Patent Publication Number: US-2020289957-A1

Title: Chromatography column with  locked packed bed and method of  packing that column

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The application claims the benefit under 35 U.S.C. § 119(e) to Provisional Patent Application No. 62/552,918 filed Aug. 31, 2017, the entire contents of which are incorporated herein by reference. 
    
    
     STATEMENT RE: FEDERALLY SPONSORED RESEARCH/DEVELOPMENT 
     Not Applicable 
     BACKGROUND 
     This application relates to a method and apparatus for packing tubular columns with chromatographic media, believed particularly suitable for high pressure liquid chromatography (HPLC) columns with media. 
     High pressure liquid chromatography (HPLC) columns contain a chromatographic media that forms a bed in the column with the bed located between two porous members such as screens or frits, typically made of glass, ceramic or metal. The media bed may be packed or unpacked. U.S. Pat. No. 5,186,826 is an example of an unpacked column where the column has enough head volume to allow the media to be manually shaken before each use. 
     For HPLC and UHPLC columns, voids in the chromatographic bed and non-uniformities in the bed can degrade performance of the column. For HPLC and UHPLC columns, packed beds with no void volumes are especially desirable and placing the bed in compression helps reduce such void volumes. Packed media beds may be formed by taking a column with a first porous member in one end, filling the column with media which is restrained by the first porous member, placing a second porous member on the other end of the media, and physically compressing the media between the two porous members using a ram or other compressing mechanism. U.S. Pat. No. 5,893,971 is one example of axial compression packing which retains a packing piston and mechanism in the column. That packing method causes an unnecessarily long and heavy column with costly hardware remaining in the column, all of which are not desirable. 
     The use of axial compression to compress the chromatographic beds of media works much better with non-porous substrates, but may work poorly with porous media and superficially porous (core-shell) media which lacks the compressive strength of non-porous media. This is especially true of silica media which crushes under sufficiently high compressive pressures and produces fragments or fines that can create non-uniformities in flow through the packed chromatographic bed or possibly clog the downstream porous member on the column. The porous and superficially porous chromatographic particles are often packed using slurry packing, in which a first porous member (e.g., frit) is placed in the downstream end of the column and a slurry of chromatographic media suspended in a fluid carrier (preferably a solvent), flows into the opposing, open end of the column, with the carrier fluid passing through the downstream porous member so the column fills with media packed by the flow of slurry and the pressure of the carrier fluid. When the column is filled to the desired degree, the slurry flow and fluid flow are stopped and a second, porous member is placed on the upstream end of the column. 
     Because the chromatography particles will not pass through the porous members, the upstream porous member is removed during slurry packing and is placed against the packed chromatography bed after slurry packing is complete. Unfortunately, when the fluid packing pressure is released the compressed bed expands so that before the upstream porous member can be added to the column, the bed expands and releases at least some of the compression achieved by the slurry packing pressure. This is described in U.S. Pat. No. 7,339,410, which removes the media extruded beyond the end of the column and adds a stepped porous member to slightly compress the bed adjacent the porous member. While that patent and others describe various ways to try and reduce the adverse effects of the bed expansion using slurry packing, simpler, faster and more consistent ways to pack the column bed are still needed. Thus, for a variety of reasons, simpler, faster and less expensive ways to reduce and retain the bed expansion of a packed chromatography bed are greatly desirable. 
     To address some of these difficulties with slurry packing, packing methods and resulting columns have been developed that use slurry packing but retain only the piston in the column, while leaving the majority of the packing mechanism outside of the column, as in U.S. Pat. No. 7,674,383. But this packing mechanism requires many parts and a locking system to retain the piston against the packed bed without releasing the pressure. There is thus still a need for a simple, less costly way to consistently pack these columns with chromatography media while maintaining a desirable compression on the chromatographic bed and increasing the density of the packed bed. 
     BRIEF SUMMARY 
     A chromatography column is provided that has a tubular body with opposing upstream and downstream ends with upstream and downstream end fittings connected to the tubular body. The tubular body has an internal bore extending along a longitudinal axis of the tubular body and column. The column includes a retaining plug permanently fixed to the upstream end of the tubular body and blocking one end of the bore to prevent fluid passage past the retaining plug. The retaining plug preferably has a single fluid passage extending therethrough along the longitudinal axis, but the passage need not be so located or limited in so number. The retaining plug has opposing upstream and downstream ends with the fluid passage having a first diameter at the upstream end of the retaining plug and a second diameter opening at the downstream end of the plug. The column also has an upstream porous member upstream of the retaining plug and extending across the bore. The column has a downstream porous member extending across the bore at a location adjacent the downstream end of the tubular body. The tubular body has a continuous wall between the retaining plug and the downstream porous member (e.g., frit) so the bore bounds the flow path between the retaining plug and the downstream porous member (e.g., frit). Chromatographic media extends from the upstream porous member, through the passage in the retaining plug, to the downstream porous member, with no voids therein. The portion of the chromatographic media between the retaining plug and the downstream porous member is under compression and forms a bed of packed media. 
     Numerous variations of the fluid passage are available. The first diameter of the fluid passage may be the same as the second diameter. But the first, upstream diameter of the passage is advantageously larger than the second, downstream diameter of the passage. An upstream end fitting may hold the upstream porous member from moving in the upstream direction along the longitudinal axis. The fluid passage preferably has a conical portion on the upstream end of the retaining plug, which conical portion converges in a downstream direction to a smaller diameter. Further, the conical portion may extend from the upstream end to the downstream end of the retaining plug. The fluid passage may optionally have a cylindrical portion located downstream of the conical portion and joining an upstream end of the cylindrical portion. The fluid passage may have a diverging conical portion that expands in diameter toward the downstream end of the retainer plug. The conical portion may be in fluid communication with a concave recess in the downstream end of the retaining plug, which recess abuts the chromatographic media. The fluid passage may include a concave recess. The concave recess may include a recess having a cross-section taken along the longitudinal axis that comprises one of a parabolic shape or a circular shape. The first, upstream opening of the passage may be smaller in diameter than the second, downstream opening, but it believed preferable to have the larger opening of the passage on the upstream end of the passage. 
     In further variations, the downstream end of the fluid passage may have an opening that is about ⅕ to about ⅓ the diameter of the bore for openings up to about 14 mm in diameter. The downstream end of the fluid passage may alternatively have an opening that is about ½ to ¾ the diameter of the bore. The retaining member may optionally have a plurality of fluid passages, each having a minimum passage diameter, which minimum diameters are preferably but optionally the same. The chromatographic media may further comprise a bed of porous particles, or a bed of superficially porous particles, or a bed of nonporous particles, or a bed of polymer particles, or a bed of silica particles, or a bed of hybrid organic/inorganic particles. 
     There is also advantageously provided an improved method of packing a chromatography column having a tubular body with a bore defining space for a packed bed of chromatographic media located between a downstream porous member filtering all flow through the bore and preferably extending across the bore and an upstream retaining plug fastened to an upstream end of the tubular body and blocking flow through the bore except for a fluid passage through the retaining plug. The method may include the steps of passing a slurry of solvent and chromatographic particles through the fluid passage under a predetermined packing pressure greater than 100 psi to form a packed bed of chromatographic media between the downstream porous member and the retaining plug. The fluid passage preferably has an upstream opening on an upstream face of the retaining plug and a downstream opening on a downstream face of the retaining plug, with the upstream and downstream openings being centered on a longitudinal axis of the tubular body. The retaining plug is permanently fastened to the tubular body. 
     The method also includes stopping the flow of the slurry and solvent when a compressed chromatographic media bed is formed between the retaining plug and the downstream porous member and when compressed media particles fill the fluid passage and possibly any portion of the column upstream of the retaining plug. The bed may expand into the downstream opening of the fluid passage when flow of slurry and solvent is stopped. After the flow of slurry is stopped, an upstream porous member is placed over the bore at a location upstream of the retaining plug and in contact with the chromatographic media with no void spaces between the upstream porous member and the adjacent chromatographic media. The method also includes the step of fastening the upstream porous member to the column. The retaining plug restrains expansion of the bed downstream of the plug and the upstream porous member restrains expansion of the media in the passage. 
     In further variations of the method, the fastening step preferably includes urging a frit retainer against the upstream porous member and fastening the frit retainer to the tubular body to restrain motion of that porous member. The variations also include using a retaining plug having the upstream opening larger in diameter than the downstream opening. The downstream opening is smaller than the bore through the tubular body and the portion of the retaining plug adjacent the downstream opening preferably restrains bed expansion of at least part of the packed chromatographic bed in a direction along the longitudinal axis. The packing pressure is preferably between about 10,000 psi and about 25,000 psi. The chromatographic media used in the method may include a porous media with a pore size of at least 2 nm, or superficially porous particles, or porous particles. The chromatographic bed preferably has no void volumes. 
     The method may further include the step of scraping off any media extruded above the top surface of the retaining plug before the placing step and after the fastening step. 
     There is also provided a chromatography column packed by one or more of the above methods. Such a packed column is believed to have improved density of the packed chromatography bed compared to columns packed without the retaining plug, and are also believed to have improved interlocking of the chromatographic media, as well as other advantages described herein. The packed column may include a packed chromatography bed of porous silica particles with a pore size of at least 2 nm, or it may include packed polymer particles, or packed silica particles, or a packed bed of porous particles, or a packed bed of superficially porous particles. 
     There is also provided a chromatography column that includes a tubular body having opposing upstream and downstream ends and a cylindrical bore extending along a longitudinal axis of the tubular body between the upstream and downstream ends. A retaining plug having an outer periphery is permanently fixed to the bore adjacent the upstream end of the tubular body in a fluid tight manner. The retaining plug may have a single fluid passage extending through the retaining plug and along the longitudinal axis. The fluid passage has an upstream opening and a downstream opening. The column also has a downstream porous member extending across the bore adjacent the downstream end of the tubular body. The downstream porous member is configured to block the passage of chromatographic media while allowing liquid and gas to pass through the downstream member. The column also includes a downstream end cap connecting the downstream porous member to the downstream end of the tubular body. 
     In further variations, the entire tubular body is preferably metal. The fluid passage may have various configurations, including a configuration where the downstream end of the fluid passage has a diameter that is smaller than a diameter of the upstream end of the passage. That is, the upstream opening is larger in diameter than the downstream opening. The retaining plug preferably has an upper surface that is flush with the upstream end of the tubular body. The retaining plug may also have a hat shaped cross-section with a downstream portion extending inside the bore of the tubular body and an upstream portion extending over an end of the tubular body and permanently fastened thereto. The column may have an upstream porous member upstream of and urged toward the retaining plug by an upstream end cap that urges a frit retainer toward the porous plug. The fluid passage is preferably filled with chromatographic media and the bore between the downstream porous member and the retaining plug are preferably filled with chromatographic media under compression. The column is preferably filled with silica chromatographic media, or polymer chromatographic media, or porous chromatographic media, or superficially porous chromatographic media. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other advantages and features of the invention will be better appreciated in view of the following drawings and descriptions in which like numbers refer to like parts throughout, and in which: 
         FIG. 1  is a cross-sectional view of a chromatography column with a retaining plug in a recess at one end of a packed chromatography bed; 
         FIG. 2  is a cross-sectional view of chromatography column with a T-shaped retaining plug at one end of a packed chromatography bed; 
         FIG. 3  is an enlarged view taken along section  3 - 3  of  FIG. 2 ; 
         FIG. 4  is an enlarged view taken along section  4 - 4  of  FIG. 1 ; 
         FIGS. 5A-5I  are cross sections of retaining plugs showing different shapes of passages through the retaining plug; 
         FIG. 6A  is a sectional view of a chromatography column with a compression end fitting; and 
         FIG. 6B  is an enlarged sectional view taken along  6 B- 6 B of  FIG. 6A . 
     
    
    
     DETAILED DESCRIPTION 
     As used herein, the following parts have the following part numbers:  9 —tubular body;  10 —column;  12 —threads;  14 —first porous member;  16 —second porous member;  18 —retaining plug;  20 —first end fitting;  21 —shoulder;  22 —second end fitting;  23 —media;  24 —media bed;  26 —passage through retaining plug  18 ;  28 —sealing rings;  30 —frit retainer;  40 —inlet portion;  42 —cylindrical portion;  44 —outlet portion;  46 —spherical inlet;  48 —parabolic inlet;  56 —shoulder;  58 —smaller diameter portion;  60 —larger diameter portion;  66 —compression end fitting;  68 —nut;  70 —sidewall;  72 —bottom;  74 A—threads;  76 —ferrule;  77 —conical surface;  78 —fitting body;  80 —gripping surface;  82 —connector passage; and  84 —conical surface. 
     Referring to  FIGS. 1-4 , a chromatography column  10  having a tubular body  9  with a longitudinal axis  11 , and optional external threads  12  on opposing first and second ends of the tubular body  9 . As used herein, the upstream and downstream directions refer to the directions relative to the flow during packing of the column. The flow direction of the packed column  10  during use may differ from the packing flow directions. The relative directions of up and down, upper and lower, above and below, top and bottom, refer to the directions of the parts when the column  10  is in the vertical direction during packing, with the upstream end pointing away from the earth. 
     The packed column  10  has first and second porous members  14 ,  16  at the respective first (upstream) and second (downstream) ends of the tubular body and column. A retaining plug  18  is shown inside the tubular body  9  and fixed to the first end of the tube  9 , with a fluid passage  26  having various possible shapes (as discussed later) extending through the plug  18 . A first end fitting  20  holds the first porous member  14  in place and preferably urges the porous member  14  toward the upstream end of the retaining plug  18 , preferably by engaging threads  12  on body  9  to move that end fitting and porous member toward the fixed retaining plug  18 . A second end fitting  22  engages corresponding threads on the second, downstream end of the tube  9 . The first and second end fittings  20 ,  22  preferably provide fluid connections between the column  10  and various chromatography related instruments, analytical instruments and packing equipment. Appropriate sealing rings  28 , typically O-rings  28 , may optionally be used as needed to prevent fluid leakage, with frit retainers  30  typically held in position inside each end fittings  20 ,  22  to facilitate fluid connections with equipment, such as analytical equipment and possibly packing equipment. The end fittings  20 ,  22  are also typically of metal. 
     The porous members  14 ,  16  are typically metal, glass or ceramic filters or frits that allow liquids and gases to pass but prevent passage of chromatographic particles. The retaining plugs  18 , are typically of two general types, the first of which is shown in  FIGS. 1 and 4 , having a generally circular outer periphery that bounds opposing, flat, top and bottom sides and results in a disk-shaped part. The resulting disk shape fits into a mating cylindrical recess in the upstream end of the tubular body  9  as in  FIGS. 1, 4, 5A-5E and 5G-5I . The second general type is a T-shaped cross-sectional shape ( FIGS. 2, 4 and 5F ) with a larger diameter on top and a smaller diameter on the bottom and results in a stepped—diameter, disk—shaped part with flat shoulder  21  ( FIG. 5F ) joining the two peripheral sides. In use, the resulting stepped—shaped disk fits the smaller, downstream disk into a mating, cylindrical recess in the upstream end of the body  9  with the larger diameter portion of the disk extending over an end of the tubular body  9  or possibly into a stepped-recess (not shown) in the end of the body  9  as in  FIGS. 2 and 3 . One or more corners of the retaining plug  18  may be broken or chamfered to remove sharp corners and ensure a smooth fit with mating recesses and adjoining surfaces. 
     The fluid passage  26  may extend axially through the retaining plug  18  and is in fluid communication with and preferably axially aligned with the axis  11  of bore  9  and column  10 . When packed with media  23  to and through at least part of the passage  26  to form the chromatographic bed  24  below the retaining plug  18 , the column  10  has the retaining plug  18  abutting a slurry-packed media bed  24  so the first porous member  14  and the retaining plug  18  restrain axial expansion of that slurry-packed media bed  24 . Some media from the bed  24  usually extrudes into the fluid passage  26  when the packing pressure is removed and the media in passage  26  also expands when released from the packing pressure. The first porous member  14  is upstream of the retaining plug  18  and restrains expansion of the media  23  in the passage  26  when the member  14  is held in place. The main and predominant portion of the slurry packed media bed  24  is between the retaining plug  18  and the second, downstream porous member  16  so the media bed  24  is held in compression in the column by the downstream porous member  16  and retaining plug  18 . The compression of the bed of media does not include the atmospheric pressure. The fluid passage  26  is also packed with chromatographic media  23  but it is typically not at the same pressure as the packed bed  24 . In some configurations, it is possible that a very short distance of the tubular body  9  located upstream of the top surface of the retaining plug  18  is also filled with chromatographic media, but it will be at the same, lower compaction pressure as the passage  26 . The media particles  23  may be silica or polymer particles, and may be non-porous particles, superficially porous (core-shell) particles, or porous particles, silica gel or modified silica gel, surface modified silica gel, silica gel based chromatographic media, or organic/inorganic hybrid particles. As used herein, porous media refers to media that is totally porous and does not include core-shell media or superficially porous media. 
     The tubular body  9  has a continuous sidewall between the retaining plug  18  and the downstream porous member  14 . Thus, the bore through the body  9  defines the outer periphery of the flow path between the retaining plug  18  and the downstream porous member  14 . Alternately phrased, the column preferably has no lateral fluid paths in fluid communication with the bore through the column  9  at any location between the retaining plug  18  and the downstream porous member  14 . Thus, all chromatographic media forming the packed bed  24  passes through the passage  26  of the retaining plug  18 . When packed, the upstream porous member  14   
     Because void spaces in the chromatographic media are undesirable, it is preferable to have the upstream porous member  14  abut and at least slightly compress the chromatographic media in the passage  26  and any such media upstream of the retaining plug  18 , but preferably without breaking the media and generating fines. Because the pressure that fractures the chromatographic media  23  will vary with the media type and properties of the particular media used, the amount of media between the top of the retaining plug  18  and the bottom of the porous member  14  will vary. Thus, the resulting distance between the top of the retaining plug  18  and the bottom of the upstream porous member  14  may vary if any packing media  23  is located between the upstream surface of the retaining plug  18  and the downstream surface of the upstream porous member  14 . 
     Porous media has a lower compression strength than does superficially porous media, and superficially porous media has a lower compression strength than does non-porous media. The basic material of the media also affects compressive strength and the resulting bed expansion, as for example, polymer media is more compressible than silica media and a compressed bed of polymer media may thus compress more and expand more than a bed of silica media of comparable particle size. The larger the pore size of porous media the lower the compression strength. The thickness of the shell, the pore volume and the size of the pores (created by the particles in the shell of a superficially porous material) also affect the compression strength of superficially porous particles. The packing pressure also varies, with higher pressures being used to pack smaller diameter particles, and used to achieve higher densities in the packed media bed  24 . 
     Advantageously, the top or upstream end of the retaining plug  18  is preferably flush with the top or upstream end of the tubular body  9  of column  10 . The slurry packing process typically provides an excess of chromatographic media  23 . The excess media above the upstream end of the column can be scraped off and a porous plug  14  and end fitting  20  can be fastened to the column  10  and body  9 , or if the upstream end of the porous plug is flush with the end of body  9  the extruded media can be scraped off laterally before the porous member  14  is fastened over the media and passage  26  to restrain further extrusion and loss of pressure on the packed bed. This is usually achieved by screwing the end fitting  20  onto the threads  12  on the body  9  of column  10 , forcing the porous member  14  against the chromatographic media and often slightly compressing the media in the passage  26  and/or above the retaining plug  18 , as the fitting  20  is tightened. It is believed desirable that the distance between the upstream porous member  14  and the top of the retaining plug  18  be as small as possible without generating an unacceptable amount or volume or number of fines when the porous member  14  is moved into position to maintain as much of the packing pressure established by the retaining plug  18  as possible. Sometimes the porous member  14  is sized to fit just inside the bore of the tubular body  9  either as a disc the size of the bore, or as a stepped portion of a two-diameter disk with the smaller diameter fitting inside the bore and the larger diameter abutting a shoulder on the tubular body. 
     Thus, there is preferably no media  23  between the top or upstream porous member  12  and the top of the retaining plug  18  and any media between the upstream and downstream porous members  12 ,  14  is compressed, with the chromatographic bed  24  being held in greater compression by the retaining plug  18  than the media in the passage  26 . The downstream facing surface of the retaining plug restrains the bulk of the chromatographic bed  24  from moving axially upstream, with some media being extruded through the downstream opening of the passage  26  into that passage. It is believed that the media in the passage  26  is compressed between the upstream retaining member  12  and the packed bed  24  at a lower pressure or compression or density than the packed bed  24 . 
     The top surface of the retaining plug  18  may be downstream of the top end of the tubular body  9 , and if so it is preferably within a few millimeters of the top end. Preferably, the top surface is flush with the end of body  9  so removal of media  23  is simplified and achieved by scraping the extruded media off to one side. In either configuration with the retainer plug  18  flush with the top end of body  9  or recessed slightly downstream from that top end of body  9 , the thickness of any layer of media  23  above the retaining plug  18 , if any, is preferably such that tightening the first, upstream end fitting  20  to position the upstream porous member  14  does not crush any media  23  that is above the retaining plug  18  and below the upstream porous member  14  and thus creates no fines. Advantageously, the upstream porous member  14  abuts the top surface of the retaining plug  18 , and optionally, the upstream porous member  14  is configured and located to fit slightly inside the upstream opening of the passage  26 . 
     The passage  26  through the retaining plug  18  is filled with media  23  and the size and shape of that passage may affect the pressure on the media within that passage when the upstream porous member  14  is fastened to the column  10 . It is desirable to crush as little of the particles of media  23  as practical and generate as few fines as possible while maintaining a sufficient pressure on the bed  24  and passage  26  to minimize expansion of the packed bed. It is also believed desirable to have the axial length of the retaining plug  18  be as short as possible. 
     Referring to  FIGS. 5A through 5I , the passage  26  through the retaining plug  18  may have various configurations, with specific configurations referred to as passage  26   a  through  26   i , and the retaining plugs in general referred to a retaining plug  18 . The passage  26  through the retaining plug  18  is preferably wider at its upstream end and narrower at its downstream end so as to funnel the media  23  through the retaining plug  18  without clogging the passage during high pressure packing used to form the packed media bed  24  with no voids and a more densely packed bed than if the plug  18  is not used. The walls of the passage  26  are thus inclined but need not be straight as they are with a conical passage  26   a  ( FIG. 5A ), and may instead be curved (convex or concave) as is passages  26   g  and  FIG. 5G . The minimum cross-sectional area of this passage  26   a  is determined by the area at the smallest diameter of the conical passage  26   a , at the downstream end of the retaining plug  18 . 
     Straight sided walls on the passage  26  are believed preferable from a manufacturing viewpoint because they are easier to make, although curved walls resulting from controlled movement of a spherical grinder are also believed suitable. The walls of the passage  26  are preferably smooth to facilitate passage of the slurry through the passage during high pressure packing, with walls polished to a surface finish of less than RA 8 micro inches believed desirable. 
     It is believed preferable to have a conical passage  26  extending from the upstream surface of the retaining plug  18  to the downstream surface of that plug. The passage  26  is discussed further below, but as an example, a cone  26   a  having an included angle of about 20° is believed suitable, when the retaining plug  18  is about 1.5 to 3 mm long measured along axis  11 , and the column is about 4.6 mm in diameter. The larger, upstream diameter of the conical passage  26   a  is about 3 mm while the smaller, downstream diameter cone is about 1.5 mm when the retaining plug is about 1.5 mm in length measured along axis  11 . For most slurries using particles sizes of 5μ or smaller, a minimum opening diameter for the passage  26  is believed to be about 1.5 mm for diameters of less than about 1 inch (about 25 mm). 
     To make the column  10 , the retaining plug  18  with fluid passage  26  may be formed as a separate part which is then fixed in position in the bore of the tubular body  9 , preferably permanently fixed in position. The retaining plug  18  is preferably of the same material as the tubular body  9 , typically stainless steel but may alternatively comprise a metal which melts at a higher temperature than body  9  to facilitate permanent bonding by melted metal without risking deformation of passage  26 . But the plug  18  may be made of other metals, including titanium, and for some applications may be a polymer, such as PEEK. For metal retaining plugs  18 , a cylinder or disk is typically formed to the desired diameter with a desired outer surface finish and the fluid passage  26  is machined and/or ground through the retaining plug  18  and then polished to a desired smoothness, preferably under RA 8 micro inches. The retaining plug  18  may be machined, drilled and/or ground when the plug is already cut to the desired length or the plug may be cut to length from a longer cylindrical rod after the fluid passage  26  is drilled and/or ground or polished. Other manufacturing sequences to form the fluid passage  26  may be used and other ways to achieve a smooth surface on the wall of the passage may be used other than mechanical polishing, including chemical finishing. If the retaining plug  18  is a polymer, the passage  26  may be molded integrally with formation of the plug, in a single casting or molding operation. 
     Once the retaining plug  18  is formed, it is fixed to the tubular body  9  so that the plug  18  does not move and cannot move from the time the column is packed to form chromatographic bed  24 , until the packed bed  24  is no longer used or needed. The columns  10  are typically stainless steel with polished internal bores extending along the axis  11  of the columns. The retaining plug  18  may be press fit into the upstream end of the column so the retaining plug  18  is held in position by an interference fit. The interference fit also provides a seal between the column and the periphery of the retaining plug  18  sufficient to withstand the operating pressure and packing pressure of the column without leaking, which pressures may be several thousand psi or kpa, as discussed later for silica gel and other particles, or several hundred psi for polymer particles. 
     As an additional step to ensure a fluid tight seal between the retaining plug  18  and the tubular body  9 , a cure-in-place adhesive or sealant may be coated on the outside of the plug  18  before it is press-fit into place. It is believed possible to have the retaining plug  18  held in place in the tubular body  9  using only suitable adhesives (which include epoxies as used herein) which cure in place when the retaining plug  18  is positioned in place in the tubular body  9 . Any fastening mechanisms using adhesives require a suitable strong and durable adhesive to last the life of the column, and one compatible with the intended use of the column. The adhesive may be used with any of the mechanical fastening mechanisms to provide an additional barrier against leakage during packing or use and/or to provide an additional fastening mechanism against movement. 
     The retaining plug  18  may also be welded or brazed into place, including friction welding. The contact length of the welded or brazed metal along the axis  11  is preferably the full length of the retaining plug  18 , but may be less, although preferably not less than 0.005 inches (0.127 mm). It is believed possible, but less desirable to have internal or external threads on the tubular body  9  mate with correspondingly located threads on the retaining plug  18 , preferably with at least two full threads engaged and more preferably with the entire exterior side of the retaining plug threaded and engaged with corresponding threads on the column. The threaded connection, as with the other types of connections, must be configured to accommodate the packing pressures and uses pressures without reducing the life of the column, e.g., through fatigue failure. If threaded connections are used the connections preferably include interference threads to lock the plug  18  relative to the body  9 , or the exterior end of the plug may be deformed (e.g., upset or staked) to fix the parts relative to each other. The end of column  9  is preferably flat relative to axis  11 , and as needed the end may be ground or otherwise finished as needed to function in the chromatography applications. It is also believed suitable to use adhesives to further ensure the leakproof seal of any threaded connection, or even and to position and fix the plug relative to the column. 
     The tubular body  9  may be configured to position the retaining plug  18  at a predetermined location along the axis  11  of the column by forming a position stop  56  ( FIG. 3 ) in the tubular body  9 . Similarly, the tubular body  9  may be configured to position the downstream porous member  16  at a predetermined location along the axis  11  in order to determine the length of the packed chromatography bed between the downstream porous member  16  and the retaining plug  18 . As seen in  FIGS. 3-4 , this position stop is preferably formed as an annular, stepped recess in one or both opposing ends of the column to form a shoulder  56  or an axial stop which the downstream end of the retaining plug  18  abuts to position the retaining plug and define the length of the packed bed  24 . The position stop  56  for the porous member  18  is thus preferably formed by drilling or reaming an annular shoulder coincident with the bore through body  9 . The same type of annular shoulder or axial stop be formed for the downstream porous member  28  or the porous member may have an outer periphery held against the end of the tubular body  9  or otherwise held in position by frit retainer  30 . 
     Referring to  FIGS. 4 and 5F , the retaining plug  18  may have a stepped cross-section having a smaller diameter portion  58  that fits into the bore of the body  9  and column  10 , and a larger diameter portion  60  that extends over at least part of the annular end of the tubular body  9 , to form a hat-shaped cross section or a T-shaped cross-section. The larger diameter portion  60  forms a flange or shoulder  21  that may rest against the end of the body  9  or a shoulder of the body  9  to limit movement along axis  11  toward the downstream end. A retaining mechanism, such as endcap  20  and frit retainer  30 , prevents the upstream porous member  14  from moving in the upstream direction along axis  11 . The downstream porous member  16  is similarly held in position inside the bore of the body  9 , or pressed against the downstream end of the body  9 , by using a retaining mechanism such as the endcap  20  and frit retainer  30 . 
     Depending on the way the retaining plug  18  is fastened to the tubular body  9 , the downstream porous member  16  may need to be installed before the retaining plug  18 , or after the plug  18 . Typically, the bore is formed in the tubular body  9  and then each opposing end of the body is machined (e.g., drilled, bored, end milled) to form a shoulder  56 . The retaining plug  18  is inserted to rest against the upstream shoulder  56  and affix the retaining plug to the body  9 , preferably by a press fit or as otherwise described herein. Usually, once the body  9  has the retaining plug  18  fixed to the boy then the downstream porous member  20  is fixed to the body. A downstream porous member  16  is inserted from the downstream end of the body  9  so it rests against the downstream shoulder and the downstream frit retainer  30  and downstream end cap  20  are attached to hold the downstream porous member in place. Any cleaning arising from fastening the retaining plug  18  to the body  9  is performed before the downstream porous member is connected to the body. 
     The empty tubular body  9  before packing has the column  10  with downstream porous member  16  and retaining plug  18  defining an empty space in the bore of the tubular body  9  of predetermined length where the packed chromatographic bed will be formed between the plug  18  and the downstream porous member  28 . For packing, the end fitting  22  and is connected to the tubular body  9  in order to form the fluid connections with the column, typically using threads  12  on the outside ends of the column. The upstream connection may be formed using end fitting  20 , but typically a separate fluid packing connection is connected to the upstream end of the column, typically using threads  12  on the upstream end of body  9 . A slurry of solvent and packing media  23  is forced through the passage  26  of retaining plug  18  with the downstream porous member  16  retaining the media  23  while allowing the solvent to pass through the downstream porous member and out the fluid connections of the downstream end fitting  22 . The packing pressure and flow of slurry is preferably maintained until a predetermined amount of media is provided to the column  10 . Typically, more media  23  is provided than is needed to fill the volume between the retaining plug  18  and downstream porous member  16  and form the desired packed chromatography bed  24  between the porous member  16  and plug  18 . The excess media fills the passage  26  and may optionally extend above the retaining plug  18 , depending on the packing process. The flow of solvent may continue after the flow of slurry containing media  23  is stopped. After the solvent flow is stopped and the slurry packing connection is removed, the body  9  will typically have some of the media extruded above the end of the tubular body  9 . That excess media is scraped off and the upstream end fitting  20  and the upstream porous member  14  is fastened to the body portion  9 , usually by threads on the end fitting mating with the threads  12  on the body portion, thus forming a completed column  10  suitable for chromatographic use. Depending on the column design, the end fitting  20  is tightened to place the upstream porous member  14  against the top surface of the retaining plug  18 , or slightly inside the bore of the tubular body, or, at or slightly inside the upstream opening of the passage  26 . 
     When the flow of packing slurry and solvent are stopped, the pressure on the packed chromatography bed  24  also stops and the bed will expand. The downstream end of the retaining plug  18  surrounding and defining the downstream opening of the passage  26  opposes that bed expansion. The part of the retaining plug surrounding the downstream opening of passage  26  and abutting the packed bed of chromatographic media is believed to stop all or substantially all the axial expansion of the packed chromatographic bed axially beneath that portion of the retaining plug abutting the media. Thus, the downstream opening of retaining plug  26  is preferably as small as possible while still allowing a large enough opening for good packing density and fast packing. Some media will usually extrude into the passage  26  through the downstream opening of passage  26 , but that most that extruded media is believed to come from the area axially below the downstream opening, and perhaps the adjacent 10%-15% of the radial distance outward of the downstream opening in the retaining plug  18  which abuts the packed bed  24 . For columns with bores of about 13 mm or less, with downstream openings having a diameter that is 10-35% of the bore diameter, substantially all (e.g., over 80%) of the media particles  23  extruded into the passage  26  are believed to come from the portion of the packed bed axially below the downstream opening of the passage  26  and the and the area surrounding that downstream opening and immediately below that opening. It is believed that a downstream opening of about 20% of the bore diameter is suitable for use and may maintain a packed bed  24 , and that a downstream opening of between about 10% to 95% of the bore diameter is believed to offer advantages in the performance of the packed column  10 . 
     It is believed that because the internal cylindrical wall of tubular body  9  is so smooth, the packed bed  24  will slide along the wall of the column and expand against or push against the downstream side of the retaining plug  18 . Any voids arising from the packing process are believed to exist, if at all, in the corners of the plug  18  by the cylindrical bore or wall of the tubular body  9  and the expansion of the packed bed  24  is believed to fill any such void volumes as may arise during packing. The resulting packed column is believed to be substantially free of voids between the upstream and downstream porous members  14 ,  16 , and to be under compression between those porous members as well as being under greater compression between the bottom or downstream end of the retaining plug  18  and the downstream porous member  16  and the. The retaining plug  18  is believed to maintain the packing pressure and resulting compression of the media bed  24 —but the amount of compression retained depends on the size of the downstream opening of passage  26  and the diameter of the column, among other factors. The upstream porous member  14  is believed to restrain the expansion of the media in the passage  26  and any media above the retaining plug. The media in the passage  26  reaches atmospheric pressure upon removal of the fluid packing apparatus and the pressure in the passage  26  depends greatly on how fast the porous member  14  and end cap  20  are connected, as the expansion of the packed bed  24  into the passage  26  causes the media to push against the connected end fitting  20  and porous member  14  which resists expansion so as to determine the packing pressure in the passage. 
     The compression of the packed bed  24  is also reflected by the density of the packed bed. Preliminary testing on five columns showed improved performance. Five 150×4.6 mm columns were packed with 1.8μ porous media, packed using a retainer plug  18  with an included angle of about 20° for the conical passage  26   a  having an upstream opening of about 3 mm and a downstream opening of about 1.5 mm and an axial length of about 1.5-3 mm. That results in a downstream opening with a diameter of about 21% the bore diameter of the column  9 . These columns were compared to five columns packed without the plug  18 . Both types of columns were packed at 11 kpsi using the same slurry packing method. A comparison of ten columns packed using the same packing methods as used for a commercially sold column using porous material shows that the five columns using the retention plug  18 , had an average density increase of 3.5% in the density of the packed bed  24  compared to five columns without the retaining plug  18 . In addition to increased bed density, the columns packed with retaining plug  18  had noticeably improved peak asymmetry and increased efficiency. 
     Additionally, columns packed with the retaining plug  18  are believed to have noticeably improved stability performance over the same dimensioned, comparable column packed using the same media and slurry packing method. The columns for the stability test were packed at 10,500 psi pressure using the above described columns, media and in the case of one test column, the retainer plug  18 . The test used 100 mM Sodium Phosphate pH 6.8 and 90/10 v/v 100 mM Sodium Phosphate pH 6.8/Isopropanol, at two different flow rates, 0.35 mL/min and 0.48 mL/min. All columns used the same sequence, which began and ended with the 100 mM Sodium Phosphate pH 6.8 at 0.35 mL/min for 20 minutes, with 441 minutes of running with 90/10 v/v 100 mM Sodium Phosphate pH 6.8/Isopropanol at the different flow rates followed by a 10 minute method with 100 mM Sodium Phosphate pH 6.8 with a linear ramp of flow from 0.48 to 0.35 mL/min, and a 10 minute flush of the column with 20/80 v/v Methanol/Water following the end of the sequence, followed by a removal of flow for eight hours. 24 injections were made at varying points during this sequence. The sequence of tests was repeated 12 times for a total column time of 105 hours. The sample was 2.50 mg/mL Thyroglobulin, 5.00 mg/ml: Immuno-Globulin G, 2.50 mg/mL Ovalbumin, 1.25 mg/mL Myoglobin, 11 mg/mL Uridine dissolved in Water, with an injection volume of 0.7 μL. The efficiency of the Uridine peak was tracked throughout as was the backpressure. Columns were considered to have failed the test after a loss of more than 25% of the original efficiency of this peak. The standard column failed after 20 column hours while the column with the retaining plug  18  lasted approximately 90 hours. 
     It is also believed that the particle packing media  23  interlocks when it forms the packed bed  24  and that the lateral interlocking of axially compressed particles cause the bed to move as a single unit so that restraining the outer periphery of the packed bed  24  from axial expansion around the smooth walls or bore through the column helps to restrain the entire bed if the retaining plug  18  extends far enough inward from the wall or bore of the column. It is believed that the smaller the size of the downstream opening of the passage  26 , the greater the interlocking and the greater the resistance to axial expansion of the packed chromatography bed  24 . The packed bed  24  may dome upstream where the passage  26  is located so that part of the packed bed  24  is extruded along axis  11  and into the passage  26 , but a predominant part of the entire packed bed  24  remains compressed and does not extrude itself through the passage  26 . 
     The rate over time at which media  23  from packed bed  24  is extruded into the passage  26  decreases quickly, but may continue at a decreasing rate depending on the slurry thickness, the size of media particles  23 , the compaction pressure of bed  24  and the slurry packing pressure, the diameter of the packed bed  24 , and the size of the opening of the passage  26  abutting the packed chromatographic bed  24 . 
     It is advantageous to stop any extrusion of media particles  23  from the packed chromatographic bed  24  into the passage  26  as soon as practical. Stopping this extrusion is achieved by the upstream porous member  14  (e.g., upstream frit) and whatever mechanism is used to hold that upstream porous member in place. The retaining plug  18  retains the bed from gross expansion and the upstream porous member is used to stop any gradual extrusion of the particles  23  of packed bed  24  through the passage  26  in that retaining plug  18 . 
     After the upstream end fitting  20  is removed (if present) and the connections for fluid packing are removed, and any extruded media is scraped off or otherwise removed, the upstream porous member  14  is then placed over the column to stop axial movement of the packing  23  extending into the passage  26  and thus limit and stop any gradual bed expansion arising from extrusion of particles  23  into passage  26 . The porous member  14  is advantageously held in position by upstream end fitting  22 , which is tightened through threads mating with column threads  12  (or other tightening and fastening mechanisms) to push the upstream porous member  14  against the media  23  above the retaining plug  18  and stop and/or limit expansion of the media  23  upstream of the retaining plug  18  and downstream of the upstream porous member  14 . In short, the end fitting  20  pushes the porous member  14  against the media  23  in and above the porous plug  18  to stop extrusion of the packed bed  24  and media  23  through the passage  26 . As the porous plug  18  stops the great majority of bed expansion and as the upstream porous member  14  stops gradual reduction of the bed expansion, the parts cooperate to fix the bed compression and to stop bed expansion. The retaining plug  18  locks-in a portion, and preferably a substantial portion, of the compression that the compressed chromatography bed  24  undergoes during fluid packing. The downstream opening in plug  18  allows the locked-in compression to be gradually released but to an extent that depends on the configuration of plug  18  and passage  26 . The upstream porous member  14  stops the bed from extruding into passage  26  to stop and limit the loss of bed compression through passage  26 , and is believed to result in a compressed bed  24  having a packing density substantially greater than the packing density in the passage  26 . 
     As noted above, the packed chromatography bed  24  is believed to slide along the smooth bore of the chromatography column so that the retaining plug  18  need not necessarily extend across the entire upstream end of the bed and restrain all or almost all of the bed compression from being reduced by bed extrusion through the passage  26 . The amount of peripheral restraint by the bottom surface of the restraining plug  18  is related to the size of the opening of passage  26  at the downstream end of the restraining plug  18 . As noted above the size of the opening for passage  26  depends on numerous factors, including slurry and solvent composition, particles size, particle type, particle interlock, bore diameter and packing pressure, among others. The passage  26  must funnel or guide the slurry through the passage without clogging yet allow enough axial restraint by the remainder of the restraining plug  18  to resist axial bed expansion as much as possible. 
     It is believed that the size of the downstream opening of passage  26  is preferably between about ⅕ and ⅓ the diameter of the bore in tubular body  9  or the outer diameter of the retaining plug  18 . Larger diameters allow faster filling and less clogging of the slurry but provide less restraint against axial bed expansion and provide a larger opening for potential extrusion of particles  23  which reduces the bed compression. It is nonetheless believed that the opening on the downstream end of the passage  26  may comprise as much as 95% of the bore diameter in the tubular body  9  during use, but that is not as preferred as it is believed to result in an undesirable amount of extrusion into the passage  26  and accompanying bed expansion. It is believed more preferable to have the size of the downstream end of the passage  26  be about 0.5 to 0.7 times the diameter of the bore through the column. More preferably, it is believed desirable to have the size of the downstream end of the passage  26  be about 0.3 to 0.5 time the diameter of the bore through the tubular body  9 . And ideally, it is believed that a diameter of the opening in the downstream passage of about 0.2 to 0.35 the diameter of the column is preferred. Smaller diameters are believed suitable but are subject to risk of clogging with thicker slurries or larger particles of media  23  when the column bore diameters are under 5 mm. For large columns  11  with bores over 1 inch (25.4 mm) in diameter, a passage  26  with an opening diameter of about 13 mm or larger are believed suitable to prevent clogging but may be too small to ensure uniform packing of the bed across the bore diameter. The slurry containing the chromatographic media particles  23  passes through these various sized openings under a packing pressure selected to form the packed chromatography bed  24 . 
     In one preferred embodiment using porous media particles  23  about 2 micron in diameter in a column having a bore diameter of about 5 mm, the upstream inlet opening of passage  26  was about 2 to 2.5 times larger than the diameter of the downstream, outlet opening of the passage, with the respective diameters being about 3 to 1.5 mm. It is believed desirable to have the upstream, inlet opening of passage  26  be about 2-5 times the diameter of the downstream, outlet opening of passage  26 , for retaining plugs  18  that are less than 10 mm thick along axis  11 . 
     Referring to  FIGS. 1-4 , the position of the retaining plug  18  in the body  9  of the column  10  as measured along the axis  11 , is preferably known and consistent to allow columns with known lengths of media beds  24  to be made. The effective length of such columns is typically determined by the distance of the packed bed of chromatographic media  24 , which corresponds to the distance between the downstream surface of the retaining plug  18  and the upstream surface of the downstream porous member  16 . The porous members  14 ,  16  are typically flat discs. The downstream end of the retaining plug  18  is preferably flat, but may have other shapes as shown in  FIGS. 5A-5I and 6 , and as described herein. 
     The inclination angle of the upstream portion of passage  26  was partially discussed above in terms of avoiding damage to the media particles  23  in the passage and above the retaining plug  18  during fluid packing. The optimum inclination angle for a conical passage  26  will vary with the media type, media size, slurry (thick or thin), solvent, downstream opening size of passage  26 , packing pressure and the thickness of the plug  18  along the axis  11 . An included angle for conical passage  26  of about 10-120 degrees is believed suitable, with angles of about 15-40 degrees believed to be more desirable, and an angle of about 20 degrees believed preferable, at least for a bore about 1-5 mm in diameter, about 2-4 mm in length, and using porous media particles  23  about 2 microns in diameter. 
     It is believed that the passage  26  may have suitable shapes other than the conical shape that extends through the entire axial thickness of the retaining plug  18  and passage  26   a  of  FIG. 5A . Various shapes believed suitable for passage  26  are shown in  FIGS. 5A through 5I  and as described herein.  FIG. 5B  shows a passage  26   b  having a converging (in a downstream direction), conical upper portion or inlet  40  in fluid communication with and joining to a cylindrical downstream portion  42  which preferably opens onto a downstream side of the retaining plug  18 . The minimum cross-sectional area of this passage  26   b  is determined by the cylindrical downstream portion  42 . 
       FIG. 5C  shows a retaining plug  18  having a fluid passage  26   c  with a converging, conical inlet  40  having a very small angle of inclination relative to the axis  11  through the retaining plug, with a downstream cylindrical portion  42  having an axial length larger than the axial length of the conical inlet  40  and exceeding a majority of the axial length of the plug  18  along axis  11 . An axial length of the conical inlet  40  of about 20-45% of the axial length of the cylindrical portion  42  is believed suitable, with a minimal angle of inclination of about 5-10° believed suitable on the conical inlet portion  40 . 
       FIG. 5D  shows a retaining plug  18  having a fluid passage  26   d  with a converging conical inlet  40  having a very large diameter and a very large angle of inclination relative to the axis  11  through the retaining plug, about 140°-170°. A downstream cylindrical portion  42  has an axial length larger than the axial length of the conical inlet  40 . In the depicted embodiment, the inlet portion  40  is shallow with an axial length about ⅕ that of the outlet portion  42 . The relative axial lengths may vary. The minimum cross-sectional area of this passage  26   e  is determined by the cylindrical downstream portion  42 . 
       FIG. 5E  shows a retaining plug  18  having a fluid passage  26   e  with a cylindrical portion  42  extending entirely through the plug, from the inlet to the outlet. This embodiment is believed suitable to restrain expansion of the packed chromatography bed  24  but is believed to require lower packing pressures and flow rates when the media  23  is more fragile or the packing pressure is high because the lack of inclined sides on the upstream entry to the passage  26   e  is believed to damage the media and generate fines under high flow rates and high packing pressures. 
     A cylindrical upstream portion as shown in  FIG. 5E  is believed suitable to combine with any of the diverging downstream openings described herein and especially with the conical downstream opening  44  shown in  FIG. 5I  described later. It is also believed suitable to use a concave opening, such as a spherical shape, or the modified spherical shape of surface  46  ( FIG. 5G ) having rounded interior corners encircling a flat bottom, or the parabolic shape of surface  48  ( FIG. 5H ) as a downstream portion of the passage  26   e . Thus, it is believed possible to use the restriction plugs shown in  FIGS. 5B-5D and 5F to 5H  in an inverted orientation with the cylindrical portion of the passage forming the upstream end of the passage. Thus, while the upstream opening of passage  26  is preferably larger than the downstream opening, it is believed that the upstream opening of passage  26  may be of smaller diameter than the diameter of the downstream opening, but less preferable for the reasons mentioned herein. 
       FIG. 5F  shows a T-shaped or hat-shaped retaining plug  18  having a fluid passage  26   f  with a converging, conical inlet  40  having a relatively large angle of inclination relative to the axis  11  through the retaining plug. The downstream cylindrical portion  42  has an axial length much smaller than the axial length of the conical inlet  40 . An axial length of the conical inlet of up to about 80-90% of the axial length of the thickness of the retaining plug along axis  11  is believed suitable. The retaining plug  18  has a larger diameter top portion  60  and a smaller diameter downstream portion or bottom portion  58 , forming shoulder or flange  21 . The shoulder  21  rests on the top of the tube  9 , or on an internal shoulder in the tube  9  (not shown) during use. The shoulder  21  and resulting larger diameter of the retaining plug provide additional options for fastening the retaining plug  18  to the column  10 . The minimum cross-sectional area of this passage  26   f  is determined by the cylindrical downstream portion  42 . 
       FIG. 5G  shows a retaining plug  18  having a concave inlet portion  46  with curved downstream corners, preferably circular corners, and more preferably with the inlet portion  46  being spherical and intersecting a cylindrical downstream portion  42 . Thus, a cross-section of the passage  26   g , has an inlet portion with rounded corners or a spherical inlet portion  46 . The minimum cross-sectional area of this passage  26   g  is determined by the cross-sectional area of the cylindrical downstream portion  42 . 
       FIG. 5H  shows a retaining plug  18  having a concave recess that advantageously forms a parabolic shaped inlet portion  48  with a cylindrical downstream portion  42  in the fluid passage  26   h . Thus, the cross-section of the passage  26   h  forms a parabola shape in the inlet  48 . As with the other variations of the passage  26 , the relative length of the upstream and downstream portions along axis  11  may vary. 
     Referring to  FIG. 5I , the insert plug  18  has passage  26   i  having a converging conical inlet passage  40 , a downstream (and intermediate) cylindrical passage  42 , and a further downstream, diverging conical outlet portion  44 . The minimum cross-sectional area of this passage  26   i  is determined by the cross-sectional area of the cylindrical passage  42 . The surface finish on the diverging conical portion  44  is preferably rougher than the surface finish on the converging conical inlet passage  40  and the intermediate portion  42 , and may be rougher by a factor of at least 10 but preferably not so rough as to create turbulent flow during fluid packing and not so rough as to damage the abutting surfaces of media particles  23  and generate fines. The increased surface roughness on the portion of passage  26  facing the packed bed  24  is believed to help reduce the expansion of the media bed  24  through the intermediate portion  42  while the smoother surface on the portion of the passage  26  facing upstream is believed to facilitate the slurry flow through the passage  26  and into the tubular body  9  to form the packed bed  24 . 
     The converging conical portion  40  is configured much as described above except extending a shorter distance along axis  11 . The intermediate cylindrical portion  42  is believed to help avoid any sharp edges that may damage media particles  23  as they pass through the retaining plug  38 . The diverging conical portion  44  is believed to help disperse the slurry of media and achieve better packing of columns, especially columns with a bore diameters that are large. The use of multiple passages  26  as discussed later may help alleviate packing issues arising from a single passage or from the shape of the passage  26 . The diverging conical portion  44  is also believed to reduce expansion of the packed bed  24  after the fluid packing pressure is stopped. The inclined surface of the downstream conical portion  44  is believed to offer resistance to upstream movement along axis  11  while directing particles inward to increase the interlocking of the media particles  23  and clog the conical portion  44 . It is important that the downstream conical portion  44  be unmoving in the axial direction to minimize expansion of the packed bed  24  when the packing fluid pressure is stopped. It is also important that the retaining plug  18  be unobstructed and open to flow of the slurry and packing media  23  for packing to avoid clogging the passage  26  during packing. 
     The relative dimensions of each conical portion  40 ,  44  vary with the same parameters discussed above regarding conical passage  26 . The diameter of the intermediate portion  42  is believed to depend on the same factors as the downstream opening of passage  26 . The diameter of the intermediate portion  42  is preferably about the same size as the downstream opening in passage  26 , although it is believed that the intermediate portion  42  may slightly smaller (5-20% smaller in diameter) than the downstream opening in passage  26  for the same column. The maximum diameter of the downstream conical portion  44  is preferably smaller than the maximum diameter of the upstream conical portion  42 . It is believed that the inclination angle of the downstream, conical portion  44  should be greater than the inclination angle of the upstream conical portion  42  so as the bed expansion pushes media particles  23  inward toward axis  11  more than it does axially along axis  11 , thus increasing particle interlock and clogging the passage  36 . In the depicted embodiment, the upstream conical portion  40  has opposing walls inclined at an included angle of about 50° while the downstream conical portion  44  has opposing walls inclined at an inclined angle of about 80°. When the downstream walls are inclined at an over 90 degrees included angle, then the particles move inward toward axis  11  more than they move axially along axis  11 , and that increased lateral movement may increase the particle interlock and clogging of the passage  26 . Thus, it is believed advantageous to have the included angle of any downstream portion of the passage  26  being 90° or greater than 90°. As the included angle approaches 180° the inward movement toward axis  11  decreases. Inclined angles of about 45°—80° relative to axis  11  for conical walls forming the downstream portion of passage  26  are believed suitable, and this corresponds to an included angle of about 90°-160°. 
     The concept of using a restraining plug  18  to lock the bed in a compressed, axial position and restrain axial expansion of the bed is believed applicable to columns  10  that vary in diameter from a small bore a fraction of an inch in diameter (e.g., 0.040 inches or about 1 mm) to columns that are several inches in diameter (e.g., 4 inches or about 100 mm). The thickness of the retaining plug  18  along axis  11  is believed suitable to vary from about 0.004 inches (for smaller diameter bores of a few mm) to about 0.7 inches (for larger, 4-inch diameter bores). Columns with an internal diameter smaller than 1 mm are believed unsuitable with this method and apparatus. For retaining plugs  18  having the passage  26   i , it is believed suitable to have the plug  38  about 0.004 inches thick along axis  11 , with the upstream diameter of the upstream cone  40  about the same as the bore diameter of the column, although it is believed preferable that the cone diameter be slightly smaller than the bore diameter so for a 0.005 inch bore a cone opening of 0.0045 is believed preferable. The downstream opening of cone  44  could have the same dimensions as the upstream cone  40 . On the other extreme, for bore of about 4 inches in tubular body  9 , a retaining plug with passage  26   i  with a maximum diameter of about four inches on the upstream conical portion  44  for a four inch bore in the tubular body  9  is believed suitable, with a maximum diameter of about 3.5 inches for the intermediate cylindrical portion  42 , with the diameter of the downstream conical portion  44  being the same as the upstream portion. 
     In all the variations of retaining plug  18 , the passage  26  and especially the upstream portion of passage  26  is filled with media particles  23  during the packing process and that media is restrained from further expansion by the upper porous member  14 . Thus, the retaining plug  18  is believed to retain the majority of the expansion of bed  24  while the porous member  14  retains expansion of media  23  in passage  2 , which in turn is believed to limit expansion of bed  24  by blocking expansion through the downstream opening of passage  26 . 
     The depicted first end fitting  20  of  FIG. 1  is threaded onto the end of the column body  9  to urge the frit retainer  18  against the end of the column or body  9  or the retaining plug  18  against the annular shoulder  56  or annular end of the body  9  sufficiently to form a fluid tight seal suitable for use. As shown in  FIGS. 6A-6B , a compression end fitting may also be used. It is also believed suitable for either end fitting  20 ,  22  to comprise a compression end fitting  66 , or any other end fitting. The compression end fitting  66  has a hollow, annular nut  68  with a sidewall  70  (preferably cylindrical). One end of the nut  68  is open and the other end or bottom  72  has a centrally located hole that is slightly larger than the outer periphery of the body  9 . The chromatography column body  9  is preferably cylindrical and the hole in the bottom  72  is preferably circular. The sidewall  70  is threaded and the figures show the inward facing side of the sidewall  70  as having threads  74 A, but the outer side could be threaded. 
     A ferrule  76  is placed inside the collar  68  and rests on the bottom  72 . The ferrule  76  has a central hole so it can slide over the outer periphery of the body  9 . The ferrule has a conical shaped outer surface  77  that tapers inward toward the longitudinal axis of the body  9 , and thus has a triangular cross section with the base at or adjacent bottom  72  and the tip of the triangle extending toward the open end of the collar  68 . A fitting body  78  has a gripping surface  80  at a first end and a threaded surface  74 B at its second end with the threaded surface sized so threads  74 B threadingly engage threads  74 A during use. A cylindrical cavity in the second end of the fitting body  78  receives the mating end of the body  9  that passes through the hole in bottom  72  and passes through ferrule  72 . A threaded inlet and associated passage  82  extend along a central axis of the fitting body  78  to connect to the liquid chromatography equipment. A conical end  84  is formed on the second end of the fitting body and shaped to receive the conical outer surface  77  of the ferrule  76 . 
     The nut  68  and fitting body  78  are rotated relative to each other causing mating threads  74 A,  74   b  to move the nut and fitting body toward each other. As the threads  74 A,  74 B rotate relative to each other, the mating conical surfaces  84 ,  77  on the fitting body and on the ferrule, compress the ferrule  72  against the body  9  to grip the body  9  and fix the nut  68  in position along the length of the body  9 . As the threads  74 A,  75 B rotate relative to each other, the top of the cavity in the fitting body  78  contacts the first porous member  14  and urges the first porous member  14  against the retaining plug  18  and toward the end of the body  9  to form a fluid tight seal sufficient for use of the chromatography column. The ferrule  72  is preferably of a suitable plastic, polymer or elastomer. The compression connection uses mating threads to move two inclined surfaces relative to each other along an axis and thus to clamp one inclined surface to a column. The general operation of a compression connection is believed known, but the adaptation for use to form a seal using the porous member  14  and the retaining plug  18  is believed to be new. 
     A similar construction is provided on the compression fitting at the second end of the column and body  9  and the description is not repeated. The second compression fitting, however, does not have a plug  18  and thus the fitting body  78  urges the second porous member  16  against the end of the body  9  to form the fluid tight seal sufficient for chromatographic use. 
     The method and apparatus disclosed herein are believed especially suited for use with HPLC columns and for UHPLC columns (ultra high pressure liquid chromatography columns), which each require metal bodies. HPLC media beds  24  typically use media particles of above 2μ (up to 20μ or more depending on the application), and are typically packed at pressures from 50 to about 10,000 or 12,000 psi. UHPLC media beds typically use media particles of 2μ or smaller and are typically packed from about 10-10,000 psi to about 25,000 psi. The retaining plug  18  is preferably constructed so that at these normal packing pressures the retaining plug  18  does not dome upwards. With smaller diameter columns  10  this doming is usually not an issue but when the column bores become larger as with 3-4 inch diameter columns, any doming of the plug  18  can noticeably affect the density of the packed bed  24 . Thus, the retainer member  18  is preferably constructed stiff enough to avoid doming and fastened to the column  10  in a way to avoid doming, as well as to avoid axial movement of the retaining plug  18  that would reduce the packing pressure and reduce the bed density. 
     Locating the passage  26  in the center of the retaining plug helps relieve the maximum doming force on the retaining plug  18 , and that also allows the retaining plugs to be maintained thinner in the axial direction than would be the case if the passage  26  were not present. Further, the laterally inward direction in which the downstream end of the retaining plug  18  may direct the media particles  28  and the increased interlocking of the media particles in the media bed  24  are also believed to help reduce the strength needed for the retaining plug  18  and thus to also reduce the axial thickness of that retaining plug. 
     The retaining plug  18  and the associated methods and apparatus described herein are especially useful for HPLC and UHPLC, and to accommodate the high pressures the body  9 , plug  18 , frit retainer  30  and end caps  20 ,  22  are preferably made of metal and more preferably made of stainless steel. The materials used will be suitable for the pressures and forces involved and the required connections. Thus, for example, a retaining plug permanently fastened to body  9  will be of suitable material for such permanent fastening and will withstand the desired packing pressures and operating pressures, including pressures exerted by the chromatographic bed  24  on the bottom of the plug  18  and any forces from the upstream porous member  14 . For lower pressure applications, suitable plastics may be used. Thus, the method and apparatus described herein are also believed suitable for use with chromatography columns using plastic bodies, including solid phase extraction (SPE). The packing pressures believed most suitable for the method and apparatus described herein are believed to be above 100 psi and preferably several hundred psi for polymer particles, and more preferably from about 5,000 psi to 30,000 psi for silica and non-polymer particles, using current technology. The method and apparatus are believed suitable under even higher pressures. 
     The above method and apparatus use a retaining plug  18  permanently fixed to the body  9 , with the upstream porous member  14  abutting the plug  18  and/or media particles  23  in the passage  26  and plug  18  and being added after slurry packing is completed. It is believed possible, but less preferable, that the retaining plug  18  need not be permanently fixed to the body  9  but can be stable during packing and allowed to move slightly downstream thereafter so that the retaining plug  18  and adjoining upstream porous member  14  can be moved downstream so the plug  18  further compresses the packed chromatographic bed  24  while the porous member  14  maintains the media in the passage  26  in compression to limit further media extrusion into the passage  26  during the movement of the retaining plug  18  after slurry packing. This post-packing can be achieved, for example, by using a retaining plug  18  having threads engaging mating threads on the body  9  so that rotation of the threaded retaining plug  18  moves the plug to further compress the packed bed  24 . Prongs on the upstream end fitting  20  may pass through the upstream porous member  14  and engage recesses or peripheral shoulders on the retaining plug  18  to rotate the plug  18 . Alternatively, the porous member may be press-fit into the bore of body  9  with sufficient force to maintain its axial position during packing, with the upstream end fitting  20  mating with threads on the column  10  or body  9  to urge the upstream frit retainer  30  against the retaining plug directly or through the upstream porous member  14 , with sufficient force to move the retaining plug  18  downstream to compress the packed bed of chromatographic media  24 . The axial movement of the retaining plug  18  and porous member  14  needed to compensate for the loss of bed compression caused by extrusion of media particles into and/or through the passage  26 , is believed to be small, and measured in a fraction of a mm for columns less than about 12 mm in bore diameter and a few mm for larger, four-inch diameter columns. 
     The above description uses a single passage  26  through the retaining plug  18 . It is believed suitable, and for larger diameter columns may be preferable, to use more than one passage  26 . For small diameter bores in columns  10 , it is impractical to use multiple passages  26  and not needed for achieving a uniformly packed chromatographic bed. For larger diameter columns, the use of multiple passages  26  is believed to increase the interlocking of media particles  23  in the packed bed  24 , is believed to increase the retention of the bed compression and reduce the extrusion of media into passage  26  after the slurry packing flow is stopped. If multiple passages are used, it is believed advantageous to have the passages symmetrically located about longitudinal axis  11  and the shapes described herein are believed suitable. Thus, two passages  26  would be on diametrically opposing sides of the body  9  with passage centerlines the same radial distance from axis  11  and spaced 180° apart, while three passages  26  would be spaced 120° apart and located the same radial distance from axis  11 . Four passages  26  could be spaced either 90° apart at the same distance from axis  11 , or could have a center passage  26  centered on axis  11  with three, equally spaced outer passages  26 . Retaining members  18  with larger numbers of passages  26  may have them in single rings or in concentric rings about axis  11 , with or without a central passage on the axis  11 . 
     If multiple passages  26  are used, it is believed that the retaining plug  18  may cover the entire bore of the body  9  and column  10 , with each passage  26  having an overall smaller diameter portion in the passage than is possible than if a single passage  26  were used. If multiple passages are used the minimum diameter of the passage  26  must be large enough to prevent clogging. For 1.6μ diameter porous particles in a 4.5 mm diameter bore, a downstream opening in conical passage  26   a  of about 1.5 mm in diameter is believed suitable, while an opening diameter of 1 mm is believed subject to clogging. The 1.5 mm minimum opening diameter comprises about 9% of the cross-sectional area of the bore for the column  10  with a 4.5 mm diameter bore. It is believed that the minimum cross-sectional area of each of the multiple passages  26 , when combined may be about 9-20% of the cross-sectional area of the bore of the column  10  and body  9 , and preferable about 10-15% of that cross-sectional area, and more preferably about 12% of that cross-sectional area—for bore diameters up to at least 13 mm. 
     As the bore diameter of the column  10  increases, it is believed useful to use multiple passages  26  for fluid packing of the chromatographic bed  24  in order to more uniformly pack the bed and to create the packed bed faster. A number of small diameter passages  26  is believed to result in a more uniformly packed bed  24  and the smaller diameter passages  26  are believed to retain more of the bed compression, than a single, larger diameter passage. The number of passages  26 , the shape and minimum passage diameters, and the location of the passages, will vary with the various factors discussed above, including particle size, particle type, column diameter and packing pressure, among others. Thus, it is believed that if multiple passages  26 , are used, each having a smaller minimum passage diameter than required to achieve the same flow of packing slurry through a single passage, then a larger portion of the bed compression may be retained by the retaining plug  18 , than if a single retaining plug  18  were used or than if no retaining plug were used in the slurry packing process. Multiple passages  26  are believed especially suitable for columns  10  with bore diameters of about 20 mm-100 mm, or larger, especially when packing porous media particles and/or when using packing pressures of over 20,000 psi. 
     The number of passages  26  used in a single retaining member  18  is believed to vary, depending most greatly on the diameter of the column bore and the media particles  23  being packed to form the chromatographic bed  24 . For example, the area of a 1.5 mm minimum diameter passage  26   a  is about 1.8 mm 2  whereas the area of a 100 mm bore diameter (about 4 inch diameter) is about 8,100 mm 2 . One passage  26   a  of 1.5 mm minimum diameter is believed suitable for a bore up about 4-10 mm diameter using 1.6μ porous particles  23  and that minimum diameter comprises about 14% of the area of the 4 mm diameter bore and about 2% of the area of the 10 mm diameter bore. In comparison, ten passages  26   a  each with minimum diameters of 1.5 mm will comprise about 170 mm 2  or about 2% of the area of a 100 mm diameter bore, while providing a large area to retain expansion of the compressed bed  24  formed by fluid packing. Thus, it is believed that numerous small diameter passages  26  of about 1.5-10 mm diameter, are believed suitable for fluid packing of column diameters up to about 100 mm (about 4 inches) in bore diameter, while retaining a majority (over 50%) of the bed compression, or even a substantial portion of 80-90% of the bed compression. 
     The above described packing methods are believed to result in upstream to downstream arrangement of the upstream porous member  12 , the retaining plug  18  and its at least one passage filled with media  23 , the packed bed of chromatographic media  24  and the downstream porous member  14 , with no void spaces between the upstream and downstream porous members  12 ,  14 , and with all media between the porous members  12 ,  14  being compressed. The retaining plug  18  is interposed between the upstream and downstream porous members  12 ,  14 , with the retaining plug  18  at least partially restrain the downstream packed bed  24  of chromatographic media from expanding and decreasing the density of the slurry-packed bed, while the passage(s)  26  allow slurry packing of the chromatographic bed  24 . This is believed to differ greatly from the prior art which compressed the chromatography bed between upstream and downstream fits, with no intervening structure. Further, when the retaining plug  18  is fixed to the tubular body  9  or column  10  during slurry packing, a substantial portion of the bed compression is believed to be retained by retaining member  18  without the need to axially compress the packed bed with a piston or other movable device. 
     The volume of the media in the passage(s)  26  is very small relative to the chromatographic bed  24 , preferably about 0.5 to 5% of the volume of media in the bed  24 . The media  23  in the passage(s)  26  is compressed at a first density or first compression pressure while the media forming the packed chromatography bed  24  is compressed at a second density or second compression pressure which is greater than the first density or first compression pressure. Thus, all chromatographic media between the porous members  12 ,  14  is believed to be compressed with the chromatographic bed  24  being more compressed or more densely packed than if the column had been packed without using the retaining plug  18 . 
     The passages  26  are described as having various circular cross-sectional shapes, such as cones, cylinders, spheres, etc. Such shapes are typically easier to accurately form in metal parts. But the passages  26  need not be of circular-cross-sectional shapes and the passages  26  may thus be square, triangular, hexagonal, oval, elliptical or other shapes. 
     As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. 
     The above description is given by way of example, and not limitation. Given the above disclosure, one skilled in the art could devise variations that are within the scope and spirit of the invention, including various ways of fastening the retaining plug  18  to the body  9  so the retaining plug  18  does not move or deform axially, and various ways of retaining the plug  18  immovable relative to the body  9  during fluid packing while allowing movement of the retaining plug relative to the body  9  after packing to offset the loss of compression due to extrusion through the passage(s). Further, the various features of this invention can be used alone, or in varying combinations with each other and are not intended to be limited to the specific combination described herein. Thus, the invention is not to be limited by the illustrated embodiments.