CN112121466A - Chromatographic column device and chromatographic column packing method - Google Patents
Chromatographic column device and chromatographic column packing method Download PDFInfo
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- CN112121466A CN112121466A CN202010966206.5A CN202010966206A CN112121466A CN 112121466 A CN112121466 A CN 112121466A CN 202010966206 A CN202010966206 A CN 202010966206A CN 112121466 A CN112121466 A CN 112121466A
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Abstract
The invention relates to the field of chromatographic columns, and discloses a chromatographic column device and a chromatographic column packing method. The chromatographic column device comprises a chromatographic column tube, a column head and a bottom plate, wherein the column head is arranged in the chromatographic column tube in a sealing mode and can move up and down along the axial direction of the chromatographic column tube, the column head is provided with a first upper channel for communicating the upper side and the lower side of the column head, the bottom plate is arranged at the bottom end of the chromatographic column tube in a sealing mode, the bottom plate is provided with a first lower channel and a second lower channel which are respectively communicated with the upper side and the lower side of the bottom plate, the bottom plate is further provided with a third lower channel for communicating the lower side of the bottom plate with the second lower channel, the second lower channel is arranged to have a first state which is communicated with the upper side of the bottom plate and is disconnected with the third lower channel and a second state which is communicated with the upper side of the bottom plate, a yielding structure is formed on the inner wall. The invention can realize positive pressure mounting of the column and overcome various defects generated by negative pressure mounting in the prior art.
Description
Technical Field
The invention relates to the technical field of chromatographic columns, in particular to a chromatographic column device and a chromatographic column packing method.
Background
In the process of separating and purifying protein, a chromatographic column cannot be used, but the filling quality of the chromatographic column directly influences the distribution effect, and further influences the recovery rate and purity of the product.
At present, the filling method of the domestic and foreign industrial-grade chromatographic column mainly comprises an inhalation type and a displacement type. Displacement works well when packing small diameter columns, but works poorly when the column diameter is large (typically over 800mm in diameter). Therefore, the widely adopted filling method is of a suction type, namely, the column head of the chromatographic column is pressed down to the bottom of the chromatographic column, and the homogenate liquid which is uniformly mixed in advance is sucked into the chromatographic column in a piston mode.
However, the above-mentioned suction filling method has many drawbacks: because the homogenate is sucked into the chromatographic column in a negative pressure mode, on the first hand, bubbles are easily generated under the negative pressure condition, the final distribution effect is influenced, and the utilization rate of the filler is reduced; on the second hand, the rising speed of the column head is slow (generally below 20 mm/min), the column packing time is long, and the longer time can cause the filler to be settled, the column efficiency is reduced, and the distribution effect is poor; in a third aspect, the refining concentration is required to be high, typically below 50%, which tends to result in not too much total refining volume being sucked up, resulting in a lower bed height and less suitable production process for higher bed requirements. In addition, the suction or discharge of the filler is in an extrusion form, so that the filler is easily damaged, the filler loss rate is high, and the replacement cost is high.
Disclosure of Invention
The invention aims to provide a chromatographic column device and a chromatographic column packing method aiming at the problems, so as to realize positive pressure packing of a column and overcome various defects generated by negative pressure column packing in the prior art.
In order to achieve the above object, the present invention provides a chromatography column apparatus, including a chromatography column tube, a column head and a bottom plate, wherein the column head is hermetically disposed in the chromatography column tube and can move up and down along an axial direction of the chromatography column tube, the column head is provided with a first upper channel communicating upper and lower sides of the column head, the bottom plate is hermetically disposed at a bottom end of the chromatography column tube, the bottom plate is provided with a first lower channel and a second lower channel respectively communicating upper and lower sides of the bottom plate, the bottom plate is further provided with a third lower channel communicating a lower side of the bottom plate with the second lower channel, the second lower channel is configured to have a first state communicating with an upper side of the bottom plate and disconnecting from the third lower channel and a second state communicating with the third lower channel and disconnecting from the upper side of the bottom plate, and an abdicating structure is formed on an inner wall surface of the chromatography column tube, the abdicating structure is arranged to be capable of communicating the upper side and the lower side of the column head when the column head moves to the abdicating structure.
Optionally, the chromatography column apparatus comprises a pump disposed in communication with the second lower channel.
Optionally, the offset structure is formed by an upward chamfer formed on an inner wall surface of the top end of the chromatography column and/or a downward chamfer formed on an inner wall surface of the bottom end of the chromatography column.
Optionally, the chromatography column device comprises a movable member, and the movable member is disposed in the second lower channel and can realize the transition of the second lower channel between the first state and the second state by moving or rotating.
Optionally, the second lower channel includes a first section, a second section, a third section and a fourth section which are sequentially communicated from top to bottom, an inner diameter of the third section is larger than an inner diameter of the second section, an inner diameter of the second section is larger than the inner diameter of the first section and the inner diameter of the fourth section, and the third lower channel is configured to be communicated with the third section;
the moving part is a second lower pipe which is coaxially inserted into the second lower channel and can move along the axial direction of the second lower channel, the second lower pipe comprises a first pipe section, a second pipe section and a third pipe section which are sequentially connected from top to bottom, the outer diameter of the second pipe section is larger than that of the first pipe section, the outer diameter of the first pipe section is larger than that of the third pipe section, the outer diameter of the first pipe section is matched with the inner diameter of the second section, the outer diameter of the third pipe section is matched with that of the fourth section, a first connecting hole extending from the top surface of the first pipe section to the bottom surface of the second pipe section is arranged on the second lower pipe, a plug is arranged at the top end of the first pipe section, a pipe cavity for communicating the second lower pipe with the inner diameter of the second section is arranged on the plug, and the outer diameter of the plug is larger than that of the first section and smaller than that of the second section, the axial length from the top end of the plug to the bottom end of the second pipe section is smaller than the axial length from the top end of the second section to the bottom end of the third section.
Optionally, the column head is provided with a second upper channel which is communicated with the upper side and the lower side of the column head, and the inner diameter of the second upper channel is larger than that of the first upper channel.
Optionally, the stud is provided with a third upper channel communicating the stud upper side with the second upper channel, the second upper channel being arranged to have a third state communicating with the stud lower side and disconnected from the third upper channel and a fourth state communicating with the third upper channel and disconnected from the stud lower side.
Optionally, each channel is connected with a valve for controlling the on-off of the channel.
Optionally, the chromatography column apparatus comprises a control unit arranged to be able to control the movement of the column head.
Optionally, the chromatography column apparatus comprises a pressure detector arranged to detect a pressure within the chromatography column.
In another aspect, the present invention provides a method for packing a chromatographic column, the method comprising the steps of:
s1: injecting a predetermined amount of purified water into the chromatography column tube;
s2: pressing down the column head to discharge air bubbles to the chromatographic column tube and the column head;
s3: pumping homogenate into the chromatographic column tube, and synchronously moving the column head and pumping the homogenate so as to keep positive pressure in the chromatographic column tube;
s4: and (4) performing column pressing on the chromatography column tube.
Optionally, in step S3, the pressure inside the chromatography column tube is maintained at 0.1bar-0.3 bar.
Optionally, the column compression method in step S4 specifically includes: the column head is depressed to a predetermined bed height.
Optionally, the method is performed using the chromatography column apparatus described above, and the method further comprises bubbling the second lower channel and the third lower channel before the step S3.
Through the technical scheme, when the chromatographic column device is used for loading columns, positive column loading can be realized, and the device has the advantages of simplicity in operation, high column loading speed, no bubble generation, low requirement on concentration of homogenate, high column loading column bed height, good repeatability and the like, and can save a large amount of time, labor and material resources.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram of the structure of one embodiment of the chromatography column apparatus of the present invention;
FIG. 2 is a partial cross-sectional view of the bottom plate of FIG. 1, wherein the second lower channel is in a first state;
FIG. 3 is a schematic view of the second lower passageway of FIG. 2 in a second state;
FIG. 4 is a partial cross-sectional view of the stud of FIG. 1, with the second upper channel in a third state;
FIG. 5 is a schematic view of the second upper channel of FIG. 4 in a fourth state;
FIG. 6 is a graph showing the results of column efficiency tests in which the column packing height was 17.3cm by the column packing method of the present invention;
FIG. 7 is a graph showing the results of column efficiency tests in which the column packing height was 19.5cm by the column packing method of the present invention.
Description of the reference numerals
10-chromatography column, 11-abdicating structure, 20-column head, 21-first upper channel, 22-second upper channel, 23-third upper channel, 24-first upper tube, 241-first upper valve, 25-second upper tube, 251-second upper valve, 26-third upper tube, 261-third upper valve, 27-sealing ring, 30-bottom plate, 31-first lower channel, 32-second lower channel, 321-first section, 322-second section, 323-third section, 324-fourth section, 33-third lower channel, 34-first lower tube, 341-first lower valve, 35-second lower tube, 351-first tube section, 352-second tube section, 353-third tube section, 354-first connecting hole, 355-plug, 356-second communication hole, 357-second lower valve, 36-third lower tube, 361-third lower valve, 40-pump.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
In the present invention, unless otherwise specified, the use of directional terms such as "upper, lower, top, bottom" generally refers to the orientation shown in FIG. 1. "inner and outer" refer to the inner and outer contours of the respective component itself.
In one aspect, the present invention provides a chromatography column apparatus, comprising a chromatography column tube 10, the column head 20 is provided with a first upper channel 21 which is communicated with the upper side and the lower side of the column head 20, the bottom plate 30 is arranged at the bottom end of the chromatography column tube 10 in a sealing manner, the bottom plate 30 is provided with a first lower channel 31 and a second lower channel 32 which are respectively communicated with the upper side and the lower side of the bottom plate 30, the bottom plate 30 is further provided with a third lower channel 33 which is communicated with the lower side of the bottom plate 30 and the second lower channel 32, the second lower channel 32 is set to have a first state which is communicated with the upper side of the bottom plate 30 and disconnected with the third lower channel 33 and a second state which is communicated with the third lower channel 33 and disconnected with the upper side of the bottom plate 30, the yielding structure 11 is formed on the inner wall surface of the chromatography column tube 10, and the yielding structure 11 is set to be capable of being communicated with the upper side and the lower side of the column head 20 when the column head 20 moves to.
In the above, it should be noted that the column head 20 disposed in the chromatography column 10 divides the tube cavity of the chromatography column 10 into an upper chamber and a lower chamber, the upper side of the column head 20 is the upper chamber, and the lower side of the column head 20 is the lower chamber. The upper side of the bottom plate 30 is the lower chamber and the lower side of the bottom plate 30 is the external environment. The top of the chromatography column 10 is open, that is, the upper chamber is communicated with the external environment, and the lower chamber is a closed space.
In the above, the arrangement of the first upper channel 21, the first lower channel 31 and the second lower channel 32 can discharge the substance in the lower chamber or introduce the substance into the lower chamber, thereby realizing the column installation of the chromatography column tube 10. In addition, the provision of the third lower passage 33, and the provision of the second lower passage 32 to have the first state and the second state, enables the injection of the packing or the like during the column packing and the air bubbles of the inner passages (e.g., the second lower passage 32, the third lower passage 33) of the bottom plate 30. The provision of the abdicating structure 11 enables the air bubbles to be evacuated from the periphery of the post-head 20.
Through the technical scheme, when the chromatographic column device is used for loading columns, positive column loading can be realized, and the device has the advantages of simplicity in operation, high column loading speed, no bubble generation, low requirement on concentration of homogenate, high column loading column bed height, good repeatability and the like, and can save a large amount of time, labor and material resources.
In the present invention, referring to fig. 1, the stud 20 may include a cylindrical main body and a sealing ring 27 disposed on the outer periphery of the main body, and a groove for the sealing ring 27 to be inserted into may be formed on the outer periphery of the main body.
In the present invention, the chromatography column apparatus further comprises a pump 40, and the pump 40 is provided in communication with the second lower channel 32. During column packing, the pump 40 can be used to pump packing into the lower chamber of the chromatography column 10 through the second lower channel 32, and the positive pressure in the chromatography column 10 can be maintained by controlling the pumping speed. Wherein the pump 40 may be a pneumatic pump.
In the present invention, the abdicating structure 11 can have various embodiments as long as the upper and lower sides of the column head 20 can be communicated when the column head 20 moves to the abdicating structure 11. For example, the avoiding structure 11 may be an annular groove formed on the inner wall surface of the chromatography column tube 10, when the column head 20 moves to the annular groove, there is a gap between the inner surface of the annular groove and the outer surface of the column head 20, and the gap can communicate the upper side and the lower side of the column head 20.
According to a preferred embodiment of the abdicating structure 11 of the invention, the abdicating structure 11 can be formed by an upward chamfer formed on the inner wall surface of the top end of the chromatography column tube 10, i.e. the inner diameter of the top end of the chromatography column tube 10 gradually increases upwards, forming an annular groove with a triangular cross-section (see the top end of the chromatography column tube 10 in fig. 1). In this case, the column tube 10 should be filled with purified water (described later) when the column is installed.
According to another preferred embodiment of the abdicating structure 11 of the invention, the abdicating structure 11 can be formed by a downward chamfer formed on the inner wall surface of the bottom end of the chromatography column tube 10, i.e. the inner diameter of the bottom end of the chromatography column tube 10 is gradually increased downwards to form an annular groove with a triangular cross section (see the bottom end of the chromatography column tube 10 in fig. 1). In this case, the purified water level in the chromatography column tube 10 should be above the abdicating structure 11 when the column is installed.
In other embodiments, two offset structures 11 can be disposed on the chromatography column 10, one being the upward chamfer and the other being the downward chamfer (as shown in fig. 1).
In the present invention, the chromatography column apparatus may include a movable member that may be disposed in the second lower channel 32 and may be capable of realizing the transition of the second lower channel 32 between the first state and the second state by moving or rotating. The movable member may have various embodiments as long as the function thereof can be achieved.
For the implementation manner of the rotation, according to an embodiment of the present invention, the movable member may be a rotatable valve plate disposed at a connection position of the second lower channel 32 and the third lower channel 33, and the second lower channel 32 is switched between the first state and the second state by controlling the rotation of the valve plate to block the second lower channel 32 or the third lower channel 33.
As for the implementation of the movement, according to an embodiment of the present invention, the movable member is a second lower pipe 35 coaxially inserted into the second lower channel 32 and capable of moving along the axial direction of the second lower channel 32, the second lower pipe 35 is provided with a blocking portion, a first communicating hole 354 and a second communicating hole 356, the blocking portion is provided at the top end of the second lower pipe 35 and capable of blocking the top end port of the second lower channel 32, the first communicating hole 354 is provided on the pipe wall of the second lower pipe 35 and capable of communicating the third lower channel 33 with the second lower channel 32, and the second communicating hole 356 is provided on the pipe wall of the second lower pipe 35 to communicate the lumen of the second lower pipe 35 with the second lower channel 32. When the second down pipe 35 moves down to the port of the first communication hole 354 for communication with the third lower passage 33 is blocked by the inner wall face of the second down pipe 35, the blocking portion is away from the top end port of the second lower passage 32, so that the second lower passage 32 is in the first state; when the second down tube 35 is moved up to the stopper portion to block the tip end port of the second lower passage 32, the port of the first communication hole 354 is exposed to communicate with the third lower passage 33, so that the second lower passage 32 is in the second state. By moving the second down tube 35 up and down, the transition of the second lower channel 32 between the first state and the second state can be achieved.
Further, according to an embodiment of the present invention, the second lower passage 32 may include a small diameter section and a large diameter section which are sequentially communicated from top to bottom, the third lower passage 33 is provided to be communicated with the large diameter section, the second lower tube 35 may include a main tube having an outer diameter adapted to an inner diameter of the large diameter section, and a plug 355 connected to a top end of the main tube, the outer diameter of the plug 355 being larger than the inner diameter of the small diameter section and smaller than the inner diameter of the large diameter section, the plug 355 may include a top wall which may serve as the blocking portion and a peripheral wall, one port of the first communication hole 354 may be located on a top end surface of the main tube, the other port of the first communication hole 354 may be located on an outer wall surface of the main tube, and the second communication hole 356 may be a radial through hole opened on the peripheral wall of the plug 355. The first and second communication holes 354 and 356 may be one or more, respectively.
According to another embodiment of the present invention, as shown in fig. 2, the second lower channel 32 may include a first section 321, a second section 322, a third section 323, and a fourth section 324 which are sequentially communicated from top to bottom, the third section 323 has an inner diameter larger than that of the second section 322, the second section 322 has an inner diameter larger than that of the first section 321 and that of the fourth section 324, and the third lower channel 33 is disposed to be communicated with the third section 323; the second lower tube 35 comprises a first tube section 351 connected in series from top to bottom, the outer diameter of the second pipe section 352 is larger than that of the first pipe section 351, the outer diameter of the first pipe section 351 is larger than that of the third pipe section 353, the outer diameter of the first pipe section 351 is matched with the inner diameter of the second section 322, the outer diameter of the third pipe section 353 is matched with that of the fourth section 324, a first communication hole 354 extending from the top surface of the first pipe section 351 to the bottom surface of the second pipe section 352 is formed in the second lower pipe 35, a plug 355 is arranged at the top end of the first pipe section 351, a second communication hole 356 communicating a pipe cavity of the second lower pipe 35 with the second section 322 is formed in the plug 355, the outer diameter of the plug 355 is larger than that of the first section 321 and smaller than that of the second section 322, and the axial length from the top end of the plug 355 to the bottom end of the second pipe section 352 is smaller than that of the second section 322 and that of the third.
With the above arrangement, plug 355 and second pipe section 352 can move within second section 322 and third section 323 during the up and down movement of second lower pipe 35, thereby enabling second lower passageway 32 to transition between the first state and the second state. When the second lower path 32 is in the first state, as shown in fig. 2, the second lower tube 35 moves downward until the bottom surface of the second pipe 352 abuts against the bottom surface of the third section 323, and the communication between the second lower path 32 and the third lower path 33 is blocked, and the lumen of the second lower tube 35 communicates with the second section 322 through the second communication hole 356 of the stopper 355, and further communicates with the first section 321 and the lower chamber. When the second lower channel 32 is in the second state, as shown in fig. 3, the second lower tube 35 moves up until the top surface of the stopper 355 abuts against the top surface of the second section 322, and the communication between the first section 351 and the second section 352 is blocked, and the third lower channel 33 communicates with the second section 322 through the first communication hole 354 and further communicates with the lumen of the second lower tube 35 through the second communication hole 356.
In the above, it should be noted that the first communication hole 354 may have any shape, for example, a straight line shape, a curved line shape, a spiral shape, etc., as long as one end opening is located at a side close to the third lower channel 33 to communicate with the third lower channel 33, and the other end opening is located at the top surface of the first pipe section 351 to communicate with the second section 322. The entire inner diameter of the second lower tube 35 may be constant such that the first tube section 351 and the second tube section 352 may have a thicker tube wall to facilitate the provision of the first communication hole 354.
In the present invention, the column head 20 may be provided with a second upper passage 22 communicating upper and lower sides of the column head 20. The inner diameter of the first upper channel 21 may be set smaller than the inner diameter of the second upper channel 22 (see fig. 4), so that the first upper channel 21 may serve as an upper chromatography channel. Of course, the inner diameter of the first lower passage 31 may also be set smaller than the inner diameter of the second lower passage 32 (see fig. 2), so that the first lower passage 31 may serve as a lower chromatography passage.
Further, the post head 20 may be further provided with a third upper passage 23 communicating an upper side of the post head 20 with the second upper passage 22, and the second upper passage 22 may be provided to have a third state communicating with a lower side of the post head 20 to be disconnected from the third upper passage 23 and a fourth state communicating with the third upper passage 23 to be disconnected from the lower side of the post head 20. The second upper channel 22 and the third upper channel 23 are identical in structure and fit with the second lower channel 32 and the third lower channel 33. The second upper channel 22 may be internally provided with a movable member (e.g., the second upper tube 25) like the second lower channel 32 to enable the transition of the second upper channel 22 between the third state and the fourth state (see fig. 4 and 5), which will not be described herein.
According to the invention, the second upper channel 22 and the third upper channel 23 are arranged on the column head 20, so that the second upper channel 22 can be sterilized and cleaned by sodium hydroxide and the pipeline can be washed clean by purified water by communicating the second upper channel 22 with the third upper channel 23 (in a working state shown in figure 5) during column unloading operation; after the cleaning, the column is in the working state shown in fig. 4, purified water is pumped into the chromatography column tube 10 through the second upper channel 22 to flush and recover the filler in the column, so that the phenomenon that the filler is extruded and damaged in the above-mentioned manner of sucking and assembling the column and disassembling the column is avoided, and the column can be thoroughly cleaned.
In the present invention, the respective channels provided on the stud 20 and the base plate 30 are made on-off controllable. Specifically, the chromatography column apparatus may further include a first upper tube 24 communicating with the first upper passage 21, a third upper tube 26 communicating with the third upper passage 23, a first lower tube 34 communicating with the first lower passage 31, and a third lower tube 36 communicating with the third lower passage 33, as shown in fig. 1, for example. The second upper tube 25 may extend out of the stud 20 and the second lower tube 35 may extend out of the base plate 30. The first upper pipe 24 is provided with a first upper valve 241 for controlling the on-off of the first upper pipe, the second upper pipe 25 is provided with a second upper valve 251 for controlling the on-off of the second upper pipe, the third upper pipe 26 is provided with a third upper valve 261 for controlling the on-off of the third upper pipe, the first lower pipe 34 is provided with a first lower valve 341 for controlling the on-off of the first lower pipe, the second lower pipe 35 is provided with a second lower valve 357 for controlling the on-off of the second lower pipe, and the third lower pipe 36 is provided with a third lower valve 361 for controlling the on-off of.
In the present invention, in order to realize automatic control of the chromatography column apparatus and increase the column loading speed, the chromatography column apparatus may further include a control unit, the control unit may be configured to control the movement of the column head 20, the control unit may be further configured to control the operation of the pump 40, and the control unit may be further configured to control the opening and closing of the above-mentioned valves.
In addition, the chromatography column apparatus may further comprise a pressure detector arranged to detect the pressure inside the chromatography column 10 (i.e. the lower chamber). This facilitates the movement of the adjustment head 20 and the operation of the pump 40 according to the pressure detected by said pressure detector.
Preferably, the pressure detector may be arranged to be able to transmit detected pressure information to the control unit, which may control the operation of the pump 40 and the movement of the column head 20 in dependence of the pressure information.
In another aspect, the present invention provides a method for packing a chromatographic column, the method comprising the steps of:
s1: injecting a predetermined amount of purified water into the chromatography column 10;
s2: pressing the column cap 20 to discharge air bubbles to the chromatographic column tube 10 and the column cap 20;
s3: pumping the homogenate into the chromatographic column tube 10, and synchronously moving the column cap 20 and the homogenate so as to keep positive pressure in the chromatographic column tube 10;
s4: the chromatography column 10 is subjected to column compression.
In step S1, the purified water is injected into the column 10 in various ways, preferably from the bottom of the column 10, and the height of the injected purified water is 5cm at the lowest and 3cm at the highest from the top surface of the column 10.
In step S2, by pressing down the column head 20 to be in contact with the water surface of the purified water, air bubbles in the lower chamber of the chromatography column 10 can be eliminated; by continuously pressing the column cap 20, air bubbles in and around the column cap 20 can be removed; the pressing speed of the column cap 20 can be 10-20 mm/min.
In step S3, the pressure inside the chromatography column 10 may be kept at a weak positive pressure, for example, the pressure inside the chromatography column 10 may be kept at 0.1bar to 0.3 bar; the column cap 20 can move upwards at a speed of 30-50 mm/min.
In step S4, the pressing speed of the column cap 20 is preferably 5-20 mm/min; the column pressing method specifically comprises the following steps: the column head 20 is depressed to a predetermined bed height.
Where the packing volume is known, the bed height h can be calculated, i.e. h ═ packing volume/chromatography column cross-sectional area/compressibility. In the case of an unknown packing volume, the packing height can be recorded when the column head 20 is depressed until the homogenate is completely stratified, then the bed height can be calculated at a slightly smaller compressibility, e.g., a normal compressibility of 1.15, then the bed height can be calculated at a compressibility of 1.1 or 1.12 (the compressibility can preferably be anywhere between 1.1 and 1.15 depending on the packing characteristics), and finally the column head 20 is depressed again to the calculated bed height.
The chromatographic column packing method of the present invention can be carried out by using the above-mentioned chromatographic column device.
An embodiment of the method for packing a chromatography column according to the present invention will be described in detail with reference to FIG. 1.
Type of filler: cellulose matrix gel Cellufine GCL-2000, average particle size 90 um;
volume of the filler: 100L;
concentration of homogenate: 60 percent;
specification of a chromatographic column tube: 800X 550 mm.
In fig. 1, each valve is initially closed, and the method comprises:
injecting purified water: opening a first upper valve 241 and a first lower valve 341, injecting purified water into the chromatographic column tube 10 from the first lower channel 31 until the liquid level is 10cm, and then closing the first lower valve 341;
and (3) removing air bubbles in the chromatographic column tube 10: opening the second upper valve 251, enabling the second upper channel 22 to be in a third state, controlling the column head 20 to be pressed down at the speed of 15mm/min, discharging bubbles in the lower chamber of the chromatographic column tube 10 through the first upper channel 21 and the second upper channel 22 when the column head 20 is pressed down to be in contact with the water surface, and closing the second upper valve 251 after the bubbles are discharged;
air bubbles are removed from the column head 20: the column head 20 is pressed down continuously, and when the column head 20 descends to the yielding structure 11 at the bottom end, water overflows upwards from the periphery of the column head 20, so that bubbles in and around the column head 20 are exhausted;
eliminating air bubbles inside the bottom plate 30: setting the second lower path 32 in the second state, opening the second lower valve 357 and the third lower valve 361, simultaneously placing the second lower tube 35 and the third lower tube 36 into the homogenate, starting the pump 40, circulating the homogenate, exhausting bubbles in the second lower path 32 and the third lower path 33, then setting the second lower path 32 in the first state and closing the third lower valve 361;
injecting a homogenate: controlling the column head 20 to move upwards at a speed of 50mm/min, simultaneously controlling the pump 40 to start to operate, injecting the homogenate into the chromatographic column tube 10 through the second lower channel 32, detecting and feeding back pressure to the control unit through the pressure detector in the pulp pumping process, adjusting the pump control pressure of the pump 40, ensuring that the pressure in the chromatographic column tube 10 is 0.1bar-0.3bar (in the pulp injection process, water can be discharged from the first upper channel 21), and closing the first upper valve 241 and the second lower valve 357 after the homogenate is completely injected;
column pressing: the first lower valve 341 is opened and the column head 20 is controlled to be pressed down at a speed of 15mm/min until the predetermined bed height (h ═ 100 × 103cm3/5.024×103cm217.3cm, where 1.15 is the compressibility of the packing), water may drain from the first lower channel 31 during depression of the column head 20;
column efficiency test: testing the linear speed of 30 cm/h; equilibrating the column with 0.1mol/LNaCl solution; the test was carried out with a 0.8mol/L NaCl solution.
The results of the column efficiency test can be seen in FIG. 6, where the theoretical plate number N/m is 7152 and the symmetry As is 1.05.
For comparison, the data of the prior art are given: the theoretical plate number N/m is 3300-5500, As is 0.8-1.5; the decision for the data is: the higher the theoretical plate number, the better, the more the symmetry As approaches 1, the better. According to the test results, the theoretical plate number and symmetry of the invention are good.
FIG. 7 shows the results of the same test conducted by adjusting the height of the column bed to 19.5cm by the same column packing method, wherein the theoretical plate number N/m was 7435 and the symmetry As was 1.07. Therefore, the repeatability and the stability of the column packing method are better.
By adopting the full-automatic synchronous positive column pressing method, the suction of the filler is completed in a mode that the column head 20 moves upwards and the pump 40 is started synchronously, the weak positive pressure in the column is maintained, the filler can be sucked into the chromatographic column tube 10 quickly, the filler is not easy to damage, and the filler loss rate is extremely low. The column installing method is simple to operate, and can be started by one key by arranging the control unit; the repeatability is good, and the efficiency is high; no negative pressure is generated in the whole process, no air bubbles are generated, and the distribution effect is good; because the auxiliary power of the external pump is used for synchronous operation, the column head rises quickly, the homogenate liquid basically has no sedimentation, and the column efficiency is high; the requirement on the concentration of the homogenate is low (can reach 80 percent), and the packed column bed is high; can save a large amount of time, manpower and material resources.
The preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. The chromatographic column device is characterized by comprising a chromatographic column tube (10), a column head (20) and a bottom plate (30), wherein the column head (20) is arranged in the chromatographic column tube (10) in a sealing manner and can move up and down along the axial direction of the chromatographic column tube (10), the column head (20) is provided with a first upper channel (21) communicated with the upper side and the lower side of the column head (20), the bottom plate (30) is arranged at the bottom end of the chromatographic column tube (10) in a sealing manner, the bottom plate (30) is provided with a first lower channel (31) and a second lower channel (32) respectively communicated with the upper side and the lower side of the bottom plate (30), the bottom plate (30) is further provided with a third lower channel (33) communicated with the lower side of the bottom plate (30) and the second lower channel (32), and the second lower channel (32) is arranged to have a first state communicated with the upper side of the bottom plate (30) and disconnected with the third lower channel (33) and a first state communicated with the third lower channel (33 And with the second state of bottom plate (30) upside disconnection, be formed with abdicating structure (11) on the internal wall face of chromatography column tube (10), abdicating structure (11) sets up to be able to when column cap (20) moves to abdicating structure (11) department the upper and lower both sides of column cap (20) communicate.
2. The chromatography column device according to claim 1, comprising a pump (40), said pump (40) being arranged in communication with said second lower channel (32); and/or
The abdicating structure (11) is formed by an upward chamfer formed on the inner wall surface of the top end of the chromatography column tube (10) and/or a downward chamfer formed on the inner wall surface of the bottom end of the chromatography column tube (10).
3. Chromatography column device according to claim 1, characterized in that it comprises a movable member which is arranged in the second lower channel (32) and which is able to effect a transition of the second lower channel (32) between the first state and the second state by moving or rotating.
4. The chromatography column device of claim 3,
the second lower channel (32) comprises a first section (321), a second section (322), a third section (323) and a fourth section (324) which are sequentially communicated from top to bottom, the inner diameter of the third section (323) is larger than that of the second section (322), the inner diameter of the second section (322) is larger than that of the first section (321) and that of the fourth section (324), and the third lower channel (33) is communicated with the third section (323);
the movable piece is a second lower pipe (35) which is coaxially inserted into the second lower channel (32) and can move along the axial direction of the second lower channel (32), the second lower pipe (35) comprises a first pipe section (351), a second pipe section (352) and a third pipe section (353) which are sequentially connected from top to bottom, the outer diameter of the second pipe section (352) is larger than that of the first pipe section (351), the outer diameter of the first pipe section (351) is larger than that of the third pipe section (353), the outer diameter of the first pipe section (351) is matched with the inner diameter of the second pipe section (322), the outer diameter of the third pipe section (353) is matched with that of the fourth pipe section (324), a first connecting hole (354) extending from the top surface of the first pipe section (351) to the bottom surface of the second pipe section (352) is formed in the second lower pipe section (35), and a plug (355) is arranged at the top end of the first pipe section (351), the plug (355) is provided with a second communication hole (356) for communicating the tube cavity of the second lower tube (35) and the second section (322), the outer diameter of the plug (355) is larger than the inner diameter of the first section (321) and smaller than the inner diameter of the second section (322), and the axial length from the top end of the plug (355) to the bottom end of the second section (352) is smaller than the axial length from the top end of the second section (322) to the bottom end of the third section (323).
5. Chromatography column arrangement according to any one of claims 1-4, characterized in that the column head (20) is provided with a second upper channel (22) communicating the upper and lower sides of the column head (20), the inner diameter of the second upper channel (22) being larger than the inner diameter of the first upper channel (21).
6. The chromatography column device of claim 5,
the column head (20) is provided with a third upper passage (23) which communicates the upper side of the column head (20) with the second upper passage (22), and the second upper passage (22) is arranged to have a third state of communicating with the lower side of the column head (20) and being disconnected from the third upper passage (23) and a fourth state of communicating with the third upper passage (23) and being disconnected from the lower side of the column head (20); and/or
And each channel is connected with a valve for controlling the on-off of the channel.
7. The chromatography column device of claim 6,
the chromatography column device comprises a control unit arranged to be able to control the movement of the column head (20); and/or
The chromatography column arrangement comprises a pressure detector arranged for detecting a pressure within the chromatography column tube (10).
8. A method of packing a chromatography column, comprising the steps of:
s1: injecting a predetermined amount of purified water into the chromatography column tube (10);
s2: pressing down the column head (20) to exhaust air bubbles from the chromatography column tube (10) and the column head (20);
s3: pumping homogenate into the chromatographic column tube (10), and synchronously moving the column cap (20) and the homogenate so as to keep positive pressure in the chromatographic column tube (10);
s4: and (3) performing column pressing on the chromatographic column tube (10).
9. The chromatography column packing method according to claim 8, wherein in the step S3, the pressure inside the chromatography column tube (10) is maintained at 0.1bar-0.3 bar; and/or
The column pressing method in the step S4 specifically includes: the column head (20) is depressed to a predetermined bed height.
10. The chromatography column packing method according to claim 8 or 9, wherein the method is performed using the chromatography column device according to any one of claims 1 to 7, and further comprising bubbling the second lower channel (32) and the third lower channel (33) before the step S3.
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| CN114033853A (en) * | 2021-10-29 | 2022-02-11 | 江苏汉邦科技有限公司 | Chromatographic column and three-position air cylinder |
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