Abstract:
An apparatus for use in liquid chromatography involves a column filled with media bounded axially by first and second porous plates. The column has an inlet end with an inflow region between the inlet end and the first porous plate. The column also has an outlet end with an outflow region between the outlet end and the second porous plate. The inflow region may be substantially longer than the outflow region. The porous plates may be moveable under pressure so as to compress the media.

Description:
[0001]    The present Application is a continuation of U.S. patent Ser. No. 09/687,801, filed on Oct. 13, 2000. U.S. patent Ser. No. 09/687,801 is a continuation-in-part of U.S. patent Ser. No. 09/137,019 filed Aug. 20, 1998, now U.S. Pat. No. 6,132,605 (which is a continuation-in-part of U.S. patent Ser. No. 08/968,287 filed Nov. 12, 1997, now U.S. Pat. No. 6,086,766) and a continuation-in-part of U.S. patent Ser. No. 09/548,261 filed Apr. 12, 2000, now U.S. Pat. No. 6,294,087 (which is a divisional application of U.S. patent Ser. No. 09/137,278 filed Aug. 20, 1998, now U.S. Pat. No. 6,139,733) and a continuation-in-part of U.S. patent Ser. No. 09/548,214 filed Apr. 12, 2000, now U.S. Pat. No. 6,221,252 (which is a divisional application of U.S. patent Ser. No. 09/137,278 filed Aug. 20, 1998, now U.S. Pat. No. 6,139,733). 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    In one aspect, the invention relates to columns used in liquid chromatography. In another aspect, the invention relates to introducing a sample into a chromatography column. The invention, in yet another aspect, relates to an apparatus and method for sealing the openings of vessels; in particular, the apparatus relates to sealing the openings of liquid chromatography cartridges.  
           [0003]    Liquid chromatography is a technique for separating the individual compounds that exist in a subject sample. In employing the technique, the subject sample is carried in a liquid, called a mobile phase. The mobile phase carrying the subject sample is caused to migrate through a media, called a stationary phase. Different compounds will have differing rates of migration through the media, which effects the separation of the components in the subject sample. Liquid chromatography is commonly performed with reusable columns or with disposable cartridges, both of which are usually cylindrical, in which the media bed is bounded axially by porous plates, or plates containing defined flow paths, through which the mobile phase will flow. (See U.S. Pat. No. 4,250,035 to McDonald et al.) Chromatography systems demand that a reliable seal be created between the column and the head through which the mobile phase enters. This is a particular problem in chromatography systems that employ disposable cartridges in which precise tolerances and careful machining that are customarily required for the creation of effective seals will increase manufacturing costs. Methods of sealing liquid chromatography cartridges typically require forcing a sealing head onto or into the cartridge. Some prior art techniques involve forcing a one-piece sealing head with an elastomeric o-ring, connected radially or to an end of the sealing head, into the cartridge. Alternately, a tapered sealing head can be forced into a cartridge without employing an o-ring to create a seal. Other prior art techniques employ o-rings or knife-edges (see U.S. Pat. No. 5,601,708 to Leavesley) oriented axially on a sealing head that create seals with the edges of the cartridges at their open ends.  
           [0004]    Chromatography columns and cartridges also demand a close contact between the sealing heads and the media bed. Prior art sealing techniques often create gaps between the sealing head and the media bed. Even small gaps can reduce the resolution of distinct components that can be achieved. At the entrance of a column or cartridge, gaps between the sealing head and the porous plate or media will allow the subject sample to disperse and become diluted. At the exit of a column or cartridge, gaps will create a volume in which distinct fractions of the subject sample, which were separated during migration through the media, can blend back together. Thus, gaps at either end of the media bed can degrade the analytic performance of chromatography columns or cartridges.  
           [0005]    When chemists optimize liquid chromatographic separations conditions, they may need to dissolve the sample mixture in a dissolution solvent which may be nonideal for elution. This can result in poor separation and poor recovery of desired components.  
           [0006]    One solution to this problem is to pre-absorb the sample onto a media prior to chromatography. This involves dissolving the sample mixture in a suitable solvent and adding an amount of a dry media (usually similar to the media being used for the separation) to this solution. The dissolution solvent is then evaporated off, usually using a rotary evaporator, leaving the sample mixture dry, and absorbed to the media. The pre-absorbed media is then placed at the head of a pre-packed glass, metal or plastic chromatography column, and the optimized chromatographic solvent would flow through the pre-absorbed media and then through the column of separation media. This method has the potential hazard of the operator coming into contact with the dry powdery media both before and after the addition of the sample. This method also can lead to poor separations and recovery.  
         SUMMARY OF THE INVENTION  
         [0007]    In one aspect, the invention in general relates to sealing a chromatography cartridge containing a media bed that forms a stop. The apparatus involves a sealing head that includes a first head piece, a second head piece, and an elastomeric sealing member. The first head piece includes a first compression face and a contact face adapted to contact said stop. The second head piece includes a second compression face and a compression force receiving member. The elastomeric sealing member is at least partially situated between the first and second compression faces. The first head piece, the second head piece, and the elastomeric sealing member are sized to slide easily into a chromatography cartridge having interior walls and containing a chromatography media bed that is bounded axially by porous plates.  
           [0008]    In operation, the sealing head is inserted into the cartridge. Friction between the elastomeric sealing member and the cartridge is minimized when the elastomeric sealing member is in an uncompressed state during insertion. After the head has been inserted into the cartridge, the elastomeric sealing member is compressed between the first and second compression faces, by moving the second head piece toward the media bed so that the contact face presses against the stop. Compression of the elastomeric sealing member causes the elastomeric sealing member to expand laterally so that it presses against the interior walls of the cartridge and creates a seal.  
           [0009]    The sealing apparatus may be removed from the cartridge by raising the second head piece relative to the first head piece, thus reducing the compression forces on the elastomeric sealing member and lessening its lateral expansion. This reduces the frictional contact between the elastomeric sealing member and the interior walls of the cartridge, so that the sealing member and the first and second head pieces may be more easily removed.  
           [0010]    By providing a second sealing apparatus, it is possible to seal both ends of a chromatography cartridge in the manner described above.  
           [0011]    In the preferred embodiments, the first head piece contains a body portion and an outwardly-extending shoulder. The first compression face is located on the shoulder. Additionally, the first and second head pieces are shaped and sized so that the second head piece may slidably receive the body of the first head piece. The first head piece defines a flow path for the passage of a fluid, such as a mobile phase for chromatography. The contact face on the first head piece includes a slight conical concavity that helps to distribute a mobile phase uniformly to the media bed.  
           [0012]    In operating the preferred embodiments, the contact face of the first head piece presses against the porous plate while the elastomeric sealing member is compressed axially. This axial compression causes the elastomeric sealing member to expand laterally, thus forming a seal with the interior walls cartridge.  
           [0013]    In another aspect, the invention in general relates to sealing chromatography columns containing a first media bed, by creating a plurality of seals. The apparatus involves a sealing head that includes a first head piece, a second head piece, an intermediate head piece, and a plurality of elastomeric sealing members. The first head piece has a body portion and a shoulder that extends farther radially than the body portion. The second head piece and the intermediate head piece define central openings through which the body portion of the first head piece is slidably fitted. The elastomeric sealing members are annular and they circumscribe the body portion of the first head piece. A first elastomeric sealing member is between the shoulder and the intermediate head piece. A second elastomeric sealing member is between the second head piece and the intermediate head piece.  
           [0014]    The first and second head pieces, the intermediate head piece, and the elastomeric sealing members are sized to slide easily into the chromatography column.  
           [0015]    In operation, the apparatus is inserted into the column. Friction between the elastomeric sealing members and the column is minimized when the elastomeric sealing members are in an uncompressed state during insertion. After the head has been inserted into the column, the elastomeric sealing members are compressed, by moving the first and second head pieces relative to each other, so that the second head piece moves toward the shoulder of the first head piece. The first elastomeric sealing member is compressed between the shoulder and the intermediate head piece. The second elastomeric sealing member is compressed between the intermediate head piece and the second head piece. Compression of the elastomeric sealing members causes them to expand laterally so that they press against the interior walls of the column and create seals.  
           [0016]    In alternate preferred embodiments, an inner cartridge fits into an open end of the column. Preferably, the inner cartridge is a sample module. The sample module contains a subject sample. The sample module may also contain chromatography solvents and a chromatography media of either the same or different composition that is used in the column. The shoulder of the first head piece and the first elastomeric sealing member are sized to fit slidably into the sample module, when the first elastomeric sealing member is uncompressed. When a compressive force is applied to the sealing head, the first elastic sealing member forms a seal with the sample module and the second elastic sealing member forms a seal with the column.  
           [0017]    Embodiments of these aspects of the invention may include one or more of the following advantages. Insertion of the sealing apparatus into a chromatography cartridge creates minimal friction between the sealing head and the interior walls of the cartridge. Use of the sealing apparatus does not require close tolerances that create a precise fit between the sealing head and the cartridge. A small amount of force is required to create a seal with chromatography cartridges, relative to prior art methods. The sealing apparatus can be easily removed from a chromatography cartridge, so that the cartridge can be replaced. Because use of the apparatus can minimize frictional shear forces during insertion into a chromatography cartridge, wear on elastomeric sealing members is reduced. Using the apparatus can create a high quality seal. Using the apparatus can minimize the gap that is created between a sealing head and the media bed or porous plate, when sealing a chromatography cartridge. Using the apparatus can axially compress the media bed while sealing a chromatography cartridge. The apparatus has the ability to create a seal by the relative motion of two pieces that compress an elastomeric sealing member without exerting axial force on the media itself. The apparatus can also create a seal by pressing the sealing head against a rigid stop within the column. The apparatus can be adapted for sealing chromatography cartridges having media beds of varying lengths and distances from the opening of the cartridge. The invention may be used with any type of column, including disposable pre-packed cartridges, columns with flexible walls, and columns made of, e.g., glass, steel, or a synthetic material. By employing a sample module that is fitted and sealed within the column a subject sample may be analyzed with two different media in one column. The sample module and the column may employ either porous plates or plates having drilled channels to bound the media beds. The outwardly extending shoulder may have any shape that allows it to exert a compressive force on an elastomeric sealing member.  
           [0018]    In an alternative aspect, the invention features, in general, a chromatography sample module including a flow-through member having an inlet and an outlet and chromatography media within the flow-through member. A sample is added to the media, and the module, with the sample carried therein, can then be connected to a separation column.  
           [0019]    Preferably the chromatography sample module is a tubular member that is sized to fit within the end of a chromatography column that is used for separation of the sample contained on the media in the module. Alternatively, the module can be connected to the chromatography separation column by a flow line. The sample in the dissolution solvent can be added to the sample module, and then the dissolution solvent can be evaporated. Alternatively, the sample in the dissolution solvent can be added to the sample module as a liquid without evaporation.  
           [0020]    In yet another alternative aspect the invention features a rack of sample modules arranged in an array.  
           [0021]    Embodiments of these aspects invention may include one or more of the following advantages. The samples can be easily introduced into separation columns. Various solvents can be used for separation and dissolution of the sample, permitting optimization of the separation procedure. Samples are easily preprocessed, and the operator is not exposed to the media before or after adding the sample. A large number of samples can be prepared for processing at one time, facilitating the carrying out of multiple separations at one time.  
           [0022]    Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0023]    [0023]FIG. 1 is a diagrammatic cross-sectional view of a sealing apparatus according to the invention and a chromatography cartridge.  
         [0024]    [0024]FIG. 2 is a diagrammatic cross-sectional view of a sealing apparatus according to the invention.  
         [0025]    [0025]FIG. 3 is a diagrammatic cross-sectional view of a sealing apparatus according to the invention showing the formation of a seal with a chromatography cartridge.  
         [0026]    [0026]FIG. 4A is a diagrammatic vertical-sectional view of a sealing apparatus according to the invention.  
         [0027]    [0027]FIG. 4B is a diagrammatic vertical-sectional view of a sealing apparatus according to the invention and a chromatography column.  
         [0028]    [0028]FIG. 4C is a diagrammatic vertical-sectional view of a sealing apparatus according to the invention showing the formation of seals with a chromatography column.  
         [0029]    [0029]FIG. 5A is a diagrammatic exploded vertical-sectional view of an alternate sealing apparatus according to the invention with a chromatography column.  
         [0030]    [0030]FIG. 5B is a diagrammatic vertical-sectional view of an alternate sealing apparatus according to the invention showing the formation of a seal with a chromatography column.  
         [0031]    [0031]FIG. 6 is a schematic diagram of a chromatography system according to the invention.  
         [0032]    [0032]FIG. 7 is a vertical sectional view of a chromatography sample module used in the FIG. 6 system.  
         [0033]    [0033]FIG. 8 is a plan view of a rack containing a plurality of the FIG. 7 sample modules in an array.  
         [0034]    [0034]FIG. 9 is an elevation of the FIG. 8 rack and modules. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0035]    Referring to FIGS.  1 - 3 , there is shown a sealing apparatus employing sealing head  2 , which includes first head piece  4 , having body  6  with longitudinal axis  8 . First head piece  4  has outwardly-extending shoulder  10 , first compression face  12  that is located on shoulder  10 , and contact face  14 . Part of contact face  14  has a slightly conical shape or other concavity (exaggerated in FIGS. 2 and 3). First head piece  4  defines flow path  16  along axis  8 . Second head piece  18 , which includes second compression face  20  and a compression force receiving member extending thereabove, is sized to slidably receive body  6  of first head piece  4 . Elastomeric sealing member  22  is at least partially situated between first compression face  12  and second compression face  20 .  
         [0036]    Sealing head  2  and its components are sized to fit slidably into cartridge  24 , having chamfered edges  26 , and chromatography media  28  bounded axially by porous plates which form a rigid stop within the cartridge.  
         [0037]    The connection of the apparatus is shown in FIGS. 1 and 3. As shown in FIG. 1, first head piece  4 , elastomeric sealing member  22 , and second head piece  18  are oriented so that they may slide into cartridge  24 . As shown in FIG. 3, after first head piece  4 , elastomeric sealing member  22 , and second head piece  18  are situated within cartridge  24 , and contact face  14  is stopped at porous plate  18 , additional downward force on second head piece  18  causes first head piece  4  and second head piece  18  to be moved toward each other, thus axially compressing elastomeric sealing member  22  between first compression face  12  and second compression face  20 . The axial compression of elastomeric sealing member  22  causes it to expand laterally and press against cartridge  24 , thus forming a seal.  
         [0038]    As shown in FIG. 3, insertion of sealing head  2  can create a close connection between contact face  14  and porous plate  30 . Compressing elastomeric sealing member  22  between first compression face  12  and second compression face  20 , by pressing second head piece  18  against elastomeric sealing member  22 , which in turn presses first head piece  4  against porous plate  30 , can maintain a close connection between contact face  14  and porous plate  30  while forming a seal between sealing head  2  and cartridge  24 .  
         [0039]    In the preferred combination, sealing head  2  is used to seal cartridge  24  having flexible walls. In other combinations, sealing head  2  is part of a containment structure assembly for receiving cartridge  24 . The containment structure assembly may be pressurizable, such as a pressure vessel assembly that receives cartridge  24 , compresses the flexible walls of cartridge  24 , and provides uniform packing for chromatography media  28  therein. A containment structure of this type is discussed in U.S. Pat. No. 4,250,035 to McDonald, et al., which is incorporated herein by reference. Another type of pressurizable containment structure is discussed in U.S. Pat. No.  5 , 601 , 708  to Leavesley, which is also incorporated herein by reference. In other combinations, sealing head  2  is used to seal cartridge  24  having rigid walls.  
         [0040]    The radial displacement of the outer surface of elastomeric sealing member  22  accommodates a range of inner diameters of cartridge  24 , permitting effective seals while relaxing tolerances for the inner diameter of cartridge  24  to ±0.005″.  
         [0041]    Referring to FIGS.  4 A- 4 C, there is shown an alternate preferred embodiment of a sealing apparatus employing sealing head  210 , which includes first head piece  212 , second head piece  224 , intermediate head piece  228 , and first and second annular elastomeric sealing members  234 ,  236 .  
         [0042]    First head piece  212  has body  214  with longitudinal axis  216 . First head piece  212  has outwardly extending shoulder  218 , and contact face  220 . Part of contact face  220  has a slightly conical shape or other concavity. First head piece  212  defines flow path  222  along axis  216 .  
         [0043]    Body  214  of first head piece  212  fits slidably through second head piece  224 , intermediate head piece  228 , and through first and second elastomeric sealing members  234 ,  236 .  
         [0044]    First elastomeric sealing member  234  is adjacent to both shoulder  218  and intermediate head piece  228 . Second elastomeric sealing member  236  is adjacent to both intermediate head piece  228  and second head piece  224 .  
         [0045]    Sealing head  210  and its components are sized to fit slidably into column  238 , having chamfered edges  240 , filled with chromatography media bed  242 , which is bounded axially by porous plates  244 ,  245 .  
         [0046]    The connection of the apparatus is shown in FIGS. 4B and 4C. As shown in FIG. 4B, first head piece  212 , second head piece  224 , intermediate head piece  228 , and elastomeric sealing members  234 ,  236  are oriented so that they may slide into column  238 . As shown in FIG. 4C, after sealing head  210  is situated within column  224 , first head piece  212  and second head piece  224  are moved relative to each other. This relative movement axially compresses first elastomeric sealing member  234  between shoulder  218  and intermediate head piece  228  and also compresses second elastomeric sealing member  236  between intermediate head piece  228  and second head piece  224 . The axial compression of elastomeric sealing members  234 ,  236  causes them to expand laterally and press against the interior surface of column  238 , thus forming seals.  
         [0047]    As shown in FIG. 4C, insertion of sealing head  210  can create a close connection between contact face  220  and porous plate  244 . Compressing elastomeric sealing members  234 ,  236 , by pressing second head piece  224  against second elastomeric sealing member  236 , which in turn presses second elastomeric sealing member  236  against intermediate head piece  228 , intermediate head piece  228  against first elastomeric sealing member  234 , first elastomeric sealing member against shoulder  218 , and contact face  220  against porous plate  244 , can maintain a close connection between contact face  220  and porous plate  244  while forming a seal between sealing head  210  and column  238 .  
         [0048]    Referring to FIG. 6, there is shown chromatography system  310  which includes a source of solvent  312 , pump  314 , sample module  150 , liquid chromatography column  138 , and sample collection vessel  320 . In this system, the sample to be analyzed is preabsorbed onto media in sample module  150  prior to pumping solvent into module  150  and into chromatography column  138  to perform the separation procedure.  
         [0049]    Referring to FIG. 2, it is seen that sample module  150  includes cylindrical plastic tube  152 , porous plates  156 ,  157  (made of inert plastic porous frits), and chromatography media  154  (only partially shown in the figures) between porous plates  156 ,  157 .  
         [0050]    As appears from FIGS. 5A and 5B, sample module  150  is designed to fit within chromatography column  138  at the entrance thereof and to be sealably connected to the sealing head. Tube  152  is designed to fit within column  138  with minimal space between the two; in particular, there is 0.000″ to 0.010″ of radial clearance.  
         [0051]    Sample module  150  can be filled with media that is the same as or is different from the media of chromatography column  138 . The sample is dissolved in the required solvent and added to the top of sample module  150 , where it is drawn into the media by capillary action. This dissolution solvent is then removed by placing sample module  150  in a vacuum chamber. Heat may also be applied.  
         [0052]    After sample module  150  has dried, it can be placed directly inside separation column  138  so that the lower porous plate  157  is an in intimate contact with the surface of the separation media or with a porous plate within the separation column on top of the separation media.  
         [0053]    Alternatively, sample module  150  can be placed in a remote tube connected by a solvent line. Alternatively, the sample can be dissolved in a separation solvent (or a weaker solvent), and added to module  150 . The wet module can then be loaded into the column or into a remote tube.  
         [0054]    Examples of the types of complex samples where this technique has particularly advantageous use include synthetic organic reaction mixtures and natural product extracts, (e.g., from fermentation broths or plants). These samples often need to be dissolved in a solvent not compatible with the optimized separation solvent. Solvents are organized according to their “solvent strength,” where hexanes have a value close to zero, and methanol has a value of 0.95. Optimized separation eluents often have a lower solvent strength; e.g., hexane:ethylacetate 1:1 has a solvent strength of 0.295. If the sample needs to be dissolved in a strong solvent such as methanol, there will be a solvent strength difference of 0.655 seen initially after loading the sample onto the column, and this will impair the separation of the sample. If the sample dissolved in methanol is instead preadsorbed to the media in the sample module and dried, the sample will not face this impairment during separation.  
         [0055]    Referring to FIGS. 8 and 9, sample modules  150  can be supplied in racks  332 , and a whole rack of sample modules  150  can be efficiently prepared at one time rather than one at a time.  
         [0056]    [0056]FIGS. 5A and 5B show the placement of a module  150  in a column  138  and the sealing of the module  150  and column  138  to a sealing head used to deliver solvent. Sealing head  110  has first head piece  112 , second head piece  124 , intermediate head piece  128 , and first and second annular elastomeric sealing members  134 ,  136 .  
         [0057]    First head piece  112  has body  114  with longitudinal axis  116 . First head piece  112  has outwardly extending shoulder  118 , and contact face  120 . Part of contact face  120  has a slightly conical shape or other concavity. First head piece  112  defines flow path  122  along axis  116 .  
         [0058]    Body  114  of first head piece  112  fits slidably through central openings in second head piece  124 , intermediate head piece  128 , and first and second elastomeric sealing members  134 ,  136 .  
         [0059]    Second head piece  124  has outwardly extending compression force receiving member  146 . Intermediate head piece  128  has narrow portion  148  distal from second head piece  124 .  
         [0060]    First elastomeric sealing member  134  is adjacent to both shoulder  118  and narrow portion  148  of intermediate head piece  128 . Second elastomeric sealing member  36  is adjacent to both intermediate head piece  128  and second head piece  124 .  
         [0061]    Sample module  150  has tube  152  which contains second chromatography media bed  154  bounded axially by second porous plates  156 ,  157 . The outer diameter of tube  152  is sized so that sample module  150  fits into column  138 . The inner diameter of tube  152  is sized so that it may slidably receive shoulder  118 , first elastomeric sealing member  134 , and narrow portion  148  of intermediate head piece  128 .  
         [0062]    Intermediate head piece  128 , second elastomeric sealing member  136 , and second head piece  124  are sized to fit slidably into column  138 , having chamfered edges  140 , filled with first chromatography media bed  142 , which is bounded axially by first porous plates  144 .  
         [0063]    Referring to FIG. 5B seals are formed with the apparatus by inserting sample module  150  into column  138  so that second porous plate  157  abuts first porous plate  144 . Sealing head  110  is then inserted into column  138  and tube  152  of sample module  150 , so that shoulder  118 , first elastomeric sealing member  134 , and narrow portion  148  are within tube  152 , and contact face  120  abuts second porous plate  156 . Sealing head  110  extends far enough into column  138  so that second elastomeric sealing member  136  opposes the inner surface of column  138 .  
         [0064]    Downward compressive force applied to outwardly extending compression force receiving member  146  causes second head piece  124  to slide relative to first head piece  112  and transmits compressive force to second elastomeric sealing member  136 , intermediate head piece  128 , first elastomeric sealing member  134 , shoulder  118 , second porous plate  156 , first media bed  154 , second porous plate  157 , first porous plate  144 , and first media bed  142 . The compressive force causes first and second elastomeric sealing members  134 ,  136  to expand radially so that first elastomeric sealing member  134  forms a seal with tube  152  and second elastomeric sealing member  136  forms a seal with column  138 .  
         [0065]    The seals are released by applying an upward force to second head piece  124 , thereby reducing the compressive force on the components of sealing head  110  and reducing the radial expansion of elastomeric sealing members  134 ,  136 .  
         [0066]    Preferably, cartridge  24  and tube  152  are made of high-density polyethylene. However, columns and cartridges may be constructed of other materials, including glass or stainless steel. Preferably, elastomeric sealing member  22  is made of a fluorocarbon polymer, such as that sold under the trade name CHEMRAZ.