stationary phase.
Another object of this invention is to provide a
chromatography column which has a reduced pressure
; drop, enhanced flow and enhanced capacity.
A further object of this invention is to provide a
chromatography column having essentially no determined
diametric size limitationO which can be quickly and
relatively inexpensively manufactured.
A still further object of this invention is to
provide a chromatography column which resolves the
uneven fluid flow problems encountered when attempting
to scale up analytical columns to preparative and
production columns.
Yet another object of the present invention is to
provide a solid stationary phase for liquid chromato-
graphy which ensures that substantially all of the
chromatographic media is utilized.
A further object of the invention is to provide an
inexpensive, high quality chromatographic column which
can be a disposable item in many, perhaps most, pro-
cessing situations.
- The foregoing objects of this invention are
accomplished by a chromatography column for effecting
chromatographic separation of at least two components
of a sample flowing through the column. The column
comprises a housinq and at least one solid stationary
phase within the housing. The stationary phase has
~chromatographic functionality and is effective for
chromatographic separation. Means are provided for
:
'
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25~
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distributing the sample through the stationary phase
and for collecting the sample after the sample has
flowed through the stationary phase. The stationary
phase comprises:
(a) a plurality of layers of a swellable matrix in
sheet form having chromatographic functionality and
being effecting for chromatographic separation; and
(b) a spacer means between each layer for
permittlng controlled swelling thereof and enhancing
the distribution of sample flowing through the
stationary phase.
According to a still further broad aspect of the
present invention, the stationary phase comprises one
or more layers of a swellable fibrous matrix having
chromatographic functionality. The stationary phase
also comprises a spacer means between each layer for
permitting controlled swelling thereof and enhancing
the distribution of sample flowing through the
stationary phase. The stationary phase and the
radially outwardly expanding stationary phase chamber
coact to provide substantially uniform radial
distribution of sample across the stationary phase.
The solid stationary phase may be fabricated into a
cartridge form for placement in the housing. A
plurality of cartridges may be used either in series
or parallel flow configuration in a single housing.
In one embodiment, the chromatography column for
effecting chromatographic separation of at least two
components of a sample flowing therethrough comprises
a housing and at least one solid stationary phase
having chromatographic functionality and effective
for chromatographic separation within said housing.
The housing comprises an inlet member having a sample
inlet and a distribution means in communication with
said sample inlet means, said distribution means
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- 13a -
substantially uniformly distributing said sample
therethrough, and an outlet housing member having a
sample outlet means and a sample collection means in
communication with said sample outlet means. In one
preferred embodiment, the sample inlet means and
sample outlet means form a radially outwardly
expanding stationary phase chamber.
The stationary phase comprises one or more layers
of a swellable fibrous matrix in sheet form, each
B
62~i~
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layer having chromatographic functionality and being
effective for chromatographic separation. Where the
stationary phase comprises a plurality o~ layers, said
layers may be separated from each other by a spacer
means, said spacer means permitting ~ontrolled
swelling of said layers of swellable fibrous matrix in
sheet form. However, the stationary phase and housing
coact to provide substantially uniform radial distri-
bution of the sample.
Further characteristics, features and advantages
of the invention, as well as other objects and util-
ities, will become readily apparent to those skilled
in the art from consideration of the invention as
described herein and illustrated by the following
drawings:
Figure 1 is a partial sectional view of a
side elevation of one embodiment of the chromatography
column of this invention;
Figure 2 is an enlarged cross-sectional view
taken along line 2-2 of Figure l;
Figure 3 is a perspective view of the core
with a portion of the solid stationary phase broken
away therefrom showing the spirally wound
chromatographic media and spacer means therebetween.
Figure 4 is a cross-sectional ~iew of another
embodiment of the invention wherein the chromatography
column is in disc configuration.
Figure 5 is a top plan view of the inlet
housing member of the invention embodiment in disc
configuration.
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-
Figure 6 is a top plan view of the outlet
housing member of the invention embodiment in disc
configuration.
Figure 7 is a top plan view of one embodiment
of the stationary phase of the invention column in
disc configuration.
Figure 8 is a side elevation of one embodi-
ment of the stati-onary phase of the invention column
in disc configuration.
Figure 9 is a cross-sectional view of one
embodiment of the stationary phase of the invention
column in disc configuration depicting a plurality of
layers of separ~tion media and spacer means interposed
between adjacent layers of said separation media,
prior to the sonic welding of the peripheral edges.
Figure 10 is a cross-sectional view of a
preferred configuration for the invention column in
disc configuration. In this configuration, the hous-
ing in disc configuration forms a radially outwardly
expanding chamber. A portion of the spacer means is
removed for clarity.
The solid stationary phase in this invention
comprises a swellable fibrous matrix in sheet form.
Preferably, this sheet is homogenous or substantially
homogenous, which in effect means that the stationary
phase is of a uniform or substantially uniform struc-
ture and/or composition.
Referring to the drawings, wherein like character
references indicate like parts, Figures 1 through 3
depict one embodiment of the chromatography column of
~ Ei2~;~7
-16-
this invention. Referring to Figure 1, the column,
which may be in cartridge form, generally designated
10, is comprised of a cylindrical stationary phase 12,
and cylindrical tube 13, which form a cylindrical
chamber 14 which acts as a housing for the stationary
phase 12. The solid stationary phase 12 can be
inserted into chamber 14 formed by a glass, metal or
polymeric tube or cylinder 13 having a diameter
somewhat larger than the external diameter of the
stationary phase 12. Suitable fluid admission, col-
lection and monitoring sys~ems can also be employed
with the column as in conventional analytical and
preparative columns. The stationary phase 12 is
positioned within the chamber 14 and preferably has a
longitudinal axis 16 coaxial with the axis of the
cylindrical chamber 14~ Optionally, a plurality of
cartridges may be placed in a single housing in vari-
ous configurations to effect parallel and/or series
flow between the cartridges (not shown). The solid
stationary phase has chromatographic functionality and
is effective for chromatographic separation.
Referring to Figures 2 and 3, the stationary
phase 12 is constructed of a swellable fibrous matrix
18, usually hydrophilic swellable, in sheet form which
is the active media for chromatographic separation.
The chromatographic media in sheet form 18 is sand-
wiched between a single non-woven mesh 22 or plurality
of mesh. The composite sheet of chromatography media
18 and mesh 22, preferably non-woven, is spirally
wound around a cylindrical core 24 having a longi-
tudinal axis 16 to form a plurality of layers around
the axis 16. The core 24 is provided with a plurality
of longitudinal and axially oriented channels 21 for
~9$2S~7
-17-
directing the liquid into circumferential channels 23
which are in fluid communication with core 24. The
mesh 22, due to the openness and thickness thereof,
acts as a spacer means between each layer of media 18
which permits the controlled swelling of the media and
enhances the distribution of the sample flowing
through the stationary phase 12. The cylindrical core
24 is provided with apertures 26 near the top thereof
for the flow of sample from the circumferential chan-
nels 23 into the open interior of the core~
Referring to Figure 1, the wound composite sheet
18 and 22 and core 24 are then capped by stationary
phase end caps 32 and 34. The stationary phase end
caps 32 and 34 of this subassembly are sealed by ther-
moplastic fusion to the core 24 and also to the ends
of the composites 18 and 22. The subassembly, com-
prising 18, 22, 24, 32 and 34 is then slipped into
chamber 14. The cylinder end cap 36 is then thermo-
plastically fused to the top edge 3~ of cylinder 13.
The fluid or sample 42 can thus flow radially from the
outside through the solid stationary phase to the open
channel 21 of core 24, since the interior and exterior
are completely separated by the solid stationary phase
and sealed off by stationary phase end caps 32 and 34.
The preformed stationary phase end caps 32 and 34
are preferably applied to the cylindrical solid
stationary phase 12 by heating an inside face of the
thermoplastic stationary phase end cap to a tempera-
ture sufficient to soften a sufficient amount of the
stationary phase end cap to form a thermoplastic seal
with the ends of the core 24 and composite sheet 18
and 22. All of the edges are then embedded into the
~9~
softened material. The softened material is then
hardened, typically by ambient condi~ions, to form a
thermoplastic sealing relationship between the sealing
surface of the stationary phase end caps 32 and 34,
the core 24 and the ends of the solid stationary phase
12 to form a leak-proof seal~ Such methods of applying
stationary phase end caps are well known in the
filtration art. See, for example, U.S.
Patent Nos. 4,929,354 and 4,906,371
to Meyering et al. and Miller, respectively.
Optionally, the stationary phase end caps can be
molded integrally ln situ onto the solid stationary
phase.
Stationary phase end caps of thermoplastic
materials are preferred because of the ease of bond-
ing, but it is also possible to use thermo-setting
resins in a thermoplastic, ~usible or heat-softenable
stage of polymerization, until the bondings have been
effected, after which the curing of the resin can be
completed to produce a structure which can no longer
be separated. Such a structure is autoclavable with-
out dangex of destroying the fluid tight seal, the
solid stationary phase 1~, and the stationary phase
end caps 32 and 34. Thermoplastic resins whose
softening point is sufficiently high so that they are
not softened under sterilizing autoclaving conditions
are preferred for biomedical use. Exemplary of the
plastic materials which can be used are polyolefins.
Referring to Figure 1, the preferred column 10 has
a stationary phase end cap 34 on one end which does
not open to the exterior of the subassembly 18, 22,
24, 32, and 34 but is closed off. This stationary
~2962~i~
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phase end cap 34 can nest on the bottom end wall 44 of
cylinder 13 while still permitting the flow of sample
42 into chamber 14 around the outside of stationary
phase 12, or this lower stationary phase end cap 34 of
the subassembly 18, 22, Z4, 32 and 34 is in spaced
apart relationship from the bottom end wall 44 of
cylinder 13, thus permitting the flow of sample 42
into the chamber 14.
The upper end of cartridge 40 has a cylinder end
cap 36 which is in fluid communication with channels
21 of cylindrical core 24 thus permitting the flow of
fluid from the outer periphery of cylindrical core 24
to the center of core 24 to the outside of cylinder
end cap 36. The cylinder end cap 36 has molded
thereon fitting 48 for fluid connection through a
collection means (not shown).
Referring to Figure 2, prior to winding the
chromatography media 18 on the core 24, the exterior
surface of core 24 may be completely wrapped with a
scrim material 20. Additionally, after winding the
chromatography media 18 on the core 24, the exterior
surface thereof may be completely wrapped with mesh
material 22.
~ igures 4 through 10 depict another embodiment of
the chromatography column of this invention, the embo-
diment wherein the column is in disc configuration,
again wherein like character references indicate like
parts.
Referring to Figures 4-10, the column in disc
configuration, generally designated 110, comprises an
inlet housing member 112, an outlet housing member
114, and a stationary phase 116.
The inlet housing member 112 comprises a sample
nlet meanL 118, baffle means 120, and sample
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distribution means 122. The sample inlet means 118 is
in communication with the sample distribution means
122.
The sample distribution means 122 comprises plural
radial distribution channels or grooves 130 and plural
concentric distribution ~hannels 140, the radial dis-
tribution grooves 130 and concentric distribution
channels 140 being in communication with each other
and with inlet means 118. Radial distribution grooves
130 comprise distribution groove bottom portions lying
in a plane represented by line Pl in Fig. 4 and Pl in
Fig. 10, and distribution groove wall portions 134a
and 134~. Concentric distribution channels 140 com-
prise concentric distribution channel b~ttom portions
142, concentric distribution channel wall portions
144a and 144b, and concentric distribution channel
apex portions 146.
Optionally, the inlet housing member 112 may con-
tain a venting means 150, the function and operation
of which will be defined below. The venting means is
in communication with a chamber 152. Chamber 152 is
formed by inlet housing member 112 and outlet housing
member 114 ~see Figs. 4 and 10). Chamber 15Z contains
the stationary phase 116.
The outlet housing member 114 comprises a sample
collection means 154 and sample outlet means 156,
sample collection means 154 being in communication
with sample outlet means }56.
Sample collection means 154 comprises plural
radial collection grooves 160 and plural concentric
collection channels 170. Radial collection grooves
160 and concentric collection channels 170 ~re in
:
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-21-
communication with each other and with sample outlet
means 156.
Radial collection grooves 160 comprise radial
collection groove bottom portions lying in a plane
represented by line P2 in Fig. 4 and P2 in Fig. 10,
and radial groove wall portions 164a and 164b. Con-
centric collection channels 170 comprise concentric
collection channel bottom portions 172, concentric
collection channel side wall portions 174a and 174b
and concentric collection channel apex portions ~76.
Stationary phase 116 has chromatographic function-
ality and is effective for chromatographic separation.
Referring to Figures 7, 8 and 9 in particular, the
stationary phase 116 may comprise a plurality of
layers of a swellable fibrous matrix 180 in sheet
form, having chromatographic functionality and being
effective for chromatographic separation, and a spacer
means 182 between each adjacent layer of swellable
fibrous matrix 180. This configuration is best shown
in Figure 9, a cross-sectional view of one embodiment
of the separation phase 16.
The swellable fibrous matrix 180 is preferably
hydrophilic swellable and comprises the active media
for chromatographic separation. The spacer means 182
may be typically a woven or non-woven mesh similar to
mesh 22 of Figures 2 and 3 above and is further
described below. The mesh, due to the openness and
thickness thereof, acts as a spacer means hetween each
layer of swellable fibrous matrix 180 and permits the
controlled expansion thereof without closing off the
porous structure of the media, thereby enhancing the
distribution of the sample flowing through the sta-
tionary phase 116.
~l2~i2~i~
-22-
As may be seen from Figure 8, a typical manner of
conforming the stationary phase 116 is to produce a
"sandwich" of alternating layers of swellable fibrous
matrix in sheet form and layers of spacer means, with
the periphery of the sandwich compressed into a fluid
tight configuration 184. Typically, the peripheral
edges of alternating discs of swellable fibrous matrix
180 and spacer means 182 are joined. Preferably, the
fibrous matrix 180 contains ox has bonded therein a
thermoplastic polymeric material. Similarly, in a
preferred embodiment, spacer means 182 also is made of
or contains thermoplastic polymeric materials. In
this configuration, the edges may be uniformly joined
by appropriate heat treating, e.g. sonic welding. As
may be seen from Figure 4, in a preferred embodiment,
the fluid tight peripheral configuration 184 is itself
contained in a fluid tight, hermetic seal formed by
the mating edges 186 and 188 of, respectively, the
inlet housing member 112 and the outlet housing member
114. In this manner, sample entering through inlet
means 118 must pass through stationary phase 116 prior
to exiting through outlet means 156.
The disc configured chromatography column of
Figures 4-9 is formed using conventional and well
known fabrication techniques. Typically, the station-
ary phase 116, a preformed i'sandwich" of alternating
layers of swellable fibrous matrix and spacer means,
with peripheral edges sonically welded and configured
as in Figure 8, is placed in inlet housing 112 and
outlet housing member 114 is placed thereover. Subse-
quently, the mating ed~es 186 of the inlet housing
member 188 and of the outlet housing ~iember 190 are
joined to form an airtight and fluid tight seal. In
~ ~9qE~2S~
-23-
one embodiment, the edges are sealed by sonically
welding same, the technique described in Branson Sonic
Power Company, Danbury, Connecticut, Information Sheet
PW-3, 1971~
Vent means 150, as mentioned above, represents an
optional configuration of the disc embodiment of the
column. Its purpose is to allow air in the column to
exit the column during use. Typically, vent m~ans 150
is adapted to be sealed off when all air has been
removed from the system. In an alternative embodi-
ment, vent means 150 contains a hydrophobic media
which will allow the passage of gases but not liquids,
as disclosed in V~S. Patent 4,113,627~
In a preferred embodiment, depicted by Figure 10,
chamber 152 is radially outwardly expanding. By the
term "radially outwardly expanding" is meant that the
volume at the interior chamber is less than the volume
at the periphery of the chamber. In this configura-
tion, rèferring to Figure 10, the distance between
distxibution means 112 and collection means 114 at the
interior, dl, is less than the distance between dis-
tribution means 112 and collection means 114 at the
periphery d2.
Because the stationary phase 116 is hydrophilic
swellable, sample solution on contact with separation
phase 116 causes the separation phase to swell. As the
separation phase swells, the pressure differential
between the inlet and outlet sides of the separation
media increases, thereby restricting sample flow-
through. By designing a housing as described above,
i.e. in radially outwardly expanding co~nfiguration,
~ ''
3L~9~i%S~
-24-
the pressure differential between the inlet and outlet
sides of the stationary phase decreases towards the
periphery, thereby maximizing utilization of the
chromatographic separation function of the stationary
phase and substantially increasing the adsorption
capacity of a given unit.
In another preferred embodiment, also depicted in
Figure 10, the volume of each succeeding concentric
distribution channel 140 and concentric collection
channel 170 increases from the intexior to the periph-
ery of the chromatographic column. In this manner,
clogging of the channels by the swelling of the hydro-
philic swellable stationary phase is vitiated, thereby
promoting uniform distribution of sample and maximum
utili~ation of column capacity.
In the embodiment depicted in Figure 10, lines A,
A', C and C' are lines which represent cross-sectional
view of parallel planes which are perpendicular to the
longitudinal axis L of the chromatography column.
Lines B and B', respectively, represent cross-section-
al views of planes which are substantially tangent to
the apices 146 and 176 of concentric distribution
channels 140 and concentric collection channels 170.
Planes ~ and B' form angles ~ and ~' with planes A
and A'. Thus, planes B and B', at angles ~ and ~ ' to
planes A and A', respectivelyt define a radially
outwardly expanding chamber 152, which in turn defines
the limits of expansion of stationary phase 116. As
described above, the optimal configuration for the
radially outwardly expanding embodiment is such that
stationary phase 116, in maximally swelled status, is
just touching the most peripheral apices 146 and 176.
~L296Z5~
-25-
It is to be understood that angles ~ and ~ ' may be
the same or different and may vary with the number ~f
layers of swellable fibrous matrix and the particular
matrix in use. Typically, a and ~ ' are about
2~
Lines D and D', respectively, represent cross-
sectional views of planes which contain concentric
distribution channel bottom portions 142 and concen-
tric collection channel bottom portions 172 and define
angles ~ and ~ ' with planes C and C'. Thus, planes
D and D', at angles ~ and ~ ' to planes C and C',
resRectively, de~ine the slope of the increasing depth
of channels 140 and 170. In the embodiment of Figure
10, ~ and ~ ' are typically each about 5. However,
these angles may be varied, both with respect to one
another and absolutely.
In similar manner, it is within the scope of the
present invention to configure a chromatographic
column such that radial distribution grooves 130
andJor radial collection grooves 160 increase in
volume from the interior to the periphery of the
column. Such a configuration is disclosed in U.S.
Patent No. 3,361,261b,
As is understood by those skilled in the art, it
is desirable to minimize the hold-up volume of a
chromatographic column. With this in mind, an optimal
design for a radially outwardly expanding chamber is
that where the distance d2 is such as to allow the
swellable stationary phase to swell to its maximum,
but with no unused space left. In this manner, the
pressurP differential at the periphery is minimized,
i2~ 4~
-26-
minimized, while at the same time reducing hold-up
volume to its lower limit as well. This housing con-
~iguration permits as well the use of a single layer
of fibrous matrix or a plurality of layers of fibrous
matrix with no spacer means interposed between layers.
The radially outwardly expanding chamber coacts with
the thus configured stationary phase to uniformly
distribute sample thereacross.
The present invention as conceived utilizes known
media and known media preparation techniques, specifi-
cally those decribed in the aforementioned co-pending
applications and patents. This preferred media is
~ibrous, in sheet form and generally has the char-
acteristics that it is hydrophilic swellable. The
pre~erred chromatographic media is that described in
the aforementioned Crowder, III et al. patents and Hou
and Hou et al. patents. It should be
realized, however, that this invention is applicable
to any type of swellable media in sheet form, whether
it is hydrophilic swellable or otherwise.
In order to provide a chromatographic media matrix
which is coherent and handleable, it is desirable that
at least one of the components which go into forming
the porous matrix be a long, self-bonding structural
fiber. Such fiber gives the stationary phase suffi-
cient structural integrity in both the wet "as formed"
condition and in the final dry condition. Such a
structure permits handling of the phase, in particular
a sheet, during processing and at the time of its
intended use. Preferablx, the sheets which form the
chromatographic media are formed by vacuum felting an
aqueous slurry of fibers. The sheets may also be
~2g~2~
pressure felted or felted from a non-aqueous slurry.
The sheet shows a uniform high porosity, with excel-
lent flow characteristics, and is substantially
homogeneous. In general, the media can range in
thicknesses from about 5 mils to about 150 mils (dry);
however, thicker or even thinner media may be utilized
provided the sheet can be spirally wound or layered to
produce a column which can perform as described above.
The media can swell to at least 25% this thickness,
and generally greater, e.g. two to ~our times, this
thickness.
It is important when constructing the chromato-
graphy column of this invention that the chromato-
graphic media used in the column be of uniform
thickness throughout its length and width and that the
media have a substantially uniform density throughout.
It is preferred that the layer of media be substan-
tially homogenous with respect to itself; however, for
certain applications and material, it is understood
that non-homoyenous construction may be employed.
Since the solid stationary phase is intended in
use to effect separation by maintaining a substantial
pressure differential across the solid stationary
phase, it is essential that the solid stationary phase
have a sufficient degree of compressive strength to
withstand deformation under such loads as may be
imposed upon it. Such compressive streng~h must not
only exist in the media itself but in the spacer means
and the internal core upon which the chromatography
media, or solid stationary phase is compressed.
Due to the swellability of the me~ia, a key
element of this invention is the spacer means between
1;2~3Çi2~ 7
-28-
each layer of the media and/or the coaction of the
chamber wall and the fibrous matri~. The spacer means
permits controlled expansion of the media and enhance-
ment of the distribution of sample flowing through the
stationary phase. The spacer means located between
each layer of the swellable chromatographic media pro-
vides for the distribution moveme~t of the sample as
the sample passes through the solid stationary phase.
The spacer means functions to uniformly control thick-
ness and density of the chromatographic media during
use. In addition, the spacer means can serve as a
backing or support for the layer of chromatographic
media. This latter aspect is particularly useful dur-
ing the manufacturing phase.
It is preferred that the spacer means be composed
of a material which is inert with respect to the
chromatographic process. By inert, it is meant the
material does not adversely affect the fu~ction of the
solid stationary phase.
Referring to Figures 2 and 3, the spacer means
comprises the mesh 22. Alternatively, where the
column design is as depicted in Figures 4-10, the
spacer means 18~ may also comprise a mesh, or scrim
and mesh. A scrim material can function to channel,
to a certain extent, the sample flowing through the
media and substantially evenly disperse the sample
axially and circumferentially across the meaia. The
mesh material provides spacing between the media to
permit controlled expansion thereof to prevent the
"cut-off" to flow therethrough by compression of the
permeable media and also assists in distributing or
channeling the sample 10wing through the media.
~2~6;~
,
The mesh material is an open type o~ material
having openings ranging, for general guidance, from
1/16 inch to 1/4 inch.
It should be noted that the thickness of the
spacer means, i.e. the scrim and particularly the mesh
material, and the pore size of each to be used may be
readily determined by one skilled in the art by per-
forming te~s which vary these ~actors. ~uch factors
as the openness and thickness o~ these spacer means
are highly dependent on the type of media utilized,
e.g. swellabilityl wettability, thickness, chemical
composition, etc., the flow rate of the sample through
the stationary phase, the surface area of the sta-
tionary phase, e~g. number of windings, thickness of
media, diameter of stationary phase, etc. It is thus
very dif~icult to clearly specify these variables,
other than to say that these may be determined by
either trial and error or more elaborate testing
procedures to determine the optimum paramekers.
The preferred mesh material, at this time, is
polypropylene CONWED"'~Grade TD-620)~
The overall width o the stationary phase in
accordance with the present invention can be infinite,
the actual diameter being limited only by practical
considerations such as space requirements. Since the
diameter or width of the overall column can be in-
creased without theoretical limitation, the sample
size or amount of substance to be separated in the bed
is not limited. Thus, the diameter can be increased
to separate the desired amount of sample substance to
be produced.
* Registered Trademark
A
5~
-30-
In operation, the sample is driven through the
stationary phase and separated into distinct chromato-
graphic fractions by the chromatographic media. The
spacer means induces and permits flow of this stream
as it moves through the column and therefore provides
for improved resolution and utilization of-the media's
potential capacity.
Referring to Figure 1, the sample is preferably
introduced at the bottom of the column flowing to the
outer surface of the solid stationary phase and then
flowing radially inward through the layers of chroma-
tographic media and spacer means into the channels 21
of core tube 24 and is withdrawn centrally. It is
apparentr from what has been set forth above, that the
radial flow can also be caused to circulate in the
opposite direction.
Referring to Figure 4, sample is preferably intro-
duced at the inlet 118, passes to distribution means
122, is substantially uniformly distributed over the
surface of the stationary phase 116 by radial dis-
tribution grooves 132 and concentric distribution
channels 130, and passes through radial collection
grooves 140 and concentric collection channels 170 and
exits through outlet 156.
The chromatographic columns of this invention may
be used for any of the well-known chromatographic
separations usually performed with conventional
columns. Additionally~ the columns of the present
invention may be found useful in the areas where
conventional columns are impractical.
The novel columns of this invention can be used
for separations in the analytical and preparative
fields. The columns can be connected to all common
J
-31-
types of chromatographic equipment. Several columns
or cartridges of solid stationary phase can be con-
nected in series or parallel. In large units, the
columns can contain identical or different chromato-
graphic media and can be of identical or different
length and/or diameter.
It has been found that the aforedescribed station-
ary phase produces unexpected results in that the flow
of sample through the column is enhanced without
destroying the adsorptive capacity of the media. Addi-
tionally, when protein and dye staining tests were
performed it was found that the stationary phase of
this invention provided even distribution of sample
flow therethrough without an increase in pressure drop
when compared to a stationary phase not utilizing the
spacer means described herein.
From the foregoing, it can be seen that a conveni-
ent stationary phase configuration has been invented
which is easy to install, operate r and disassemble and
is easily adaptable to any batch size or continuous
type operation by the use of multiple configurations.
Addit.ionally, the chromatography column has excellent
structural integrity.
The stationary phases decrease total processing
time and when used with the proper chromatographic
media has excellent binding capacity. The stationary
phases may be used with standard type pumps, gravity
feed, or syringes, utilizedt in their preferred mode,
at from 1 to 50 PSI, and even unde~ vacuum. The sta-
tionary phases of chromatographic media are totally
enclosed and completely self-contained to ensure
sterile conditions. Due to the fact that the solid
stationary phase is manufactured in a factory and
~ 9G2r~
assembled therein, each is virtually identical to the
other, does not vary as in previously known columns
and eliminates the dependence upon packing expertise.
Additionallyt there is no premeasuring of chromato-
graphic media, no media loss due to handling, no pack-
ing problems, no fines generation and removal within
the column and other problems associated with packing
chromatographic columns. The column is simple to
operate, does not produce any channeling by passing or
shifts in bed volume. The chromatographic stationary
phase allows scale up from milligram laboratory
quantities to megagram production quantities. The
stationary phase provides rigidity and strength and is
particularly useful as a high flow, medium pressure
matrix and is highly suitable for large scale protein
or non-protein purifications.
It has surprisingly been found that when a column
configured as in Figure 10 is employed, the actual
capacity is substantially increased over that of the
column of Figure 4. In this way, the actual capacity
more closely approximates the theoretical capacity of
the column. By configuring the column to maximize
sample distribution, minimize hold-up volume, and
maximize stationary phase utilization by creating a
differential pressure gradient which decreases from
the interior to the periphery, the useful and effec-
tive life of the column is substantially improved.
The present invention has been described ln
relation to several embodiments. Upon reading the
specification, one of ordinary skill in the art would
be able to effect varlous alterations, or changes in,
- ~2~362~
or substitutions of equivalents to the present inven-
tion as disclosed~ It is intended that the invention
as conceived be limited only by the definition of the
invention contained in the appended claims.
