CA1296257C - Chromatography column - Google Patents
Chromatography columnInfo
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- CA1296257C CA1296257C CA000505906A CA505906A CA1296257C CA 1296257 C CA1296257 C CA 1296257C CA 000505906 A CA000505906 A CA 000505906A CA 505906 A CA505906 A CA 505906A CA 1296257 C CA1296257 C CA 1296257C
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Abstract
ABSTRACT OF THE DISCLOSURE
A chromatography column for effecting chromatographic separation of at least two components of a sample flowing therethrough comprising, a hous-ing, the housing comprising, an inlet housing member and an outlet housing member, the inlet housing member and the outlet housing member defining a radially, outwardly expanding stationary phase chamber and a stationary phase within the radially outwardly expand-ing stationary phase chamber, the stationary phase chamber comprising at least one layer of a swellable fibrous matrix in sheet form, wherein the stationary phase and the radially outwardly expanding stationary phase chamber coact to provide substantially uniform radial distribution of sample across the stationary phase.
A chromatography column for effecting chromatographic separation of at least two components of a sample flowing therethrough comprising, a hous-ing, the housing comprising, an inlet housing member and an outlet housing member, the inlet housing member and the outlet housing member defining a radially, outwardly expanding stationary phase chamber and a stationary phase within the radially outwardly expand-ing stationary phase chamber, the stationary phase chamber comprising at least one layer of a swellable fibrous matrix in sheet form, wherein the stationary phase and the radially outwardly expanding stationary phase chamber coact to provide substantially uniform radial distribution of sample across the stationary phase.
Description
~2962S7 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.
~2~6i2~7 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.
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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.
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 :
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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 . ,,~
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 /~ '`
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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.
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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 -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~
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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.
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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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 ~J
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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
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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.
-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 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 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~
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 ~:$~
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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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.
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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~
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~
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~
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~
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 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~
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
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.
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.
~2~6i2~7 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 .
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.
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 . ,,~
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~
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 -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.
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 :
' ' ' - , 25~
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 ~J
~625~
- 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~
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.
-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 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 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~
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 ~:$~
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 :
~2~36~
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~
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~
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~
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~
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~
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 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~
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
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.
Claims (26)
1. A chromatography column for effecting chromatographic separation of at least two components of a sample flowing therethrough comprising:
(1) a housing, said housing comprising:
(a) an inlet housing member, and (b) an outlet housing member, said inlet housing member and said outlet housing member defining a radially, outwardly expanding sta-tionary phase chamber; and (2) a stationary phase within said radially outwardly expanding stationary phase chamber, said stationary phase comprising:
(a) one or more layers of a swellable fibrous matrix having chromatographic func-tionality, and (b) a spacer means between each layer for permitting controlled swelling thereof and enhancing the distribution of sample flowing through the stationary phase wherein said stationary phase and said radially outwardly expanding stationary phase chamber coact to provide substantially uniform radial distribution of sample across said stationary phase.
(1) a housing, said housing comprising:
(a) an inlet housing member, and (b) an outlet housing member, said inlet housing member and said outlet housing member defining a radially, outwardly expanding sta-tionary phase chamber; and (2) a stationary phase within said radially outwardly expanding stationary phase chamber, said stationary phase comprising:
(a) one or more layers of a swellable fibrous matrix having chromatographic func-tionality, and (b) a spacer means between each layer for permitting controlled swelling thereof and enhancing the distribution of sample flowing through the stationary phase wherein said stationary phase and said radially outwardly expanding stationary phase chamber coact to provide substantially uniform radial distribution of sample across said stationary phase.
2. A chromatography column for effecting chromatographic separation of at least two components of a sample flowing therethrough comprising:
(1) a housing, said housing comprising:
(a) an inlet housing member, and (b) an outlet housing member, said inlet housing member and said outlet housing member defining a stationary phase chamber; and (2) a stationary phase within said stationary phase chamber, said stationary phase comprising:
(a) a plurality of layers of a swellable fibrous matrix in sheet form having chromatographic functionality and being effective for chromatographic separation, and (b) a spacer means between each layer of said swellable fibrous matrix for permitting con-trolled swelling thereof and enhancing the distri-bution of sample flowing through said stationary phase, wherein said stationary phase chamber and said stationary phase coact to provide substantially uniform radial distribution of sample across said stationary phase.
(1) a housing, said housing comprising:
(a) an inlet housing member, and (b) an outlet housing member, said inlet housing member and said outlet housing member defining a stationary phase chamber; and (2) a stationary phase within said stationary phase chamber, said stationary phase comprising:
(a) a plurality of layers of a swellable fibrous matrix in sheet form having chromatographic functionality and being effective for chromatographic separation, and (b) a spacer means between each layer of said swellable fibrous matrix for permitting con-trolled swelling thereof and enhancing the distri-bution of sample flowing through said stationary phase, wherein said stationary phase chamber and said stationary phase coact to provide substantially uniform radial distribution of sample across said stationary phase.
3. A chromatography column for effecting chroma-tographic separation of at least two components of a sample flowing therethrough comprising:
(1) a housing, said housing comprising:
(a) an inlet housing member, and (b) an outlet housing member, said inlet housing member and said outlet housing member defining a radially, outwardly expanding stationary phase chamber; and (2) a stationary phase within said radially, outwardly expanding chamber, said stationary phase comprising:
(a) a plurality of layers of a swellable fibrous matrix in sheet form having chromatographic functionality and being effective for chromatographic separation, and (b) a spacer means between each layer of said swellable fibrous matrix for permitting con-trolled swelling thereof and enhancing the distribu-tion of sample flowing through the stationary phase, wherein said stationary phase chamber and said stationary phase coact to provide substantially uniform radial distribution of sample across said stationary phase.
(1) a housing, said housing comprising:
(a) an inlet housing member, and (b) an outlet housing member, said inlet housing member and said outlet housing member defining a radially, outwardly expanding stationary phase chamber; and (2) a stationary phase within said radially, outwardly expanding chamber, said stationary phase comprising:
(a) a plurality of layers of a swellable fibrous matrix in sheet form having chromatographic functionality and being effective for chromatographic separation, and (b) a spacer means between each layer of said swellable fibrous matrix for permitting con-trolled swelling thereof and enhancing the distribu-tion of sample flowing through the stationary phase, wherein said stationary phase chamber and said stationary phase coact to provide substantially uniform radial distribution of sample across said stationary phase.
4. The column of claim 1 wherein said inlet housing member comprises a sample inlet means and a sample distribution means, said sample inlet means in communication with said sample distribution means, and said outlet housing means comprises a sample collection means and a sample outlet means, said sample collection means in communication with said sample outlet means.
5. The column of claim 2 wherein said inlet housing member comprises a sample nlet means and a sample distribution means, said sample inlet means in communication with said sample distribution means, and said outlet housing means comprises a sample collection means and a sample outlet means, said sample collection means in communication with said sample outlet means.
6. The column of claim 3 wherein said inlet housing member comprises a sample inlet means and a sample distribution means, said sample inlet means in communication with said sample distribution means, and said outlet housing means comprises a sample collection means and a sample outlet means, said sample collection means in communication with said sample outlet means.
7. The column of claim 4 wherein said sample distribution means comprises radial distribution grooves and concentric distribution channels, said grooves and channels being in communication with each other, and said sample collection means comprises radial collection grooves and concentric collection channels, said radial collection grooves and con-centric collection channels in communication with each other.
8. The column of claim 7 wherein said sample distribution means comprises radial distribution grooves and concentric distribution channels, said grooves and channels being in communication with each other, and said sample collection means comprises radial collection grooves and concentric collection channels, said radial collection grooves and con-centric collection channels in communication with each other.
9. The column of claim 6 wherein said sample distribution means comprises radial distribution grooves and concentric distribution channels, said grooves and channels being in communication with each other and the other, and said sample collection means comprises radial collection grooves and concentric collection channels, said radial collection grooves and concentric collection channels in communication with each other.
10. The column of claim 7, 8 or 9 wherein the volume of said concentric distribution channels and/or said concentric collection channels increases from the interior to the periphery of said column.
11. The column of claim 7, 8 or 9 wherein the volume of said radial distribution grooves and/or said radial collection grooves increases from the interior to the periphery of said column.
12. The column of claim 4, 5 or 6 wherein said inlet housing member further comprises a venting means.
13. The column of claim 4, 5 or 6 wherein said swellable fibrous matrix is hydrophilic swellable.
14. The column of claim 4, 5 or 6 wherein said swellable fibrous matrix swells at least about 25% of its thickness.
15. The column of claim 4, 5 or 6 wherein said spacer means comprises:
(a) a scrim layer for channeling the sample flow through the matrix and substantially evenly dispersing the sample; or (b) a mesh layer to provide a spacing between the layers to permit controlled expansion thereof and assist in distributing the sample; or (c) (a) in combination with (b).
(a) a scrim layer for channeling the sample flow through the matrix and substantially evenly dispersing the sample; or (b) a mesh layer to provide a spacing between the layers to permit controlled expansion thereof and assist in distributing the sample; or (c) (a) in combination with (b).
16. The column of claim 2 wherein said spacer means comprises a mesh layer to provide spacing between said layers to permit controlled expansion thereof and assist in distribution of the sample.
17. The column of claim 16 wherein the peripheral edges of said stationary phase have a fluid tight, heat induced hermetic seal.
18. The column of claim 17 wherein said stationary phase comprises thermoplastic polymer to facilitate formation of said seal.
19. The column of claim 17 wherein said peripheral edges have a sonically welded seal.
20. A solid stationary phase having chromato-graphic functionality and being effective for chromatographic separation of at least two components of a sample flowing therethrough wherein the stationary phase comprises:
(a) a plurality of layers of sheets of a swellable fibrous matrix having chromatographic func-tionality and being effective for chromatographic separation, and (b) a spacer means between each layer sepa-rating the layers for permitting controlled swelling of the matrix and enhancing the distribution of sample flowing through the stationary phase by substantially evenly dispersing the sample across the matrix.
(a) a plurality of layers of sheets of a swellable fibrous matrix having chromatographic func-tionality and being effective for chromatographic separation, and (b) a spacer means between each layer sepa-rating the layers for permitting controlled swelling of the matrix and enhancing the distribution of sample flowing through the stationary phase by substantially evenly dispersing the sample across the matrix.
21. The stationary phase of claim 20 wherein the swellable matrix swells at least about 25% of its thickness.
22. The stationary phase of claim 20 wherein the swellable matrix is hydrophilic swellable.
23. The stationary phase of claim 20 wherein said spacer means comprises:
(a) a scrim layer for channeling the sample flow through the matrix and substantially evenly dispersing the sample; or (b) a mesh layer to provide a spacing between the layers to permit controlled expansion thereof and assist in distributing the sample; or (c) (a) in combination with (b).
(a) a scrim layer for channeling the sample flow through the matrix and substantially evenly dispersing the sample; or (b) a mesh layer to provide a spacing between the layers to permit controlled expansion thereof and assist in distributing the sample; or (c) (a) in combination with (b).
24. The stationary phase of claim 17 wherein the spacer means comprises said mesh layer (b).
25. The stationary phase of claim 20 wherein the peripheral edges of said plurality of layers of sheets of a swellable fibrous matrix and said spacer means are formed into a fluid-tight hermetic seal.
26. The stationary phase of claim 25 wherein at least one of said swellable fibrous matrix and said spacer means comprises a thermoplastic polymer to facilitate sealing the peripheral edges thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/723,691 US4675104A (en) | 1983-06-17 | 1985-04-16 | Chromatography column |
| US723,691 | 1985-04-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1296257C true CA1296257C (en) | 1992-02-25 |
Family
ID=24907284
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000505906A Expired - Lifetime CA1296257C (en) | 1985-04-16 | 1986-04-04 | Chromatography column |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1296257C (en) |
-
1986
- 1986-04-04 CA CA000505906A patent/CA1296257C/en not_active Expired - Lifetime
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |