WO2007121751A1

WO2007121751A1 - Double flow chromatography system

Info

Publication number: WO2007121751A1
Application number: PCT/DK2007/000191
Authority: WO
Inventors: Jan Marcussen; Peter Schultz
Original assignee: Jan Marcussen; Peter Schultz
Priority date: 2006-04-24
Filing date: 2007-04-23
Publication date: 2007-11-01

Abstract

The disclosed invention relates to a new liquid chromatographic method, wherein the flow direction of sample 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, preferably at least 1:0.2, more preferably at least 1:0.1 , even more preferably at least 1:0.04. The invention further relate 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.

Description

Title: Double Flow Chromatography System

Technical Field

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.

Background

The Russian botanist Michael Tsvet introduced the word chromatography in 1903. He was the first inventor in this area and developed methods to separate plant pigments. However, the major breakthrough was seen in 1940, when A.J.P. Martins and R.L.M. Synges published their supreme major contribution to the development of liquid chromatography. Their work resulted hereinafter in the Nobel Prize Award in chemistry in 1952.

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.

In the conventional liquid chromatographic system, the separating matrix is either packed or fluidised in a cylindrical tube, referred to as the column. In packed columns 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.

Well-established conventional liquid chromatographic systems are characterised by having a single flow direction that, from a geometrical point of view, is operated in a vertical flow direction.

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-

Y sorption chromatographic applications, compounds will initially be bound to the separating matrix in the application zone and often cause an unwanted rise in back pressure. This phenomenon is caused by the fact that available binding sides will first be occupied in the application zone and afterwards may reduce the overall flow rate through the column.

A rather high and unwanted back pressure is observed, when the length of the column is of a considerable size. The term, 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.

In affinity chromatography, a certain volume of the column matrix has a definite binding capacity. This often results in a sub-optimal utilisation of the matrix material as binding sites near the inlet are occupied first and subsequently reduce further flow to a degree, where new column material or elution buffer is needed to elute the column, even though the column contains free binding sites near the outlet.

To compensate for a reduced flow rate, due to application conditions, it is often described that it is beneficial to change the vertical flow direction from a downstream to an upstream flow direction.

As many liquid chromatographic processes are applied in purification proteins and other biomolecules, it is often desirably to keep the temperature within a certain limit. The low temperature partly prevents contamination and further minimises thermal- induced protein denaturation under the process.

To maintain an efficient cooling system in the conventional liquid chromatographic system, restrictions regarding the column diameter are limited due to temperature balances in the vertical cross section through the column under flow operation conditions. The same evidence is valid, when applying a sanitary process for sterilising the chromatographic system at an elevated temperature. In this case, the heat transfer will be directed either in a horizontal or in a vertical direction.

Furthermore, reducing the processing time in general as well as reducing buffer consumption provides major benefits in industrial processes, where chromatography is used.

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.

Accordingly, it may be summarised that there exist a constant need in the art for new column designs, new methods and new apparatuses for chromatography which have improved features in one or more areas, such as optimised utilisation of the separation matrix binding sites, providing a reduced back pressure (facilitating an increased flow rate), overall decreased time consumption (faster processing times in general), reduced buffer consumption, temperature control as well as possibilities to sterilise equipment after use.

Disclosure of the Invention

By the term "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.

By the term "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. In one embodiment 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. Referring to the column as a cylinder, the column has a "height", which is defined as the vertical distance enclosing the solid supporting ring. Furthermore, 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.

By the term "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. Preferably, the flow direction is from the periphery through the separation matrix towards the central column outlet.

By the term "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.

Surprisingly, it has been shown that 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. In this system, 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.

In one aspect 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.

Preferably, the separating matrix has a dimensional ratio between the column width and column height of at least 1 :0.2.

More preferably, the separating matrix has a dimensional ratio between the column width and column height of at least 1 :0.1.

Even more preferably, the separating matrix has a dimensional ratio between the column width and column height of at least 1 :0.04.

The higher the dimensional ratio between the column width and column height, the better separating power is obtained.

In one particular aspect 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.

By "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. Alternatively, 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.

In one aspect 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.

Thus, 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.

Brief Description of the Drawinq(s)

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). In position A the flexible parts (2) and (4) are in direct contact with the surfaces of the chromatographic column, whereby the flow is directed in an essentially horizontal direction. In 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.

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). Between the two movable parts (2) and (4), 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). Furthermore, in the second casing (9) 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. In 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. In 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.

Detailed description of the Invention

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. We have named the system "Double Flow Chromatographic System", for which DFCS is used as an abbreviation. In the following text, including examples, references will be designated to said abbreviation.

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 unique advantages, which are obtained according to the invention in comparison with conventional liquid chromatographic separation systems, are the following:

• overall reduced processing time

• optimised utilisation of available matrix binding sites in the application zone due to a unique geometrical design

• elevated flow rate • reduced back pressure

• reduced buffer consumption • more economical handling of separated samples, such as concentration and/or drying separated fractions

• possibility to install an efficient cooling system applicable in even very large columns • possibility to use said cooling system for sanitary purposes

Thus, in one aspect 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.

In one particular aspect, 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.

In another particular aspect, 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.

In yet another particular aspect, 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.

In one embodiment 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.

In one embodiment 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. In the first case, matrices suitable for liquid chromatography generally comprise derivatives of e.g. polysaccharides, acryl amide and mixtures thereof. In the second case, the supporting material is based on an inorganic support such as porous glass, silica- derivates or ceramic matrices.

In a particular aspect of the invention, 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. In this aspect, 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.

In a further aspect, the invention includes a method of liquid chromatographic separation of species present in a liquid sample comprising the steps of:

a) Applying said liquid sample to a chromatographic column having a dimensional ra- tio between the column diameter versus a 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 flow of liquid through the separation matrix, c) Optionally, applying an elution liquid, and d) Applying pressure providing an essentially vertical liquid flow through the separation matrix.

In a particular aspect of the invention, the liquid sample is applied to the peripheral part of the column. In a typical sample application mode of operation, due to the geometrical concept used, this will provide an enlarged application zone, compared to conventional liquid chromatographic systems. As a result, 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.

Thus, in one embodiment of the method according to the invention, at least one of the steps a) - d) is performed under temperature-controlled conditions. In another em- bodiment of the method according to the invention all of the steps a) - d) are performed under temperature-controlled conditions. Especially liquid chromatographic separations of proteins and other biomolecules require strict temperature control. In this embodiment of the method according to the invention the temperature is controlled to be in the range of 2 - 10⁰C. Preferably, the temperature is controlled to be 4°C.

In a number of liquid chromatographic applications, e.g. within pharmaceutical applications, sterilisation of equipment by heat treatment is essential. Thus, the method according to one aspect of the invention further comprise a step:

e) After finishing a separation process, the chromatographic column is heated to a temperature in the range of 70⁰C - 120⁰C.

Preferably, the column is heated in step e) to a temperature in the range of 9O⁰C - 11O⁰C.

In one aspect, 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. In a preferred embodiment, the outlet in the apparatus according to the invention is placed centrally with respect to the column matrix.

In order to switch the operation mode from an essentially horizontal to an essentially vertical flow direction, 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. Thus in one embodiment according to the invention 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.

In a preferred aspect of the invention, the apparatus comprises expandable sealing material (10) between the first (2) and the second part (4).

In one particular embodiment according to the invention, the apparatus further comprises means for temperature control of the column.

In one embodiment, 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.

In another embodiment, 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. Added to this, implementation of the DFCS-concept will furthermore raise possibilities to install efficient cooling and sterilising devices even in full-scale chromatographic processes.

The advantage of the DFCS-concept is based upon several factors. Among those ele- ments summarised above, the column design and the unique possibility to operate the system in either an essentially horizontal or an essentially vertical flow direction, contribute significantly to the above-mentioned advantages.

From a geometrical point of view, 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.

The low back pressure documented in the examples indicate that a higher flow rate may be utilised to accelerate the overall processing time without damaging the chromatographic matrix due to compression.

In the conventional chromatographic system it is not possible to maintain a stable temperature control during the process when the column diameter is broad. It is evident that the DFCS-concept overcomes this problem as the chromatographic column has a much larger contact area and a reduced height.

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.

In general, the packing of column material according to the invention does not differ from procedures used in conventional packaging of liquid chromatographic columns.

If the chromatographic matrix consists of fine particles, 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.

Model apparatus for liquid chromatography utilising the DFCS-concept

In this exemplary embodiment, an apparatus for liquid chromatography as described in Fig. 2 was used.

In the second casing (9), rows of bored holes containing screws for fastening the as- sembled parts were inserted and additionally rows of bored holes containing screws for positioning the mutually movable first and second part were inserted (11).

In both the first and second flexible part, a slot was inserted. Said slot function is to support a flexible sealing O-ring (10).

In this embodiment 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. Thereby, 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,

B a position, wherein the first and second flexible parts are separated from the liquid chromatographic column by liquid-filled cavities.

In this prototype 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.

After the column was properly inserted, the parts constituting the apparatus were as- sembled and screws referred to as (11) were tightened. The compressible sealing rings, coordinated with loosening and tightening of screws referred to as (11), functioned to direct the flow through the column in the desired mode. When screws are fully compressed, the flow direction into the column is in an essentially horizontal mode from the radius towards the central outlet.

After the operation mode for sample application was maintained for a period of time, 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. In this mode, 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).

In this mode of operation, the flat column design will favour a rapid elution and separation of adsorbed components and further reduce the back pressure.

All parts of the column were elaborated in stainless steel. However, other materials may be utilised. The prototype is operated manually, but it is obvious that the system may as well be operated automatically.

Experimental examples

In the following examples, 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.

After passing the chromatographic column, the solvent flow is directed to a quartz flow cell inserted in a spectrophotometer. In the following examples, 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.

All buffers are prepared according to "Data for Biochemical Research, ed. Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K.M., Oxford Science Publications 1986". Prior to connection to the system all buffers are degassed.

Prior to application all samples are filtrated through a 45 μm filter, obtained from MiIIi- pore US.

In the following examples, it is important to stress that neither attempts to optimise a certain purification strategy nor attempts to identify separated fractions have been taken.

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 quelled DEAE Sephadex, Pharmacia Sweden.

Sample for chromatographic separation comparison consisted of 100 μl bovine serum albumin (BSA) solution containing 0.05 g BSA/ml dissolved in a 10 mM deaerated Tris buffer pH 8.2.

Under sample application conditions the flow direction was directed in an essentially radial (or horizontal) direction through the separation matrix towards the central outlet in the DFCS-column.

Under elution conditions 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 following elution profile data were obtained using a 0.1 M NaCI solution at pH 4.0 as elution solution.

DFCS operated at a flow rate held at 5 ml/min. Elution characteristics:

Elution time (h) Elution volume (ml) Back pressure (bar)

Front 0.01 0.01 0.1

Peak i 0.43 130 0.1

Peak 2 5.00 1500 0.1

Peak 3 5.80 1740 0.1

Tailing 11.00 3300 0.1

Total 22.24 6670

XK 26 operated at a flow rate held at 5 ml/min. Elution characteristics:

Elution time (h) Elution volume (ml) Back pressure (bar)

Front 0.33 100 0.3

Peak i 0.83 250 0.3

Peak 2 6.50 1950 0.3

Peak 3 11.00 3300 0.3

Tailing 21.67 6500 0.3

Total 40.33 12100

As seen in this comparison between the elution profiles in the two systems, three peaks are recorded in both systems. This was unexpected as it was foreseen that two separated peaks were recorded in the conventional system due to the dynamic interaction between the monomer and the dimer fractions present in the sample. However, it is surprising that the very thin DFCS-column used in this example has the same capacity to separate the sample components as compared to the much higher conventional column.

However, the elution profiles clearly show, that the DFCS-concept in all cases give an accelerated elution with a composition corresponding to profiles recorded using a conventional chromatographic system. Elution profiles are recorded corresponding to elution time and elution volume. It is thus obvious that this invention contributes to more favourable processing conditions when handling biologically fragile specimens.

If the XK 26 column should be operated under identical back pressure conditions as in the DFCS-system, the back pressure should be reduced to 0.1 bar. To achieve such conditions, 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.

Example 2

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".

Under sample application conditions the flow direction was directed in an essentially radial (or horizontal) direction towards the central outlet in the DFCS-column.

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.

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:

Elution time (h) Elution volume (ml) Back pressure (bar)

Front 0.30 90 0.05

Peak 3.70 11 10 0.05

Tailing 8.00 2400 0.05

Total 12.00 3600

XK 26 operated at a flow rate held at 5 ml/min. Elution characteristics:

Elution time (h) Elution volume (ml) Back pressure (bar)

Front 1.70 510 0.90

Peak 7.00 2100 0.90

Tailing 15.30 4590 0.90

Total 24.00 7200

Example 3

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.

Both systems were packed with 48 ml gel and equilibrated with a 25 mM phosphate buffer pH 7.0. Samples of 4 ml desalted potato extract are prepared as described in example 2. Elution is performed with 200 mM methyl α-D- mannopyranoside, 25 mM phosphate buffer pH 7.0.

Under sample application conditions the flow direction was directed in an essential radial direction towards the central outlet.

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.

DFCS operated at a flow rate held at 5 ml/min. Elution characteristics:

Elution time (h) Elution volume (ml) Back pressure (bar)

A 4.5 1350 0.05

B 5.4 1620 0.05

C 0.3 450 0.05

D 1.5 450 0.05

Total 11.7 3870

XK 26 operated at a flow rate held at 5 ml/min. Elution characteristics:

Elution time (h) Elution volume (ml) Back pressure (bar)

A 6.6 1980 0.2-0.6

B 5.0 1500 0.6

C 3.0 900 0.6

D 3.3 990 0.5

Total 17.9 5370

In example 3, "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.

The comparison between the DFCS-system and a conventional system reported in this example shows that the two systems are in certain ways comparative under sample application conditions. However, a much higher back pressure is observed in the con- ventional system. Example 4

This example is a comparison between flow rates and back pressure when porous glass-plates are inserted into the DFCS-system. In this example 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. For an overview concerning silane coupling agents: "C. R. Lowe: "An introduction to affinity chromatography, Elsevier/ North Holland Biomedi- cal Press, 1979" may be consulted.

In this example, 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.

DFCS-svstem operated with porous glass of porosity "4":

Flow (ml/min). Back pressure (horizontal Back pressure (vertical flow) (bar) flow) (bar)

2.5 0 0

5.0 0 0

7.0 0.01 0

20.0 0.05 0

27.0 0.10 0.05

37.0 0.15 0.10

46.0 0.20 0.20

DFCS-svstem operated with porous glass of porosity "3":

Flow (ml/min). Back pressure (horizontal Back pressure (vertical flow) (bar) flow) (bar) 2.5 0 0

5.0 0 0

7.0 0.02 0

20.0 0.20 0.20

27.0 0.40 0.32

37.0 0.45 0.50

Without decimal, "0" indicates "no detectable back pressure".

Elution profiles documented in examples 1 , 2 and 3 clearly show that the DFCS- concept in all cases give an accelerated elution with a composition corresponding to profiles recorded using a conventional chromatographic system.

Elution profiles were recorded corresponding to elution time and elution volume. It is thus obvious that this invention contributes to more favourable processing conditions when handling biologically fragile specimens.

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.

In contrary to polysaccharide derivatised gels, which may be sensitive to changes in pH and ion strength conditions under various chromatographic processes, a solid porous glass or ceramic support will stay unaffected and thus contribute to a higher mechanical stability.

According to the above-mentioned examples, elution is performed in an essentially vertical direction and the fractionated components are in all examples favouring the DFCS-concept when compared with the conventional system. It is therefore concluded that there can be considerable advantages concerning not only elaborating chroma- tographic purification strategies for fragile components, but also in industrialised processes, where care has to be taken to the overall solvent consumption and processing time. This point may be correlated to economical and environmental viewpoints.

Claims

1. A method for chromatographic separating of species present in a liquid sample wherein the flow direction of sample is directed alternately in an essentially horizon- tal direction and in an essentially vertical direction through a chromatographic column having a dimensional ratio between the column width 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.

2. A method according to claim 1 comprising the steps of: a) applying said liquid sample to the chromatographic column, b) applying pressure providing essentially horizontal flow of liquid material through the separation matrix, c) optionally, applying an elution liquid, d) applying pressure providing essentially vertical flow of liquid sample ma- terial and/or the optional elution solution through the separation matrix.

3. Method according to claims 1 or 2 wherein the liquid sample is applied to the peripheral part of the column.

4. Method according to claims 2 or 3 wherein the liquid sample is eluted in step d).

5. Method according to any of claims 2 - 4 wherein at least one of the steps a) - d) is performed under temperature-controlled conditions.

6. Method according to claim 5 wherein all of the steps a) - d) are performed under temperature-controlled conditions.

7. Method according to claims 5 or 6 wherein the temperature is controlled to be in the range of 2 - 1O⁰C.

8. Method according to claim 7 wherein the temperature is controlled to be 4°C.

9. Method according to any of claims 1 - 8 further comprising a step e) wherein the chromatographic column after the separation process is heated to a temperature in the range of 70⁰C - 120⁰C.

10. Method according to claim 9 wherein the chromatographic column after the separa- tion process is heated to a temperature in the range of 9O⁰C - 110⁰C.

11. Apparatus for liquid chromatography comprising:

I. an inlet (1) for supplying a liquid sample,

II. a first part (2) having a first surface facing a chromatographic column (3) having a dimensional ratio between the column width 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 sec- ond surface facing said first surface, the first part and the second part being mutually movable in a vertical direction and,

III. 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,

IV. distributing means (6) for distributing liquid material to a peripheral part (7) of the column matrix, and

V. at least one outlet (8).

12. Apparatus according to claim 11 wherein the apparatus further comprise a second casing (9) enclosing the casing (5) and the first and second part.

13. Apparatus according to claims 11 or 12 wherein the distribution means (6) for distributing liquid material in the peripheral part of the column are provided by a peripheral channel (6) in the second part (4) connecting the inlet (1) with the peripheral part (7) of the column matrix.

14. Apparatus according to any of the claims 11 - 13 having expandable sealing material (9) between the first (2) and the second part (4).

15. Apparatus according to any of the claims 11- 14 wherein the outlet is placed centrally with respect to the column matrix.

16. Apparatus according to any of claims 11 - 15 wherein the first part (2) and the sec- ond part (4) are mutually movable between:

A. a position, wherein the first surface and the second surface are in direct contact with the separation matrix and,

B. a position, wherein at least the second surface is separated from the separation matrix by a liquid-filled cavity.

17. Apparatus according to claim 16 wherein the first part (2) and the second part (4) are mutually movable between:

B. a position, wherein the first surface and the second surface are sepa- rated from the separation matrix by liquid-filled cavities.

18. Apparatus according to any of the claims 11 - 17 further comprising means for temperature control of the matrix material.