Abstract:
Chromatography column comprising a column tube and at least one adjustable adapter assembly with a plunger that may be positioned at a plurality of longitudinal positions in the column tube, wherein the piston is arranged at the end of a threaded piston rod arranged with the threads engaged by a rotable end cap for enabling longitudinal movement of the piston by turning the end cap, and wherein the adaptor assembly comprises rotation prevention means arranged to prevent rotation of the plunger rod in response to turning of the end cap.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a filing under 35 U.S.C. 371 of international application number PCT/SE2011/050079, filed Jan. 26, 2011, published on Aug. 4, 2011 as WO 2011/093776, which claims priority to application number 172/DEL/2010 filed in India on Jan. 28, 2010. 
     FIELD OF THE INVENTION 
     The present invention relates to a column for liquid chromatography, specifically a column capable of adjustable compression of separation medium contained therein, and to a method of packing separation medium in a chromatography column 
     BACKGROUND OF THE INVENTION 
     Liquid chromatography is a process by which one or more compounds from a chemical mixture may be separated and identified. In order for a chromatographic column to be able to separate biological substances effectively, it is usual to pack fine particles of separation material as tightly and as uniformly as possible in the column tube Filling of the column, or column packing as it is often referred to, is normally effected by closing one end of the column with an outlet means which includes a filter element, and pumping a liquid suspension of the particles under pressure into the other end of the column. Whereas the pumped liquid is able to pass through the filter element essentially unobstructed, the particles are retained by the filter element, so as to build up a particle bed along the length of the tube. As the column tube is filled, the particles are pressed out towards the wall of the tube and the particle bed obtains a stable compaction state with the particles well distributed, this state being maintained during the whole of the filling process. 
     However, when the column tube has been filled with particles and pumping of the liquid suspension is terminated to enable an inlet element to be fitted to the filling-end of the tube, the stable restraining force in the particle bed is partially lost, resulting in expansion of the particle bed. Consequently, when the column tube is once again placed under pressure, disturbing heterogeneities or irregularities are liable to occur in the particle bed, such as the formation of channels and dead volumes. 
     The uniformity of the packing medium within the column has a significant effect on column performance. It is desired that the particles comprising the packing medium be perfectly arranged and completely homogeneous so that the transport liquid and the sample mixture move at uniform rates through the column Areas of loose packing medium create channels causing locally increased flow rates while areas that are partially plugged due to particle aggregation create eddies that retard the flow. Such local variations in the flow rate caused by non-uniform packing medium result in transport liquid mixing that degrades the column performance resulting in broadening of the peaks and a concomitant decrease the resolving capability of the chromatography apparatus. 
     In order to provide versatile columns, there is provided a range of columns capable of adjustable compression of separation medium contained therein. Such columns typically comprise one or two plungers that are moveable in the column tube in order to adjust the column length. Examples of prior art columns include the XK column series from GE Healthcare, and the Kronlab ECOplus columns from YMC Europe GMBH. 
     SUMMARY OF THE INVENTION 
     The object of the invention is to provide a new column for liquid chromatography and a method of packing a chromatography column, which overcomes one or more drawbacks of the prior art. This is achieved by the column for liquid chromatography and the method of packing a chromatography column as defined in the independent claim. 
     One advantage with such a column for liquid chromatography is that it is. 
     Another advantage is that Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples while indicating preferred embodiments of the invention are given by way of illustration only. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description below. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       These and other advantages of the present invention will become more apparent as the following description is read in conjunction with the accompanying drawings, wherein: 
         FIG. 1  shows a column for liquid chromatography in accordance with an embodiment of the invention. 
         FIG. 2  shows the column of  FIG. 1  in cross-section. 
         FIG. 3  is a partial cross-sectional view of one end of the column of  FIG. 1 . 
         FIG. 4  is a partial exploded view of an end piece assembly. 
         FIG. 5  is a disassembled column of  FIG. 1 . 
         FIG. 6  is an exploded view of an adaptor assembly of the column of  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The presently preferred embodiments of the invention are described with reference to the drawings, where like components are identified with the same numerals. The descriptions of the preferred embodiments are exemplary and are not intended to limit the scope of the invention. 
       FIG. 1  shows a column  10  for liquid chromatography in accordance with an embodiment of the invention. The column  10  comprises a column tube  20  and two adapters  30  with tubing  40  at each end for connection to valves, pumps or monitors (not shown). 
       FIG. 2  shows the column of  FIG. 1  in cross section. In the disclosed embodiment, the adapters  30  are essentially identical and are both of “plunger-type” with a plunger  70  allowing adjustment of the bed height from both ends.  FIG. 3  is an enlarged view of an adaptor assembly  30  and one end of the column tubing assembly  50 , and  FIG. 4  is an exploded view of the end cap  110  and the end sleeve  100 . As the columns are equipped with two such adapters  30 , a large range of bed heights between the plungers  70  can be obtained. The column further comprises a column tube  50  comprised of a material that is inert to the solvents and samples that may be run through the column, such as glass, stainless steel or the like. The column tube  50  may be of any suitable diameter, depending on the desired column capacity and in one embodiment it is coaxially surrounded by a protection tube  60  which acts as a safety barrier in case of failure of the column tube  50  if it accidentally should be exposed to too high pressure and break. The column tube  50  and the protection tube  60  ate interconnected at each end by a tubing end piece  90  arranged to hold the column tube  50  and protection tube  60  in position with respect to each other. The tubing end pieces  90  further provides sealed connection interfaces for the adaptors  30  to the column tube  50 . In the disclosed embodiment, the tubing end pieces  90  are firmly attached to the protection tube  60  by threads  92 , and the column tube  50  is held in position by a compression seal  93 . In other embodiments, the protection tube  60  may be omitted and the tubing end piece  90  or the adaptor  30  may be attached directly on the column tube  50 . In the disclosed embodiment, the upper tubing end piece  90  is provided with a column holder section  91  arranged to facilitate holding of the column assembly  10 . 
     The adapter  30  comprises an end sleeve  100 , an end cap  110  and a plunger assembly  70  with an anti-rotator plate  120 . The end sleeve  100  is arranged to be firmly attached to the tubing end piece  90  by threads  94  and to fixate an anti rotation plate  120  in a fixed position there between. The end cap  110  is rotably attached to the end sleeve  100  by a retaining ring  111  and it comprises a central through hole for receiving the plunger assembly  70 . The plunger assembly  70  comprises an elongated threaded plunger rod  80  for applying packing force on the bed. The end cap  110  comprises spring loaded release buttons  130   a  and  130   b  with threaded lock surfaces  133  that are formed to mate the threads of the plunger rod  80  to allow adjustment of the position of the plunger assembly  70  with respect to the end sleeve  100  by turning the end cap  110 . Alternatively, the position of the plunger assembly  70  can be adjusted by pressing the release buttons to disengage the lock surfaces  133  from the plunger rod  80 , whereby the plunger assembly can be moved manually up or down. Manual movement of the plunger assembly  70  allows quick positioning of the plunger assembly  70  to set the desired bed height, whereas the end cap  110  may be used for fine adjustment of the bed height and for subsequent compression of the bed. The release buttons  130   a  and  130   b  are pivotally linked to the end cap  110  by button pins  132   a  and  132   b  arranged in through holes  134   a  and  134   b  in the end cap  110  and which pins  132   a  and  132   b  extends through button holes  135   a  and  135   b , respectively. The release buttons  130   a  and  130   b  are forced in the engaging direction by button springs  131   a  and  131   b . In order to allow efficient manual movement of the plunger rod  80  with respect to the release buttons  130   a  and  130   b , their mating threading is asymmetric with respect to the axis of movement of the plunger rod  80 , in that the threading is tapered in the compression direction when the plunger assembly is moved into the column tube  50  to allow compression without need to press the release buttons  130   a  and  130   b  as the tapered threading automatically disengages them from the plunger rod  80 . With respect to the counter-compression direction, the mating threading of the release buttons  130   a  and  130   b , and the plunger rod  80  is essentially transverse to provide a stiff lock. Moreover, the pivot axis of each release button  130   a  and  130   b  is arranged prior to the lock surface  133  in the compression direction of the plunger assembly  70 , whereby a force on the plunger rod  80  in the counter-compression direction effectively will tighten the interaction of the threading due to a lever action of the release buttons  130   a  and  130   b.    
     The plunger assembly  70  comprises a plunger  86  arranged to keep the bed in a compressed state and to distribute the sample fluid over the full diameter of the bed. The plunger is provided with a fine mesh net or the like to avoid bed particles from entering the fluid paths. The plunger  86  is in fluidic contact with a chromatography system (not shown) through tubing  85  arranged in the plunger rod  80 . In the disclosed embodiment, the plunger is provided with an adjustable seal  71  against the wall of the column tube  50 , disclosed more in detail with reference to  FIG. 6  below. As is mentioned above, the plunger assembly  70  may further be moved in the compression and counter-compression directions by turning the end cap  110  with respect to the end sleeve  100 . In order to avoid distortion of the bed at the interface to the plunger the end sleeve  100  is provided with an anti-rotation member  120  comprising a protrusion  122  arranged to interact with a longitudinal groove  81  in the plunger rod  80  to prevent it from rotating with respect to the column tube  50  when turning the end cap  110  to move the plunger rod  80  in the longitudinal direction. In the disclosed embodiment, the anti rotation member  122  is provided with one protrusion  122 , but in other embodiments, it may be provided with two or more protrusions, and the plunger rod  80  with a corresponding number of longitudinal grooves  81 . In the disclosed embodiment, the anti rotation member  122  is provided as a separate member that is arranged in between the end sleeve  100  and the end piece  90 , and wherein the anti rotation member  122  is provided with a rotation lock structure  121  arranged to prevent rotation of the same, when it is pressed against the end piece  90 , while allowing the end sleeve  100  to be tightly attached to the end piece by threads  94 . In the disclosed embodiment the rotation lock structure  121  is comprised of an annular teeth structure on the surface facing the end piece  90 , and wherein the corresponding surface of the end piece  90  is provided with a mating teeth structure. According to one embodiment, the anti rotation member  122  may be formed as an integral part of the end sleeve  100 .  FIG. 5  shows the column  10  with the adapters  30  removed from the column tube  20 , wherein the teeth structure of the end piece  90  is exposed. 
       FIG. 6  shows an exploded view of one adapter  30  wherein the anti-rotation member  120  is shown separately. Starting from the bottom, the plunger  86  is comprised of a mesh retaining ring  72 , a mesh  73 , a plunger distributor body  74 , a seal  71  in the form of an o-ring, and a plunger seal compression body  75 . The plunger compression body is attached to and supported by the plunger rod  80 , whereas the plunger distribution body is attached to a plunger sealing rod  84  and to fluidics tubing  85 . Above the end cap  110  the plunger sealing rod  84  is attached to a plunger sealing knob  88  by means of threads. In the assembled state the sealing knob  88  abuts the upper end of the plunger rod  80 , and when it is rotated in the sealing direction it moves the plunger sealing rod  84  and thus the plunger distribution body  74  upwards with respect to the plunger rod  80  and the plunger seal compression body  75 , whereby the seal  71  is pressed against the inner wall of the column tube  50  to achieve a tight seal there between. The upper end of the fluidics tubing is attached to suitable fluidics fittings  87  to be connected to a chromatography system. 
     Mode of Usage: 
     A. Inserting and adjusting the adapter used as bottom end piece: 
     
         
         
           
             1. Insert the bottom adapter  30  in the column tube  50 , tighten the end sleeve  100  to the end piece  90 , and adjust the plunger  86  to the desired level. 
             2. Tighten the seling knob  88  to seal the sealing O-ring  71  against the column tube wall  50 .
 
B. Packing the column:
 
             1. Remove the top adapter  30  and make sure the plunger  86  of the bottom adapter  30  is in the right place with the O-ring  71  tightened. 
             2. Pour a small amount of packing liquid into the column tube  50  and let it drain until the level of liquid in the tube is 2 to 3 mm above the plunger  86 . 
             3. Close the column outlet on the bottom adapter  30  e.g. using a stop plug. 
             4. Carefully fill chromatography medium into the column tube  50  avoiding introducing air bubbles. 
             5. Slacken the sealing O-ring  71  of the top adapter  30  and insert the plunger  86  in the column tube at an angle so that no air is trapped under the plunger  86 . 
             6. Fasten the adapter to the column tube  50  by turning the end sleeve  100  down. 
             7. Tighten the sealing knob  88  to seal the sealing O-ring  71  against the column wall  50 . 
             8. Slide the plunger  86  slowly down by turning the end cap  110  so that air in the plunger  86  and capillary tubing  85  is displaced by the eluent. 
             9. Pack the column  10  according to appropriate media instruction.
 
According to one embodiment, there of the packing may comprise one or more of the following steps:
 
             1. Flowing packing fluid through the column at a predetermined rate, such as 750 cm/h. 
             2. Consolidating the bed during a predetermined time period, such as 5 min 
             3. Registering the top of the thus compressed bed. 
             4. Terminating the flow of packing liquid 
             5. Positioning the plunger  86  at a predetermined distance, e.g. 10 mm, above the bed surface, to make sure that the bed surface is not disturbed. 
             6. Tightening the sealing knob  88  to seal the o-ring seal  71  on the plunger  86  and start turning the end cap  110  until the plunger reach the top of the compressed bed as registered. 
             7. Measuring the bed height and calculate the desired bed height by dividing the measured bed height with the predetermined packing factor. 
             8. Turn the end cap  110  until the desired bed height is reached.
 
B. Adjusting the piston  86  position using release buttons  130   a  and  130   b . To readjust the position of the piston in the column:
 
             1. Loosen the sealing knob  88  of the top adapter  30  to slacken the seal O-ring  71 . 
             2. Press in the release buttons  130   a  and  130   b  and adjust the piston  86  to a position just above the bed level. 
             3. Tighten the sealing knob  88  to obtain a good seal. 
             4. Adjust the piston position by turning the end cap  100 . 
             5. Turning of the end cap  100  can also be used for axial compression of the gel bed as described in the media instruction. If needed, a spanner may be used to turn the end cap  100 . 
           
         
       
    
     Although the present invention has been described and illustrated in detail, it is to be clearly understood that this is done by way of illustration and example only and is not to be taken by way of limitation. The scope is to be limited only by the terms of the appended claims.