WO2007121751A1 - système de chromatographie à double flux - Google Patents

système de chromatographie à double flux Download PDF

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Publication number
WO2007121751A1
WO2007121751A1 PCT/DK2007/000191 DK2007000191W WO2007121751A1 WO 2007121751 A1 WO2007121751 A1 WO 2007121751A1 DK 2007000191 W DK2007000191 W DK 2007000191W WO 2007121751 A1 WO2007121751 A1 WO 2007121751A1
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WIPO (PCT)
Prior art keywords
column
liquid
matrix
chromatographic
flow
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PCT/DK2007/000191
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English (en)
Inventor
Jan Marcussen
Peter Schultz
Original Assignee
Jan Marcussen
Peter Schultz
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Publication of WO2007121751A1 publication Critical patent/WO2007121751A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces
    • G01N30/6017—Fluid distributors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/14—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the introduction of the feed to the apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces
    • G01N30/6021—Adjustable pistons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/91—Application of the sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N2030/382—Flow patterns flow switching in a single column
    • G01N2030/385—Flow patterns flow switching in a single column by switching valves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N2030/386—Radial chromatography, i.e. with mobile phase traversing radially the stationary phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N2030/388—Elution in two different directions on one stationary phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N2030/521—Physical parameters form

Definitions

  • the disclosed invention relates to a new liquid chromatographic method.
  • the method is based on an apparatus and a column design for controlling the flow direction of a liquid sample alternately in either an essentially horizontal or in an essentially vertical direction through a chromatographic separating matrix.
  • liquid chromatography Since then liquid chromatography has become a major discipline and is routinely ap- plied in preparative and analytical chemistry. Today liquid chromatography is regarded as a keystone in pharmaceutically- and biotechnologically-based productions of highly purified end products.
  • the separating matrix is either packed or fluidised in a cylindrical tube, referred to as the column.
  • the flow is commonly operated in a downstream (vertical) direction, but may as well be directed in an upstream (vertical) direction.
  • An upstream flow direction is always used, when it is operated as a fluidised bed column.
  • the diameter and length of a conventional chromatographic column may be chosen according to specific applications. In general, however, the application zone is quite narrow due to the geometry. Whenever using such columns in any sort of liquid ad-
  • back pressure is here used as the pressure measured between the pump outlet and the column entrance.
  • Most commonly used separat- ing materials are based on various spherical polysaccharide or inorganic matrices which are often quite sensitive to compression. Therefore, a high back pressure has to be compensated by a corresponding reduction of the flow rate. This adjustment will slow down the overall processing time and will often rise the peak broadening resulting in dilution of separated fractions.
  • US5139680 discloses a chromatographic column having different separating properties in two directions. The sample flows repeatedly and alternately through the column in the two directions.
  • the column design is in other respects similar to conventional columns and is cylindrical with a ratio of the column thickness to the radial length of the column of greater than 1. Accordingly, the above drawbacks of conventional columns in terms of e.g. reduced back pressure, sub-optimal utilisation of the separation matrix binding sites, temperature control, reduced buffer consumption and reduced processing time are not addressed.
  • EP1396721 describes the manufacture of a chromatographic column, wherein the ratio of the thickness to the radial length of the column is smaller than or equal to 1. There is no mention of a chromatographic process, wherein the directional flow of samples may be changed.
  • liquid chromatography is meant a process of separating one or more species from liquid samples.
  • the separation may be carried out by means of for exam- pie adsorption or gel fractionation matrices which may separate said species in a more or less specialised way. Said separation may involve binding, retention or differential passage through the matrix based on e.g. size, charge, antibody binding etc.
  • the term liquid chromatography does not cover the general area of filtration, where a solid compound or particle is retained from a gas phase or a liquid suspension by means of a filter with a certain mesh size.
  • Gel filtration defined as matrix depending molecular weight size separation of sample components according to differences in molecular weight is considered a liquid chromatographic process.
  • column is meant a three-dimensional geometric object comprising a solid supporting structure, preferably a solid supporting ring and a separation matrix (column matrix).
  • a tightly woven net may be fixed to both sides of the supporting ring in order to support the column matrix.
  • the mesh size should allow free passage of liquids and retaining the separation matrix in the column.
  • the enclosed volume is filled with a chromatographic separation matrix material suitable for the specific chromatographic separation process. All known commercially available separation materials are compatible with the column design, but the design is not restricted to said materials.
  • a porous glass disk inserted into the solid supporting ring can serve as a permeable column-supporting matrix.
  • Said matrix may be chemically modified to resemble any known separation matrix structures. In case the matrix itself is solid, no net supporting the separation matrix is needed.
  • the column is preferably constructed as a cylinder, however, other geometrical designs can be envisaged, e.g. a sector of a circle.
  • the column has a "height", which is defined as the vertical distance enclosing the solid supporting ring.
  • the column has a diameter, which may be determined as two times the radial distance from the periphery to the central column outlet.
  • the column has two surfaces: one surface facing the inlet and one surface facing the outlet.
  • essentially horizontal flow is meant a liquid flow in a direction from the peripheral application zone towards the central outlet or, with an angle deviating less than 20° from parallel with respect to the surface of the separating column, facing the inlet.
  • the flow direction is from the periphery through the separation matrix towards the central column outlet.
  • essentially vertical flow is meant a liquid flow in a direction perpendicular or with an angle deviating less than 20° from perpendicular with respect to the surface of the separating column facing the inlet and passing in said direction through the separation matrix. The flow will be directed towards the central outlet.
  • chromatographic columns having a narrow elution distance as defined below through the vertical separating matrix are comparable with respect to separation characteristics as compared with conventional vertical chromatographic systems.
  • the narrow elution volume will give rise to a lower back pressure, as compared with conventional chromatographic systems, and thus enable the use of higher flow rates under washing and elution conditions.
  • the invention provides a unique new double-flow chromatographic system, which can be manufactured and used in a new chromatographic method.
  • the flow direction through the separating matrix is directed alternately in an essentially horizontal and in an essentially vertical flow direction through the separation matrix.
  • Such columns and methods provide beneficial improvements in more than one of the above-mentioned areas of in- terest in industrial processes, where liquid chromatography is used.
  • the disclosed invention relates to a new liquid chromatographic method, wherein the flow direction of samples is directed alternately in an essentially horizontal direction and in an essentially vertical direction through a separating matrix having a dimensional ratio between the column width and column height of at least 1 :0.5.
  • the separating matrix has a dimensional ratio between the column width and column height of at least 1 :0.2.
  • the separating matrix has a dimensional ratio between the column width and column height of at least 1 :0.1.
  • the separating matrix has a dimensional ratio between the column width and column height of at least 1 :0.04.
  • the invention provides a method for liquid chromatographic fractionation of species present in a liquid sample comprising the steps of: a) applying a liquid sample to a chromatographic column having a dimensional ratio between the column diameter and column height of at least 1:0.5, preferably at least 1 :0.2, more preferably at least 1 :0.1, even more preferably at least 1 :0.04, b) applying pressure providing an essentially horizontal liquid flow through the separation matrix, c) optionally, applying an elution liquid, and d) applying pressure providing an essentially verti- cal flow of equilibrating or elution solutions through the matrix.
  • applying pressure is meant that the liquid sample is forced through the column matrix by applying pressure, e.g. by use of a pump.
  • the directional control of the flow may e.g. be achieved by the design of the apparatus, where according to one aspect of the invention the presence or absence of a liquid filled cavity above the surface of the column determines the flow direction. When the cavity is absent, the flow is essentially horizontal, whereas the flow is essentially vertical when the cavity is present.
  • the column comprises a solid supporting ring and a separating matrix.
  • a tightly stretched net may be fixed on both sides of the solid supporting ring.
  • the cavity thus created enables the introduction of a suitable separation matrix.
  • the separation matrix consists of a solid but porous material such as a glass or ceramic supporting material. Such materials may be suitable for chemical modifications. In this case no net is needed.
  • the sample is applied to the periphery of the column, thus creating an essentially horizontal flow through the separation matrix towards the central outlet.
  • the invention further relates to an apparatus and a column design for controlling the flow direction of a liquid sample through a separating matrix in either an essentially horizontal or in an essentially vertical direction.
  • the invention provides an apparatus for liquid chromatography comprising an inlet (1) for supplying a liquid sample, a first part (2) having a first surface facing a chromatographic column (3), and a second part (4) having a second surface facing said first surface, the first part and the second part being mutually movable in a vertical direction and, a casing (5) peripherally enclosing said first and second part, whereby an enclosed cavity is defined by said casing, the first surface and the second surface of the first and second part, respectively, distributing means (6) for distributing liquid material to a peripheral part (7) of the column matrix, and at least one outlet.
  • the apparatus has two flexible parts facing the chromatographic column and by means of the inserted casings the two flexible parts will determine the presence or ab- sence of a liquid filled cavity above and below the column, and thereby direct the flow in either an essentially horizontal direction or in an essentially vertical direction towards the central outlet.
  • Fig. 1 shows a cross-sectional view of an apparatus according to one aspect of the invention.
  • the apparatus comprise an inlet (1) for applying a liquid sample, a first flexible part (2) having a first surface facing the liquid chromatographic column (3), and a sec- ond flexible part (4) having a second surface facing said chromatographic column.
  • the first part and the second part are mutually movable in a vertical direction.
  • the apparatus further comprise a casing (5) peripherally enclosing said first and second part and connecting solid column supporting ring to the casing (5). From the inlet (1) the liquid is distributed through a peripheral channel (6) for distributing liquid material to the periph- eral part (7) of the column matrix or the enclosed cavity.
  • the apparatus further comprise an outlet (8).
  • Fig. 2 shows a cross-sectional view of the prototype apparatus according to one aspect of the invention used in conducting the herein described examples.
  • the apparatus is similar to the apparatus of Fig. 1, and further comprise a second casing (9) enclosing the casing (5) and the first and second part.
  • the chromatographic column is inserted between the first (2) and the second part (4) and fixed to the casing (5).
  • expandable parts such as O-rings on each side (10) were inserted in a suitable slot. In position A these O-rings were completely compressed and the surface of the column was in direct contact with the surface of the movable part (4).
  • adjustment screws (11) were placed, for positioning the first part and the second part between the positions A, wherein the first surface and the second surface are in direct contact with the chromatographic separation column, and B, wherein at least the second surface is separated from the separation matrix by a liquid-filled cavity.
  • position A the flexible parts (2) and (4) are in direct contact with the surfaces of the chromatographic column, whereby flow is directed in an essentially horizontal direction.
  • position B the flexible parts (2) and (4) are moved with respect to the surfaces of the chromatographic column, creating a liquid-filled cavity, whereby the flow is directed in an essentially vertical direction.
  • This invention describes a hitherto unknown liquid chromatographic system, which allows the flow direction through the separation matrix to be directed alternately in an essentially horizontal and an essentially vertical direction.
  • DFCS Double Flow Chromatographic System
  • the column design is based on geometrically based viewpoints and exemplified in the following text. It is important to stress that this system does not in any way demand changes regarding purification protocols using conventional chromatographic systems, as the new system may be utilised using any known available separating materials and may even be used to develop new separating matrices. Benefits obtained by changing from a conventional chromatographic column configuration to a column configuration corresponding to this invention are summarised below.
  • the DFCS-concept is related to a new column design, where the flow direction, through the separating matrix, may be directed sequentially in either an essentially horizontal or an essentially vertical flow direction through the separating matrix.
  • the vertical mode of operation is similar to conventional liquid chromatographic systems, but results in lower back pressure as compared with conventional liquid chromatographic systems.
  • the chromatographic column comprises a solid supporting ring and two tightly woven net parts fixed and tightly stretched on both sides of the solid supporting ring.
  • the mesh size is chosen to allow free passage of liquids.
  • the flow direc- tion through the separating matrix is directed alternately in both an essentially horizontal and an essentially vertical direction.
  • the mesh functions to retain the separation matrix in the cavity between the two net surfaces.
  • the cavity between the two net surfaces is filled with a suitable separation matrix for a specific liquid chromatographic purpose. Access to said cavity during filling and removal of the separating matrix may be per- formed through a hole drilled in an angular correlation through the solid supporting ring. In this way, it gives free access to the column cavity.
  • the separating matrix may as well consist of a porous glass or ceramic plate optionally chemically treated to introduce an appropriate surface structure suitable for a specific liquid chromatographic separation. When using a glass or ceramic separating matrix developed as described, this may be inserted directly into the solid supporting ring without said woven net. Any known commercially available separation matrix materials or derivatives thereof as described, may be utilised in this aspect of the invention. If the separating matrix is an adsorption media, the unique geometry and the sample applica- tion direction in the periphery will optimise the utilization of available binding sites. In this aspect of the invention the back pressure is reduced which may be utilised to accelerate the chromatographic process at a higher flow rate.
  • the dimensional ratio between column diameters versus the column height is at least 1:0.5. Under sample application conditions the flow is directed from the periphery through the separating matrix creating an essentially horizontal flow direction through the separating matrix towards the central outlet. Under washing and elution conditions the flow is distributed through the narrow space between the column and the two flexible parts in an essentially vertical direction towards the central outlet. In both instances, a reduced back pressure is recorded.
  • the higher dimensional ratio between the column diameters versus the column height provides improvement in terms of the above specified, e.g. reduced back pressure.
  • the chromatographic column according to the invention pref- erably has a dimensional ratio between the column diameters versus the column height of at least 1:0.2.
  • the chromatographic column according to the invention preferably has a dimensional ratio between the column diameters versus the column height of at least 1 :0.1.
  • the chromatographic column according to the invention preferably has a dimensional ratio between the column diameters versus the column height of at least 1:0.04.
  • the chromatographic column according to the invention preferably has a dimensional ratio between the column diameters versus the column height of between 1 :0.5 and 1 :0.005.
  • the chromatographic column according to the invention preferably has a dimensional ratio between the column diameters versus the column height of between 1 :0.2 and 1:0.005.
  • Chromatographic media suitable for liquid chromatography may be divided into two major groups, where the supporting material is either a resin phase or a silica phase.
  • the supporting material is either a resin phase or a silica phase.
  • matrices suitable for liquid chromatography generally comprise derivatives of e.g. polysaccharides, acryl amide and mixtures thereof.
  • the supporting material is based on an inorganic support such as porous glass, silica- derivates or ceramic matrices.
  • the chromatographic column comprises a solid porous silica or ceramic supporting material to be chemically treated to develop a suitable liquid chromatographic separation matrix.
  • the chromatographic column according to the invention may comprise a separation matrix that is suitable for size exclusion chromatography when processing samples containing two or more substances with different molecular weights.
  • the invention provides special beneficial effects when alternately changing the flow direction from an essentially horizontal to an essentially vertical direction. Hereby, buffer and time consumption in the separation process are minimised.
  • the invention includes a method of liquid chromatographic separation of species present in a liquid sample comprising the steps of:
  • the liquid sample is applied to the peripheral part of the column.
  • this will provide an enlarged application zone, compared to conventional liquid chromatographic systems.
  • the back pressure during sample application is reduced, which in turn may facilitate the use of an increased flow rate.
  • Temperature control elements may be incorporated in the apparatus according to the invention.
  • the unique design of the chromatographic column and the apparatus according to the invention facilitate efficient temperature control of the material during processing. This is due to the increased surface area of the column and a reduction in the maximal distance from any point in the separation matrix to the surface resulting in a more efficient heat transfer and a more even temperature distribution through the separation matrix.
  • At least one of the steps a) - d) is performed under temperature-controlled conditions.
  • all of the steps a) - d) are performed under temperature-controlled conditions.
  • the temperature is controlled to be in the range of 2 - 10 0 C.
  • the temperature is controlled to be 4°C.
  • the method according to one aspect of the invention further comprise a step:
  • the chromatographic column is heated to a temperature in the range of 70 0 C - 120 0 C.
  • the column is heated in step e) to a temperature in the range of 9O 0 C - 11O 0 C.
  • the invention is an apparatus for liquid chromatography, said apparatus comprising: an inlet (1) for supplying a liquid sample, a first part (2) having a first surface facing the chromatographic column (3) having a dimensional ratio between the column diameter and column height of at least 1 :0.5, preferably at least 1 :0.2, more preferably at least 1 :0.1 , even more preferably at least 1 :0.04 and a second part (4) having a second surface facing the chromatographic column.
  • the two parts are mutually movable in a vertical direction.
  • the two movable parts are inserted in the casing (5) peripherally enclosing said first and second part, this part (5) also function to support the solid column supporting ring.
  • the upper flexible part is equipped with a distributing canal (6) for distributing liquid material to the peripheral part (7) of the column matrix or the liquid filled cavity, and directing the flow towards the central outlet (8).
  • the apparatus further comprises a second casing (9) enclosing the casing (5) and the first and second flexible parts.
  • the distribution means (6) for distributing liquid material to the peripheral part of the column and/or the enclosed cavity are provided by a peripheral channel in the second part (4) connecting the inlet (1) with the peripheral part (7) of the column.
  • the outlet in the apparatus according to the invention is placed centrally with respect to the column matrix.
  • the first part (2) and the second part (4) are mutually moved in a vertical direction. In one position there is essentially no space between the separating column and the first and second part. This position facilitates an essentially horizontal flow direction through the separating matrix. In another position there is a small liquid filled space between the chromatographic column and the first and second movable parts (2) and (4). This facilitates an essentially vertical flow direction through the separating matrix.
  • the first part (2) and the second part (4) are mutually movable between a position, wherein the first surface and the second surface are in direct contact with the separation matrix and, a position, wherein at least the second surface is separated from the separation matrix by a liquid- filled cavity.
  • the apparatus comprises expandable sealing material (10) between the first (2) and the second part (4).
  • the apparatus further comprises means for temperature control of the column.
  • inert woven fabrics are tightened on both sides of the solid column supporting ring (4). This configuration is chosen when the chromatographic separation matrix consists of fine particles.
  • the chromatographic matrix consists of a porous rigid material. In this case the inert woven fabrics are omitted.
  • This unique invention is shown by the examples to be able to accelerate the overall chromatographic process regarding processing time and solvent consumption.
  • the DFCS-concept is adaptable to any hitherto elaborated purification protocols by only changing the column and the process according to the invention described herein.
  • the implementation of this invention in full scale will thus benefit economically industrialised processes.
  • implementation of the DFCS-concept will furthermore raise possibilities to install efficient cooling and sterilising devices even in full-scale chromatographic processes.
  • the explanation may be correlated with the far broader adsorption zone found in the DFCS-concept as compared with the conventional system. This will contribute to a higher unhindered flow through the system under sample application, sample equilibration and sample elution conditions.
  • the optimised conditions in using the DFCS-concept are documented in the recorded reduction in back pressure as compared with a conventional system.
  • a temperature controlling system may be inserted in one or both flexible parts of the apparatus or in the casing. This system may act as a cooling system and/or a sterilising system used in sanitarian operations, where the chromatographic equipment is to be sterilised.
  • the packing of column material according to the invention does not differ from procedures used in conventional packaging of liquid chromatographic columns.
  • the column may be packed by filling the separation matrix into an assembled column through holes bored in the col- umn supporting ring.
  • the chromatographic column is conveniently inserted into the partly assembled apparatus and conveniently carried out in a suitable solvent such as the mobile phase.
  • a slot was inserted.
  • Said slot function is to support a flexible sealing O-ring (10).
  • the sealing O-rings provided force moving the first and second flexible part apart in a vertical direction when the pressure provided by the tightened screws (11) was released by loosening said screws. Positioning of the first and second part could then be achieved by tightening/loosening the screws.
  • the first part (2) and the second part (4) were mutually movable between:
  • A a position, wherein the first flexible surface and the second flexible surface are in direct contact with the liquid chromatographic column and,
  • the second part (4) was constructed as two items fasten together by screws.
  • the two items were constructed as drilled items.
  • the upper item contained a central bored hole, containing a tubing inlet (1).
  • the tubing inlet was connected to the liquid distributing canal (6) and the cavity formed by the two items created the means for distributing liquid material to a peripheral part (7) of the chromatographic column or to the liquid filled cavity.
  • a packed column was inserted into the partly assembled apparatus. This operation was carried out in a suitable buffer solution.
  • the chromatographic column was tightened to the casing (5) by inserting screws, connecting the solid column supporting ring to said casing.
  • the sample was eluded and further separated by changing the flow direction from an essentially horizontal to an essentially vertical direction, while elution solution was applied.
  • This operation mode was obtained by loosening screws, referred to as (11 ), in the upper and lower part of the column supporting body.
  • the flexible parts (2) and (4) will be moved upward and downward, and create a liquid-filled space between the chromatographic column and the first and second parts (2), and (4) will direct the flow in an essentially vertical flow direction through the chromatographic column (3).
  • the flat column design will favour a rapid elution and separation of adsorbed components and further reduce the back pressure.
  • a standard liquid chromatographic set-up is used. This consists of a precision adjustable pump delivering solvents to the system inlet. The delivery is distributed through a four-way valve operated to take precautions not to introduce air into the system. Said valve is also used for sample application purposes and for changing solvents. Between the pump outlet and the column entrance there is inserted a manometer to measure the back pressure. Measurements are recorded in bar.
  • the solvent flow is directed to a quartz flow cell inserted in a spectrophotometer.
  • recordings are measured at 280 nm. Recordings are made either as a written graph of the actual absorption corresponding to time or directly by recording the absorption numerical at said wavelength corresponding to time.
  • the column outlet is connected to a one-way valve.
  • the outlet-valve is operated when individual parts of the DFCS-concept are connected as described above.
  • the DFCS-column used in the examples is cylindrical with a diameter of 14.27 cm and a height of 0.50 cm or 0.30 cm (example 3). This means that the dimensional ratio be- tween the column width and column height is 1 :0.035 and 1 :0.021 respectively.
  • a conventional column used in comparison is the XK 26 column from Pharmacia Sweden.
  • the supplier describes the XK 26 column.
  • Example 1 Examples are given to document the difference between a conventional chromatographic system and the DFCS-system. The comparison comprises: elution volume, elution time and back pressure. All experiments are carried out at ambient temperature.
  • Example 1
  • This example is a comparison of ion exchange profiles obtained using a conventional chromatographic column XK 26 obtained from Pharmacia Sweden and a column and method according to the invention. Both systems were packed with 80 ml skad DEAE Sephadex, Pharmacia Sweden.
  • Sample for chromatographic separation comparison consisted of 100 ⁇ l bovine serum albumin (BSA) solution containing 0.05 g BSA/ml dissolved in a 10 mM deaerated Tris buffer pH 8.2.
  • BSA bovine serum albumin
  • the flow direction was directed in an essentially radial (or horizontal) direction through the separation matrix towards the central outlet in the DFCS-column.
  • the DFCS-column is operated to change the flow direction to be directed in essentially a vertical direction through the separation matrix.
  • the conventional chromatographic column XK 26 was conventionally operated under both application and elution conditions, i.e. the flow was in a vertical direction.
  • Fronts and tailing were recorded as increase or decrease in 10% recordings corresponding to the total maximal recorded absorption peaks.
  • the back pressure should be reduced to 0.1 bar.
  • the flow rate should be reduced to 1.7 ml/min. Such reduced flow rate will result in unwanted peak bordering and the overall processing time and buffer consumption will be increased.
  • This example is a comparison of ion exchange elution profiles obtained using a con- ventional chromatographic column XK 26 obtained from Pharmacia Sweden and a DFCS-column both packed with 80 ml DEAE Sephadex ion exchanger medium.
  • the samples applied to the two columns are identical in composition and consist of 5 ml desalted potato extract applied in a 25 mM phosphate buffer pH 7. Preparation of the extract is performed according to Racusen, D. and. Foote, M.: "A Major Soluble Glycoprotein of Potato Tubers, Journal of Food Biochemistry, 1980, vol. 4 p. 43-52".
  • the following data on elution profiles are obtained with a phosphate buffer 0.25 M NaCI pH 7.0.
  • the front is recorded as the point, where a 50% increase of the maximal recorder response is recorded.
  • the tail is recorded as the point, where a decrease corresponding to 50% of the maximal recorder response is obtained.
  • the peak is recorded as the average between front and tailing positions.
  • DFCS-column Under elution conditions the DFCS-column is operated in the same way as described in example 1, i.e. in an essentially vertical flow direction. DFCS-column operated at a flow rate held at 5 ml/rnin. Elution characteristics:
  • This example is a comparison between application and elution profiles obtained using a Con A Sepharose matrix obtained from Amersham Pharmacia Biotech AB in the DFCS system, and a conventional chromatographic XK 26 column as described above.
  • the DFCS-column Under elution conditions the DFCS-column is operated in an essential vertical mode of operation as described in example 1 and 2.
  • the liquid filled space above and below the column matrix material provided by loosening the screws (10) was 0.50 mm high.
  • the DFCS-column used in this example had a diameter of 14.27 cm and a height of 0.30 cm.
  • A refers to conditions under sample application, including washing with equilibrating buffer before the non-adsorbed fraction
  • B is eluted
  • C refers to conditions from initiation of buffer change from equilibrating buffer to elution buffer
  • D refers to conditions regarding elution of the adsorbed fraction.
  • This example is a comparison between flow rates and back pressure when porous glass-plates are inserted into the DFCS-system.
  • porous glass-plates were obtained from Duran, Germany. Two porosity grades, "4" and "3", are compared.
  • the chromatographic supporting material has a silanol-surface, which makes it suitable for further chemical treatments.
  • silane coupling agents "C. R. Lowe: "An introduction to affinity chromatography, Elsevier/ North Holland Biomedi- cal Press, 1979” may be consulted.
  • a column diameter of 14.27 cm with a height of 0.5 was inserted in the solid supporting ring.
  • the DFCS-system was operated in either an essentially horizontal or in an essentially vertical flow direction and compared to measured back pressure. When operated in a vertical flow direction the upper and lover space between the porous glass-plate is corrected to 0.5 mm. Measurements were performed with deionised water.
  • Example 4 shows that the back pressure is correlated to the porosity and the flow distribution being directed in an either essentially horizontal or in an essentially vertical direction through the chromatographic matrix.

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Abstract

La présente invention concerne un nouveau procédé chromatographique liquide, selon lequel la direction de flux d'un échantillon est dirigée alternativement dans une direction sensiblement horizontale et dans une direction sensiblement verticale à travers une matrice de séparation ayant un rapport dimensionnel entre la largeur de colonne et la hauteur de colonne d'au moins 1:0,5, de préférence d'au moins 1:0,2, mieux encore d'au moins 1:0,1, idéalement d'au moins 1:0,04,. L'invention concerne en outre un appareil et une conception de colonne permettant de commander la direction de flux d'un échantillon liquide à travers une matrice de séparation soit dans une direction sensiblement horizontale, soit dans une direction sensiblement verticale.
PCT/DK2007/000191 2006-04-24 2007-04-23 système de chromatographie à double flux WO2007121751A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3482376A (en) * 1968-06-26 1969-12-09 Atomic Energy Commission Apparatus for continuous chromatographic separations
EP0169951A1 (fr) * 1984-07-30 1986-02-05 Varex Corporation Système et appareil pour la chromatographie multidimensionnelle en temps réel
US4676898A (en) * 1985-11-04 1987-06-30 Sepragen Corporation Chromatography column using horizontal flow
US5139680A (en) * 1991-06-05 1992-08-18 Yuri Tarnopolsky Method for continuous multicomponent separation using anisotropic separation bed
US5433847A (en) * 1989-11-01 1995-07-18 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Radial flow chromatography
US20010032814A1 (en) * 2000-01-27 2001-10-25 Kearney Michael M. Shallow bed fluid treatment apparatus

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3482376A (en) * 1968-06-26 1969-12-09 Atomic Energy Commission Apparatus for continuous chromatographic separations
EP0169951A1 (fr) * 1984-07-30 1986-02-05 Varex Corporation Système et appareil pour la chromatographie multidimensionnelle en temps réel
US4676898A (en) * 1985-11-04 1987-06-30 Sepragen Corporation Chromatography column using horizontal flow
US5433847A (en) * 1989-11-01 1995-07-18 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Radial flow chromatography
US5139680A (en) * 1991-06-05 1992-08-18 Yuri Tarnopolsky Method for continuous multicomponent separation using anisotropic separation bed
US20010032814A1 (en) * 2000-01-27 2001-10-25 Kearney Michael M. Shallow bed fluid treatment apparatus

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