U.S. patent application number 11/879126 was filed with the patent office on 2008-01-24 for chromatography columns, systems and methods.
This patent application is currently assigned to GE HEALTHCARE BIO-SCIENCES AB. Invention is credited to Klaus Gebauer.
Application Number | 20080017580 11/879126 |
Document ID | / |
Family ID | 36998321 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080017580 |
Kind Code |
A1 |
Gebauer; Klaus |
January 24, 2008 |
Chromatography columns, systems and methods
Abstract
The present invention relates to axial flow chromatography
columns, methods for separating one or more analytes in a liquid by
the use of such columns, and systems employing such columns. The
column comprises a first port and a second port, the first port and
said second port being at essentially the same level or elevation
above the level of the bed space on the chromatography column.
Inventors: |
Gebauer; Klaus; (Uppsala,
SE) |
Correspondence
Address: |
GE HEALTHCARE BIO-SCIENCES CORP.;PATENT DEPARTMENT
800 CENTENNIAL AVENUE
PISCATAWAY
NJ
08855
US
|
Assignee: |
GE HEALTHCARE BIO-SCIENCES
AB
UPPSALA
SE
|
Family ID: |
36998321 |
Appl. No.: |
11/879126 |
Filed: |
July 16, 2007 |
Current U.S.
Class: |
210/656 ;
210/198.2 |
Current CPC
Class: |
B01D 15/22 20130101;
G01N 30/56 20130101; G01N 30/6004 20130101; B01D 15/18 20130101;
G01N 2030/565 20130101; G01N 30/6017 20130101; G01N 30/8658
20130101; G01N 30/606 20130101; B01D 15/206 20130101; G01N 30/6021
20130101; G01N 30/6052 20130101 |
Class at
Publication: |
210/656 ;
210/198.2 |
International
Class: |
B01D 15/22 20060101
B01D015/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 19, 2006 |
GB |
0614315.0 |
Claims
1. An axial flow chromatography column comprising: a housing
comprising a side wall; axially spaced first and second end units
positioned opposed to each other are separated by said side wall; a
first filter which is adjacent to said first end unit and a second
filter which is adjacent to said second end unit wherein said first
and second filters together with the side wall define an enclosed
bed space for containing a bed of particulate medium therein; the
first end unit including a first port which is in fluid
communication with said enclosed bed space for adding a liquid to
or removing a liquid from the bed space; the second end unit
including a second port which is in fluid communication with the
enclosed bed space for adding a liquid to or removing a liquid from
the bed space; and said first port and said second port are at
essentially the same level or elevation above the level of the bed
space on said chromatography column.
2. The chromatography column of claim 1, wherein the second port is
in fluid communication with the bed space by means of a hollow
member connected to a passageway in the second unit.
3. The chromatography column of claim 2, wherein said hollow member
is an integral part of the column.
4. The chromatography column of claim 2, wherein said passageway
extends from the bed space through a lateral wall of the second end
unit.
5. The chromatography column of claim 1, wherein the first end unit
additionally includes a valve means which is in fluid communication
with the enclosed bed space, the valve means includes a
longitudinal member extending through said first filter and having
a passageway therein, the valve being operably openable and
closable to allow filing of the bed space with the particulate
medium through said passageway.
6. The chromatography column of claim 5, wherein said longitudinal
member of said valve means comprises a nozzle.
7. The chromatography column of claim 6, wherein said nozzle is
fixed within the bed space or retractable to a position outwith the
bed space.
8. The chromatography column of claim 1, wherein said valve means
does not allow emptying of the bed space of particulate medium.
9. The chromatography column of claim 1, wherein said column is
pre-packed with particulate medium.
10. The chromatography column of claim 8, wherein said column is a
disposable column.
11. A method for separating one or more analytes in a liquid from
each other, comprising: applying said liquid containing said one or
more analytes to an axial chromatography column of claim 1, said
column containing a bed of particulate medium therein; eluting said
one or more analytes with a mobile phase; and collecting fractions
of said mobile phase eluting from the column.
12. A system for separating one or more analytes in a liquid from
each other, said system comprising: an inlet or inlet manifold in
fluid communication with said liquid; a pump; a chromatography
column of claim 1; and an outlet or outlet manifold.
13. The system of claim 12, further comprising a valve operably
openable and closable to allow the addition of liquid to, or the
removal of liquid from, the column bed space.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to axial chromatography
columns and methods of separating one or more analytes present in a
liquid from each other using such chromatography columns.
BACKGROUND OF THE INVENTION
[0002] Chromatography is a well-established and valuable technique
for separating chemical and biological substances and is widely
used in research and industry, finding many applications in
compound preparation, purification and analysis. There are many
different forms of chromatography, liquid chromatography being of
particular importance in the pharmaceutical and biological
industries for the preparation, purification and analysis of
proteins, peptides and nucleic acids.
[0003] A typical liquid chromatography apparatus has an upright
housing in which a bed of packing material, which is usually
particulate in nature and consists of a porous medium, rests
against a permeable retaining layer. A liquid mobile phase enters
through an inlet, for example at the top of the column, usually
through a porous, perforated filter, mesh or frit, moves through
the bed of packing material and is removed via an outlet, typically
through a second filter, mesh or frit.
[0004] Columns used in liquid chromatography typically comprise a
tubular body enclosing the porous chromatography medium through
which the carrier liquid or mobile phase flows, with separation of
substances or analytes taking place between the mobile phase and
solid phase of the porous medium. Typically, the porous medium is
enclosed in the column as a packed bed, generally formed by
consolidating a suspension of discrete particles, known as slurry
that is pumped, poured or sucked into the column, usually from a
central bore or nozzle located at one end of the column. The
production of a stable, even bed is critical to the final
separation process and optimum results are found using bores which
are centrally positioned through the column end. Systems for
producing such stable, even beds are known in the art and include,
for example, compressing the bed by means of an end unit.
[0005] Following column packing and prior to use it is necessary to
install the column by connecting it to a chromatography system,
usually comprising a pump, detectors and inlet and outlet
manifolds. When installing the column, it is essential to avoid any
draining of the column from liquid as well as to avoid introduction
of air into the column and the packed bed, respectively.
Furthermore, it may be necessary to purge the system employing the
column of any air upstream of the column. Typically, prior art
columns and systems accomplish column installation and/or purging
or venting by means of a valve (a "vent" or "purge" valve) at one
end of the column, usually at its base.
[0006] In practice, the valve is used together with the column,
which means that the column is connected and disconnected from the
system upstream of the valve. For a disposable column, the
introduction of such an additional valve is costly. By virtue of
its position, it increases both the risk of siphoning of liquid
from the column and also the likelihood of further air being
introduced into the column. Furthermore, the position of the valve
imposes constraints on locating the column within the laboratory
and can cause problems in terms of user accessibility, since access
to both the top and bottom of the column is required.
[0007] JP 63293456 (Sekisui Chemicals Co.) describes a column
holder to support columns having been prepared with a stationary
phase and being equipped with a discharge aperture at the base and
a ventilation device at the top. The column holder is designed such
that on the application of gravity or a centrifugal force, to
facilitate chromatographic separation of materials on the
stationary phase, any surplus fluid remaining above the stationary
phase can be removed without allowing air to enter the stationary
phase within the column. The discharge aperture is connected to a
conduit which is branched at a position that is at the same or a
higher level than the upper end of the stationary phase solution
and has three open ends, a first at the connection point with the
discharge aperture, a second at a position above the surface level
of the solution and a third located below the level of the
discharge aperture. The conduit is configured such that any surplus
fluid can be removed from the column through the third open end
where it is collected in an acceptor vessel.
[0008] Despite the high level of activity in the field of
chromatography over many years there is still a need for a simple
axial column that obviates the need for a vent valve or complex
column holder and reduces the risk of air entering the column
and/or liquid siphoning from it. To date, no axial chromatography
columns are available which meet this need and/or provide improved
user accessibility.
Definitions
[0009] "Analyte" shall be defined as a substance, compound or
chemical of natural or synthetic origin, or a reaction product or
derivative or metabolite thereof. For the avoidance of doubt, the
term shall include biological molecules, such as proteins,
peptides, amino acids and nucleic acids, and synthetic molecules
such as drugs and/or pro-drugs.
[0010] "Distribution channel" refers to structures through which
fluids are introduced to an enclosure or bed space for a packed bed
of chromatography medium from a cross-sectional zone.
[0011] A disposable column is characterized by a pre-treatment of
the chromatography medium in order to reduce installation and
qualification work otherwise required with non-disposable columns.
As a minimum, the pre-treatment involves the formation of the bed
of porous medium. Additional pre-treatment can be reduction of
microbiological burden, sterilization, depyrogenation etc.
[0012] Disposable column may be used as single-use columns, which
means that the user is not performing cleaning regimes that require
qualification (e.g. testing, validation, etc) of the packed bed
before repeated use.
[0013] One embodiment of a disposable column is a complete column
that is delivered pre-packed with chromatography medium.
[0014] Another embodiment of a disposable column consists of a
first device representing a frame or vessel designed to resist
pressure and load exerted on one or multiple lateral surfaces of
the packed bed during operation in order to provide dimensional
stability for a packed bed, and a second device, representing a
container, shell, cartridge, bag or the like containing the porous
medium or bed that is attached to the first device for operation.
With the latter embodiment, the porous medium is contained in the
secondary container and can be replaced while the frame is
re-usable. In this case, the degree of compression of the porous
medium required for operation may be adjusted after inserting the
contained medium into the frame (see, for example US2002/0166816,
Allen & Dawson and WO2005/009585, Sigma-Aldrich Co.).
[0015] "Level" shall be defined as a horizontal plane or specified
height.
SUMMARY OF THE INVENTION
[0016] The object of the invention is to provide a chromatography
column which overcomes the drawbacks of the prior art systems. This
is achieved by the chromatography column as defined in claim 1.
[0017] One advantage with such a chromatography column is that it
reduces the risk of siphoning from the column.
[0018] Another advantage of the invention is that it is easy and
cheap to produce, compared with existing columns that required an
adjacent purge valve. This is especially important when using the
column as a disposable column.
[0019] A further advantage of the chromatography column is that it
is easier to use than conventional columns because both the first
port and second port which provide an inlet and an outlet for
liquids such as mobile phase are at essentially the same level or
elevation above the level of the bed space on one end of the column
and thus provide improved user accessibility.
[0020] The simplicity of the design comprising few interconnected
elements is advantageous in that it reduces the requirement for
dynamic seals tightening and moving or rotating parts, as required
for a (disposable) purge valve, hence decreasing the likelihood of
leakage or contamination due to sanitary problems. These features
are especially important for disposable columns comprising a
reduction of microbiological burden during preparation (production)
of the column, as well as to maintain those conditions during
storage and shipping of the column.
[0021] Still a further advantage is that the chromatography column
is scalable in that increasing or decreasing the column size leads
to a predictable performance.
[0022] According to a first aspect of the invention, there is
provided an axial flow chromatography column comprising:
a housing comprising a side wall; opposed, axially spaced first and
second end units separated by the side wall; a first filter which
is adjacent to the first end unit and a second filter which is
adjacent to the second end unit wherein the filters together with
the side wall define an enclosed bed space for containing a bed of
particulate medium therein; the first end unit comprising a first
port which is in fluid communication with the enclosed bed space
for adding a liquid to or removing a liquid from the bed space; the
second end unit comprising a second port which is in fluid
communication with the enclosed bed space for adding a liquid to or
removing a liquid from the bed space; and characterised in that the
first port and the second port are at essentially the same level or
elevation above the level of the bed space on the chromatography
column.
[0023] In a second aspect, there is provided a method for
separating one or more analytes in a liquid from each other,
comprising applying the liquid containing the one or more analytes
to an axial chromatography column as hereinbefore described, the
column containing a bed of particulate medium therein, eluting the
one or more analytes with a mobile phase, and collecting fractions
of the mobile phase eluting from the column.
[0024] In a third aspect of the invention, there is provided a
system for separating one or more analytes in a liquid from each
other, the system comprising:
an inlet or inlet manifold in fluid communication with the liquid;
a pump; a chromatography column as hereinbefore described; and an
outlet or outlet manifold.
[0025] Embodiments of the invention are defined in the dependent
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a schematic transverse sectional view of a
chromatography column of the prior art showing the basic features
thereof.
[0027] FIG. 2a, FIG. 2b and FIG. 2c are simplified schematic
diagrams illustrating prior art (FIGS. 2a and 2b) columns and a
column in accordance with the invention (FIG. 2c).
[0028] FIG. 3 is a transverse section of a column in accordance
with the invention.
[0029] FIG. 4 is a three-dimensional schematic of a chromatography
column according to the invention.
[0030] FIG. 5a is a schematic diagram of a system using a column
known in the prior art.
[0031] FIG. 5b is a schematic diagram of a system employing a
column in accordance with the invention.
[0032] FIG. 6 is a chromatogram showing the chromatographic
separation of acetone on a chromatography column according to the
invention, both in upflow (dotted line) and downflow (solid line)
mode.
[0033] FIG. 7 describes a method for calculating the reduced plate
height and asymmetry factor from an eluted peak.
DETAILED DESCRIPTION OF THE INVENTION
[0034] FIG. 1 shows schematically the general components of a
chromatography column 1 as known from the prior art (for example,
see U.S. Pat. No. 6,524,484). The column has a cylindrical
fluid-impermeable side wall 11, e.g. of stainless steel or a
high-strength/reinforced polymeric material which may be
translucent. The open top and bottom ends of the side wall 11 are
closed by top and bottom end assemblies or units 12, 13. Each end
unit has a fluid-impermeable end plate 3 fitting sealingly to plug
the opening of the cylindrical wall 11, and preferably made of
stainless steel or high-strength engineering plastics material,
e.g. polypropylene. The end plates are backed up by metal retaining
plates 2 bearing against their outer surfaces and projecting
radially beyond the side wall as retaining flanges 22 through which
adjustable tension rods 14 are secured. These link the top and end
assemblies 12, 13 and help the construction to withstand high fluid
pressures.
[0035] Each end plate 3 has a central through-opening 31 for
communication between the exterior of the column and the packing
bed space 9 defined by the side wall 11 and end assemblies 12, 13.
Access through the opening 31 is subdivided into separate conduits,
connected externally through a connection manifold 8.
[0036] A filter layer 4, typically of filtered or woven plastics or
steel, extends across the area of the bed space 9 at the inner
surface of the end plate 3. The inner surface 35 of the end plate 3
is recessed behind the filter layer 4, e.g. conically as
illustrated, and preferably with the use of support ribs (not
indicated) supporting the filter layer 4 from behind, to define
between them a distribution channel 34. One of the communication
conduits, a mobile phase conduit 33, opens inwardly into this
distribution channel 34, as well as outwardly to a mobile phase
connector 81 of the manifold 8.
[0037] From the manifold 8, an access valve device 5 projects
inwardly through the end plate opening 31 and sealingly through a
central orifice 41 of the filter layer 4. The access valve 5,
governs the communication of one or more conduits from the manifold
8 directly to the bed space 9, i.e. bypassing the filter layer 4.
Indicated here are first and second valved conduits 51, 61 governed
by the valve 5, and connected externally through connectors 82 of
the manifold 8.
[0038] In a typical operation of the column, a packed bed of
particulate stationary phase material fills the bed space 9 between
the top and bottom filter layers 4. The valve devices 5 being
closed, a mobile phase is fed in through mobile phase connector 81
(arrow "A"), passes through conduit 33 into the distribution
channel 34 and through the filter layer 4 to elute down through the
packed bed, effecting separation of its components or analytes.
Liquid eluate passes thought the filter layer 4 of the bottom end
assembly 13 and out through the mobile phase connector 81 thereof
(arrow "B") for collection as appropriate. While this is an example
of "downflow" chromatography, in that chromatographic separation is
effected by the downward movement of the mobile phase through the
column, the skilled person will understand that separation may
alternatively be achieved by "upflow" chromatography, simply by
pumping mobile phase upwards through the column and thus reversing
the direction of flow. In this mode, mobile phase would enter the
column at connector 81 (arrow "B"), move upwards through the
stationary phase or particulate medium, and be collected from
connector 81 (arrow "A") at the top of the column.
[0039] When installing the column, it is essential to avoid any
draining of the column from liquid as well as to avoid introduction
of air into the column and the packed bed, respectively.
Furthermore, it may be necessary to purge the system employing the
column of any air upstream of the column. This is achieved by means
of a vent valve 5 which is located at the bottom of the column.
[0040] FIG. 1 and the above explanation are to illustrate general
relationships of components and a typical mode of operation. The
skilled person will understand, and it will also appear from the
following description, that other specific constructions and modes
of operation may be appropriate for different kinds of process.
[0041] FIGS. 2a and 2b show simplified and schematic
representations of the configuration of a prior art column. The
column 101 has a first port 133 and a second port 140 for the
introduction or collection of mobile phase. After installation of
the column and prior to use, air needs to be removed from the
column by means of venting/purging. This may be achieved by means
of a vent valve 105 with a vent outlet which is either an integral
part of the column 101 (as shown in FIG. 2b) or is connected to it
at a point beyond the second port 140 (not shown in FIG. 2a).
[0042] FIG. 2c is a simplified and schematic representation of a
configuration of a column 101 in accordance with the invention. The
first port 133, which is intended for the introduction or
collection of mobile phase, is located at the top of the column 101
(as with columns of the prior art). The second port 140, which is
in fluid communication with the bottom of the column, is at
essentially the same level or elevation as the first port 133. This
is achieved by means of a hollow member 160 which allows fluid such
as air or mobile phase to flow between the bottom of the column 101
and second port 140. This configuration eliminates the need for a
vent valve and reduces the risk of both siphoning from the column
and introducing air into the column.
[0043] FIG. 3 shows a transverse sectional view of a column in
accordance with the invention. The column 201 comprises a tubular
housing 211, a first end unit 212 (partially shown) and a second
end unit 213, secured together to form a fluid tight seal by means
of tension rods 214 with heads 216. First filter 204 and second
filter 206 are adjacent to the first end unit 212 and second end
unit 213, respectively. These filters 204, 206, together with side
wall 211, define a bed space 209 for containing a bed of
particulate medium.
[0044] The housing 211 and end units 212, 213 are typically
composed of stainless steel or a high-strength plastic material
such as polypropylene. In a preferred embodiment, where the column
is to be used for the separation of biologically active substances,
the material is biologically inert such that it does not elicit an
immune response in humans in accordance with United States
Pharmacopia (USP) <88> class VI. Tension rods 214, with heads
216, secure the end units 212, 213 to the housing 211 to form a
fluid-tight bed space 209 which is capable of withstanding high
operating pressures.
[0045] Valve means 220 and first port 233 are shown in the figure.
The second port 240 comprises a passageway 242 which extends
through second end unit 213 to, and is in fluid communication with
(via hollow member 260), bed space 209 from which liquid can be
added or collected. As is evident from the figure, the second port
240 is at essentially the same level or elevation as the first port
233, thus facilitating the addition and collection of mobile phase
to/from the column. This arrangement has further advantages in that
it assists in the installation of the column, decreases the risk of
siphoning and reduces the likelihood of introduction of air into
the column.
[0046] The column can be packed with particulate medium in the form
of a slurry through valve means 220, the valve means 220 comprising
a central bore 221 and nozzle 224. A bed of packed particulate
medium is obtained by conventional means well known in the art, for
example by the movement of one of the end units to compress the
bed. In FIG. 3 the nozzle 224 is shown in its retracted position
but it will be understood that it can be moved to a position within
the bed space 209 to facilitate filling of the column. A wide range
of nozzles can be used which facilitate the distribution and even
packing of slurry within the bed space. One alternative for
achieving an open/closed functionality at the packing valve and
nozzle respectively is to have a nozzle that is fixed in the bed
space (and thus not retractable) and located adjacent to a moveable
element or sleeve on the inside or outside of the nozzle that opens
and/or closes the nozzle depending upon its position. Filters 204,
206 are each positioned on the interior face of the end units 212,
213 and act to define the bed space (together with side wall 211)
and to prevent leakage of particulate medium from the bed space
209.
[0047] Mobile phase or liquid containing one or more analytes or
substances for separation on the column is added via first port
233. The liquid then passes through the filter 204 into the bed
space 209 that is packed with particulate medium (not shown).
Chromatographic separation of analyte(s) which has been introduced
onto the particulate medium in this manner is effected by
introduction of, and elution by, mobile phase. The mobile phase
will finally exit the column through second filter 206 and via
passageway 242 to second port 240. The resulting fractions of
mobile phase, which contain different analytes, can then be
collected.
[0048] It will be understood by the skilled person that the column
may be operated in either a "downflow" mode, as described above, or
in an "upflow" mode where the direction of flow of the mobile phase
is reversed such that it moves up the column. In upflow mode,
mobile phase will enter the column via second port 240, move along
passageway 242 and upwards through the bed of particulate medium in
bed space 209, to exit the column for collection at first port
233.
[0049] In the embodiment shown, hollow member 260 is an integral
part of the column. However, it will be understood that by means of
connectors and appropriate tubing made from a suitable material
(e.g. polypropylene, polyurethane, etc.) that the hollow member 260
need not be integral to the column.
[0050] The application and collection of mobile phase at the same
elevation on a single end unit simplifies use, in terms of operator
access and handling, reduces the risk of air accessing the system
and decreases the space necessary to set up the column.
[0051] The embodiment shown in FIG. 3 comprises a valve means (220)
for the introduction and/or removal of particulate medium from the
column. It will be understood that such a valve is not an essential
feature of the claimed invention as some columns (e.g. pre-packed,
disposable columns) may not require the addition or removal of
particulate medium to be performed by the end user or are prepared
(packed) with a different technique not requiring the use of such a
valve means.
[0052] FIG. 4 presents a three dimensional schematic representation
of the column of FIG. 3, from which the external features of the
column are evident. The column comprises a first end unit 312,
second end unit 313 and housing 311 which are secured together to
form a fluid-tight seal by tension rods 314 and heads 316.
Particulate medium in the form of a slurry can be introduced into
the bed space (not shown) via valve means 320. First port 333
serves as a conduit for mobile phase or liquid containing
analyte(s) to be separated on the particulate medium. Hollow member
360, which is in fluid communication with the bed space via an
outlet at the base of the column (not shown), ends in second port
340 from which appropriate fractions of mobile phase eluted from
the column may be collected. As can be seen, second port 340 is at
essentially the same level or elevation as the first port 333
through which mobile phase can be introduced (or collected). This
arrangement facilitates user operation and sample handling. In the
embodiment shown in FIG. 4, the capacity of the column is
approximately 10 liters; it will be understood that a wide range of
column capacities is possible, typically ranging from 0.1 to 2000
liters. Preferred capacities when using the column as a disposable
column are in the range of 0.5 to 50 liters.
[0053] FIGS. 5a and 5b schematically compare a system incorporating
a prior art column (FIG. 5a) having an integrated vent valve to a
system using a column in accordance with the invention (FIG. 5b).
The vent valve shown in FIG. 5a is a rotary valve type, but it will
be understood that other valve principles (pinch valves, membrane
valves, etc) may also be employed to achieve the vent valve
functionality.
[0054] Following installation of the prior art system (FIG. 5a),
air must be removed from the system by priming it. The system
comprises an inlet manifold 408, pump 470, sensors 472-476, column
valve 407, outlet manifold 409 and column 401 (the dotted rectangle
shown enclosing the column 401 and vent/purge valve 405 indicates
that the column and the vent valve are used as a combined unit such
that the vent valve is attached to the packed and primed column
when installing the column in a chromatography system). As
described above, the purpose of the vent valve is to protect the
column from draining and/or the introduction of air when installing
it in a system or when removing/disconnecting the column from a
system. The column valve 407 controls connection of the column 401
to the inlet 408 and/or outlet 409 manifolds and thus governs
whether the column 401 is "offline" or "online". In FIGS. 5a and
5b, the column valve is of a rotary valve type, but it will be
understood that other valve principles (e.g. pinch valves, membrane
valves, etc) may also be employed to achieve the column valve
functionality. Fluid connectivity with the column 401 and inlet
408/outlet 409 manifolds is controlled by means of several gateways
within the valve 407 as indicated by positions 1-4 in the diagram).
When valve 407 makes connection between positions 3 and 4 and
positions 1 and 2, the column is bypassed. When the rotary valve is
turned by 90 degrees, connection between positions 3 and 1 and in
between positions 4 and 2 is made, which means that the column is
inline or online and connected in upflow mode/flow direction. As
explained above, other valve principles (e.g. pinch valves,
membrane valves, etc) and a wide range of other valve
configurations (upflow and/or downflow modes as well as connection
of multiple columns) may be employed to achieve the column valve
functionality.
[0055] Air is removed from the system by means of vent/purge valve
405 which allows priming of the system, in particular priming of
conduit 480, and purging of any air within it.
[0056] The system shown in FIG. 5a is intended to be used in an
upflow mode; thus liquid from inlet manifold 408 enters column 401
via conduit 480 at second port 440 and moves upwards through the
packed bed (not shown) exiting at first port 420. Liquid (e.g.
mobile phase or sample containing analytes to be separated on the
column) is taken up from inlet manifold 408 and transferred to the
column 401 under pressure by means of pump 470 via column valve
407. Sensors 472-476 can be used to measure environmental, physical
and chemical conditions in the system (e.g. pressure, flow,
conductivity, temperature, pH, UV absorbance, air etc). These
sensors can be used to control the operation of the column, for
example by regulating flow rates of mobile phase through the
column. Liquid emerging from the column from first port 420 is
transferred via column valve 407 to outlet manifold 409 for
collection.
[0057] FIG. 5b exemplifies a system using a column in accordance
with the invention. The component parts are the same as described
above for FIG. 5a except that there is no vent/purge valve 405. In
this configuration the level or elevation of the first port 420 and
the second port 440 above the level of the bed space (not shown) in
the column is essentially the same, the second port 440 being
connected to the base of the column 401 by means of hollow member
460. While hollow member 460 is part of the column in accordance
with the invention, the corresponding liquid conduit 480 in FIG.
5a, which shows the prior art configuration, is part of the
chromatography system. The column according to the invention with
its hollow member 460 is already purged of air and may be ready for
use when installed in the system. Especially for disposable,
ready-to-use columns, the invention avoids the need for a
disposable purge valve delivered with each individual column, which
significantly reduces cost.
[0058] Following installation, the system is purged upstream of the
column valve 407 when the column 401 is bypassed (i.e. the column
is "offline"). When switching the column "inline", only a
negligible volume of air will remain between the column valve 407
and conduit 480.
[0059] The system shown in FIG. 5b can then be used in essentially
the same manner as described above for the prior art system. In
upflow mode, liquid will be aspirated from manifold 408 by pump 470
and directed, via valve 407, into second port 440 of column 401.
The liquid will then move up the column through a bed of
particulate medium (not shown) to exit at first port 420 and be
directed, by column valve 407, to outlet manifold 409 for
collection. Sensors 472-476 can be used to monitor environmental,
physical and chemical conditions within the system and thus to
regulate its operation.
[0060] FIG. 6 shows the chromatographic separation efficiency by
example of a tracer pulse experiment achieved on a 10 litre column
in accordance with the invention, operated in both downflow (solid
line) and upflow (dotted line) mode. The column was packed with a
bed of CAPTO.TM. Q anion exchange resin (GE Healthcare, Uppsala,
Sweden) of 85 .mu.m agarose particle diameter. The column had a
volume of 10.81, a diameter of 263 mm and a bed height of 200 mm.
Acetone (1% of packed bed volume) was used as a tracer substance
and eluted from the column using water as mobile phase and the
absorbance monitored at 280 nm. As can be seen from Table 1 below,
excellent column efficiency was observed with the 85 .mu.m agarose
medium used, either in downflow (solid line) or upflow (dotted
line) mode.
[0061] The data shown in Table 1 and FIG. 6 were obtained using a
chromatography column in accordance with the invention which
further comprises a transverse distribution channel and wherein the
outlet of the first port and the transverse distribution channel
have an asymmetric configuration. A chromatography column having
such an arrangement is the subject of the Applicant's (GE
Healthcare Bio-Sciences AB) co-pending patent application entitled
"Axial Chromatography Columns and Methods" filed on the same day
herewith as GB 0614316.8.
TABLE-US-00001 TABLE 1 Observed Acceptance Plates/meter 4430
>3700 (for 85 .mu.m) (N/m) Reduced plate height 2.5 <3.0 (h)
Peak asymmetry 1.14 0.8-1.8 (Af)
The data from Table 1 were derived from the chromatogram of FIG. 6
as described below.
[0062] As a measure for column efficiency, the reduced plate height
is determined with help of the peak width w.sub.h at half the
height of the eluted peak, as shown in FIG. 7. This procedure is an
approximation valid for the gaussian-shaped. In practice, eluted
peaks often deviate from this ideal gaussian shape and peak
skewness is described qualitatively by a so-called asymmetry factor
A.sub.f, where `leading` in the RTD is indicated by A.sub.f<1
and `tailing` by A.sub.f>1. Commonly applied acceptance criteria
for the asymmetry factor are 0.8<A.sub.f<1.5-1.8, depending
on the type of application.
h = HETP d P = L d p 1 5.54 ( w h V R ) 2 ##EQU00001## A.sub.f=b/a
(see FIG. 7)
[0063] As a rule of thumb, the characteristic dispersion of the
medium typically gives a reduced plate height in the range
h=1.5-2.0 at an optimized superficial velocity when considering the
highly porous medium used for protein chromatography in
biotechnological downstream processing. The ideal efficiency of the
medium has to be compared to the experimentally determined
efficiency of the chromatographic system, where an increase in the
reduced plate height is a result of additional dispersion from
peripherals, sample volume, bed heterogeneity and distribution
system. In practice, a typical standard installation qualification
of a chromatographic unit used in ion exchange separations of
proteins is an experimentally determined reduced plate height of
h.sub.Unit,Apparent<3.0
TABLE-US-00002 A.sub.f asymmetry factor d.sub.p particle diameter h
reduced plate height HETP height equivalent of a theoretical plate
L bed height, packed bed u.sub.s superficial velocity in packed bed
V.sub.R retention volume w.sub.h peak width at 50% of max. peak
height
[0064] It is apparent that many modifications and variations of the
invention as hereinabove set forth may be made without departing
from the spirit and scope thereof. The specific embodiments
described are given by way of example only, and the invention is
limited only by the terms of the appended claims.
* * * * *