Note: Descriptions are shown in the official language in which they were submitted.
WO 95120.127 _ PCTISE95I00051
1
CoJ.umn Arrangement
TECHNICAL FIELD
The present invention relates to a column construction which
includes a movable adaptor that can be used to deliver liquid
to or to lead liquid away from a liquid permeable bed in which
it is desired to adsorb a substance present in the incoming
liquid. The adaptor may also be used in the desorption/-
elution/washing of the bed. The adaptor is adapted particularly
for fluidized beds (= expanded particle beds).
By "movable" is meant that the adaptor can be moved in the
direction of flow applied during an adsorption/desorption/
washing process. -
A fluidized (expanded) bed consists of particles and a
through-flowing liquid that keep the particles suspended within
a given volume. Such beds have long been used in fermentation
processes, among others. In recent times the fluidized bed has
also been used as a separation medium in liquid chromatography
(WO-A-9218237 and WO-A-9200799).
Particles which have a density which is higher than the
density of a surrounding liquid can be fluidized by allowing
liquid to flow through the bed in a direction which opposes the
direction of gravity (an upwardly directed flow). When the
particles have a density which is lower than the density of the
surrounding liquid, a downwardly directed flow is required to
fluidize the bed. See WO-A-9218237 and WO-A-9200799.
A fluidized bed is considered to be- stable when each
individual particle keeps within a given cross-section of the
column (perpendicular to the flow direction). The settling
tendencies of- the particles (settling rate measured without
counterflow) depends on density and size. In the case of a-
stabilized fluidized bed, this tendency increases continuously
along the flow direction. Those particles which have the most
pronounced settling tendency position themselves furthest down
in the column. See, for instance, WO-A-9218237. - Stable
fluidized beds shall be vertical (+/-D.5°) with vertical flow
direction (+/-0.5°).
A DESCRIPTION OF THE BACKGROUND ART AND A RESUME OF THE
PROBLEMS SOLVED BY THE INVENTION
Adapters earlier- used in liquid chromatography are intended
WO 95/20.127 2 PCTISE95100051
to hold a stationary phase (bed) in place and to lead the flow
either from or to the stationary phase. Consequently, adapters
have had a distributing function on that side which is proximal
to the stationary phase, and an opening on the opposite or
distal side through which liquid is either delivered or led
away.
Movable adapters have earlier been used in chromatography in
conjunction with matrices which are packed conventionally in
columns. This movability has been achieved by applying a
controllable force on the matrix via the adaptor, either
through
* a rod which extends up through an upper end-piece on the
column; or
* a hydraulic/pneumatic pressure applied in the space between
the adaptor and an upper end-piece.
This latter alternative also requires the use of a rod which
extends up from the adaptor through the column end-piece,
although in this case in order to hold the adaptor in position
relative to the upper surface of the gel bed.
Movable adapters intended for fluidized beds and conventional
column constructions have long been marketed by Pharmacia
Biotech AB, Uppsala, Sweden. These adapters employ hydraulic
pressure in accordance with the above.
The main drawbacks of the known techniques are:
* Strict requirements are placed on the sealing surface-
between column wall and adaptor, which results in higher
manufacturing costs and a leakage. sensitive system. The risk
of leakage is affected by the tolerances on the column wall
and the adaptor $eal, sealing pressure and the resistance of
the seal to temperature and chemicals. This has led to
demands for extremely small tolerances vaith regard to the
column wall and to the adaptor.
* The sealing surface between column wall and adaptor makes it
difficult to clean the systems. Pockets readily form.
* The adaptor jams easily (easily becomes askew) when scaling-
up the column (primarily -in regard of column diameters
larger than I m).
* The height of the construction is twice that of the height
of the column (due to the rod mounted on earlier described
movable adapters).
WO 95120.127 3 ~ p ~ PCT/SE95100051
i
* It is difficult to remove air that is able to collect.
beneath the adaptor (due to tie requirement of stationary
seals on the earlier described movable adapters). -
* It is difficult to loosen agglomerated material, cells,
bacteria and gel that may_have agglomerated on the bottom -
surface of the net. These layers grow at times and form
"cakes" which impede the throughflow of liquid.
* It is necessary to lengthen the net that normally covers -
that side of the- adaptor which faces towards the matrix in
the case of larger diameters (> 1.5 m) , because those nets
that are commercially available are too small.
The invention provides improvements with regard to the
aforesaid drawbacks of known techniques.
DE 1,642,812 describes in Figure 4 an embodiment of a movable
adaptor that is intended for ion-exchange chromatography on a
compact bed. The adaptor is positioned on top of the bed and is
folloiNing movements of the bed.
BRIEF DESCRIPTION OF THE DRAWINGS
An inventive column construction and adaptor are illustrated
in the accompanying drawings, in which:.
Figure la is a side view of one embodiment of an upper adaptor.
Figure lb is a view from above of one embodiment of an upper
adaptor comprised of-sections. Each small circle represents one
section having a flow opening (the centre hole).
Figure 2 illustrates the inventive construction that was most
preferred on the date from which convention priority is claimed
applied for chromatography on a fluidized bed.
Figure 3 illustrates schematically an inventive construction
applied for chromatography on- a fluidized bed in which
suspended particles have a density which is higher than the _
density of the through-flowing liquid. The bed is shown in a -
non-expanded state prior to chromatography (Fig. 3a), in an
expanded state (Fig. 3b) and in a collapsed non-expanded state
after chromatography, in which latter state the bed is ready
for washing and/or desorption (Fig. 3c).
Fi~nre 4 illustrates schematically an inventive construction
applied for chromatography on-- a fluidized bed in which
~ suspended particles have a density which is lower than the --..
density of the through-flowing liquid. The bed is shown in a __:
W095120-127 ~~ . 4 PCTISE95100051
non-expanded state prior to- chromatography (Fig. 4a), in an
expanded state..(FiQ. 4b) and in a collapsed non-expanded state
after chromatography, and .ready for washing and/or desorption
(Fig. 4c).
Details which have mutually the same functionhave been
identified with the same reference numerals, unless otherwise
stated. The directions of the liquid flows are shown with the
aid of arrows.
DESCRIPTION OF THE INVENTION
The invention relates to a construction which includes
a. a vessel (I) which contains a liquid-permeable bed (2);
b. a bottom adaptor (3) which (I) is placed in the lower part
of the vesseland preferably defines the bottom of the
vessel, either_totally or partially, and (II) has an opening- -
(4) through which liquid can be delivered/led away to/from
the lower part of the vessel (preferably the bottom), said
opening (4) being also directed preferably towards the bed
(2) and provided with a distributing facility (5) for
distributian/collection of- liquid flow towards/from the
lower part of the bed;
c. an upper adaptor (5) which-(I) is placed in the upper part
of thevessel and preferably covers the cross-sectional area
of said vessel completely or partially and (II) has an
opening (7) through which liquid is able run from/to the
upper part of the vessel, said opening (7) also preferably
being directed towards the bed (2) and provided with a
distribution facility (8) for distribution/collection of
liquid flow towards/from the upper part of the bed; and
d. means (23, 24) for leading liquid flow to the adapters (3,
6) through the opening (4) in the lower or bottom adaptor
(3) and/or through the opening (7) in the upper adaptor (6).
According to the-inventive concept, the upper adaptor behaves
as a buoyant body when in use. This buoyancy function can be
achieved when the -density of the adaptor is lower than the
density of the through-flowing liquid, i.e. a' density lower
than 1 g/cm' in the case of water and water-miscible organic
solvents. The adaptor is thus comprised completely or partially
of material which has a density below 1 g/cm'. If the adaptor
includes material of higher density, this is compensated for _._
WO 95120427 ~ ~ ~ PCT/SE95/00051
with cells of lower compactness, e.g. air cells. Because the -
adaptor- behaves as a buoyant body, it is able to move in the
direction of flow (both with and against the flow).
According to the present invention, there can be permitted
5 between the periphery of - the- upper adaptor (the buoyant
adaptor) and the vessel wall or walls a gap which in normal
cases may be 0.01-10 mm in size. This gap provides the
advantage of avoiding "jamming", enables air beneath the
adaptor to be easily removed, enables the adaptor to be set
IO into motion in order to release-agglomerates that have caked
beneath the net, enables desired sensors (e. g. particle
sensors) to be inserted into the liquid zone, and so on. An
excessively large gap should be avoided, since such a gap may
easily result in pockets of liquid in which material is able to
collect in -an-undesirable manner. The size of an appropriate
gap will depend on the inner diameter of the column casing,
among other things.
The buoyant adaptor (see-Fig. la) used in accordance with the
invention has at least oneliquid opening (7) on its upper
side. The underside of the adaptor is usually circular in shape
(faces towards the bed/matrix) and includes a distributing
facility (8), which may have the form of one single hole or of
a number- of holes which are distributed uniformly across the __
underside of the adaptor and are in liquid communication with
the opening (7). The distributing facility, or means, may be
covered with a woven fine-mesh material (net) (9), in order to
prevent particles from the matrix/the bed clogging the
distributing facility. As illustrated in Fig. lb, the adaptor -
may be comprised of sections (18), each having a liquid
inlet/liquid outlet opening (7). The sections need not abut one-
another
Studies made hitherto have indicated that known techniques
can be utilized in the construction of the adaptor, provided
' that the requirements.of buoyancy and movability in relation to
through-flowing liquid are fulfilled.
The vessel (1) containing the liquid-permeable -bed may have
any one of a number of different geometrical shapes, although
the most common and most practical configuration is a generally
.straight tube (column), i.e. does not taper, which is
positioned vertically. The vessel may be made of glass,
W095120a27 ~ 6 PCf/SE95/00051
plastic, metal or soin~ 'other inert material.
The liquid-permeable bed is formed by packed beadsor of
particles or monoliths (continuous matrices). Porous particles
and continuous matrices are often used. Particle beds may be
fluidized (= expanded).
The liquid may either flow vertically downwards or vertically
upwards with the tolerances for fluidized beds given above. The
precise flow direction is determined by the design of the
distribution -facility in each adaptor. The distributing
direction is as a rule the same as for the openings (7) and
(4), respectively.
The devices (23 and 24) used to lead the liquid flow to/from
the upper and the lower adaptor are comprised of connections
for hoses or pipes, suitably made of plastic, glass, metal,
etc. The devices (23 and 24) are only indicated symbolically in
the figures. The flow of liquid is generated with the aid of an
appropriate pump arrangement connected to one orto both of. the
adapters by means of said devices (23 and 24).
In order to make possible wider variations in bed height,
particle density, density of-through-flowing liquid and flow
rate, the upper adaptor-(6) may be provided with means which
will enable a counterpressure, preferably a controllable
counterpressure, to be applied to the adaptor .and therewith
counteract the pressure generated by the through-flowing
liquid. This control is preferably effected pneumatically, by
constructing the space (10) above the upper adaptor (6) as a
gas chamber (most practically an air chamber) having direct
connection to the upper side of the upper adaptor. Such a gas
chamber can be created by mounting above the upper adaptor (6)
in spaced relationship therewith an. end-piece (11) which
sealingly closes the upper end of the vessel. In the case of
this embodiment, the opening (7) in the upper- adaptor (6) is
connected to an outer storage vessel or reservoir-.(not shown),
preferably through the medium of the end-piece (11) , by means '
of a liquid delivery line (12), preferably in the form of a
flexible hose or a flexible pipe-of the periscope kind. In the
illustrated case, the gas chamber (10) includes an inlet (13)
which coacts with a valve (14), preferably-an adjustable valve
for the outlet/inlet of gas (air). The inlet.(13) will normally
be arranged on the end-piece-(11). The chamber (10) may be
WO 95120x27 ~ ~ ~ ~ PCfISE95/00051
i '
connected controllably to a pressure source, preferably via the
inlet (13).
In one practical embodiment, the column is provided with a
graduated vertical level pipe (15) which extends parallel-with
the vessel (the column) and the function of which may be
combined with the function of a gel-bed sensor or some other
sensor (16) relevant to the purpose and fixedly mounted on the
adaptor. The sensor may, in turn, be connected to a signal
processing unit (17). This arrangement enables the process
taking place in the vessel to be controlled very effectively.
The adaptor -can be caused to move with the intention of
releasing or dislodging any material that has agglomerated on -_-
the adaptor, by pulsating the liquid flow through the column or -
by pulsating the air pressure in the chamber (10).
Figures 3 and 4 illustrate an inventive construction applied -
to a fluidized bed, particularly a stable fluidized bed, in
which the particles have either a higher or a lower density
than the density of the through-flowing liquid. The vessel (1)
has the form of an upstanding column. According to preferred
embodiments, the construction includes an air chamber (10)
which applies pressure onthe upper side of the upper adaptor
(6) (the buoyant adaptor) against the through-flowing liquid.
As a result of the flow of liquid through the column, the
fluidized bed (primarily a stable bed) can be generated in a
zone of the liquid located between the upper and the lower -
adaptor. -
When the particles in the fluidized bed have a density which
is higher than the density of the liquid (Fig. 3), the inlet is
effected via the lower adaptor (3) and the outlet is effected
via the upper adaptor (6), the buoyant adaptor. The reference
numeral (20) identifies the bed in a non-expanded state, while -
reference numeral (21) identifies the bed in an expanded state.
When the bed is expanded and adsorption takes place, there will
' preferably be found a particle-free liquid layer (19) between
the upper surface of the expanded bed and the upper adaptor (6,
the buoyant adaptor).
V~lhen the density of the fluidized bed particles is lower than
the density of the liquid (Fig. 4), the inlet -is effected via
the buoyant adaptor (6) and the outlet is effected via the
lower adaptor (3). The reference numeral (20) identifies the
W093/20427 ~ ~ PCTISE95/00051
8
bed in a non-expanded state, whereas the reference numeral (21)
shows the bed in an expanded state. when the bad is expanded
and adsorption takes place, there will preferably be found a
particle-free liquid layer. (I9) between the lower -surface of
the expanded bed and the bottom adaptor-(3).
The described structures may be mounted on a stand provided
with feet which can be adjusted to facilitate vertical
positioning of the-Structure. The stand may have the form of at
least three stable. pull rods which are placed uniformly around
and parallel with the walls of -the vessel (the column). The
bottom parts -of the rods are bent radially outwards from the
vessel (the column), in order to improve stability.
When using the constructions illustrated in Figures 3-4, the
process is commenced with a non-expanded bed. The vessel (1)
(the column) is filled with liquid toa predetermined level in
a first process stage. The vessel is filled with liquid which
flows in through either the bottom or the upper adaptor. The
direction of the incoming liquid flow is- determined by whether
the particles have a higher or Lower density than the density
of the incoming liquid (see above). The liquid outlet-should be
closed when filling the vessel, and the valve to the gas
chamber open. When the desired liquid level has been reached,
the flow is allowed to continue with the . outlet open and the
gas chamber closed, optionally after adjusting the rate, of
flow, until expansion of the fluidized bed is complete, for
instance when a stable fluidized bed has been reached. It is
normally endeavored to create a matrix-free liquid layer (19)
at a distance of S-30 mm between the expanded bed and the
outlet adaptor (the upper adaptor in Fig. 3 and the lower
adaptor in. Fig. 4). This. layer impedes clogging of the Qutlet
adaptor. There is no requirement to create a corresponding
layer between the inlet adaptor and the fluidized bed. The
liquid level in the vessel (column) can be lowered, by reducing
the liquid flow rate and/or increasing the gas chamber
pressure. when the flow rate is increased and/or the gas
chamber pressure decreased, a corresponding change in liquid
level is obtained. When expansion of the bed is complete, a
test .run is carried out, optionally subsequent to an
intermediate buffer exchange.
Subsequent to adsorption to the matrix, undesired material
WO 95I2D.i27 ~ ~ ~ ~ PCT/SE95/00051
9
can be washed away by passing an appropriate buffer through the _
bed with the bed fully expanded, partially expanded or in a -
non-expanded state. The extent to which the bed is expanded can
be changed by adjusting the pressure and/or changing the flow
rate.
Desorption is effected with a desorption buffer, suitably
with the bed in a non-expanded state and with the buffer
flowing in a direction opposite to the direction in-.which
adsorption took place. Prior to desorption, the bed is allowed
to return to its non-expanded state, whereafter the flow is
reversed and adjusted so that a thin particle-free liquid layer
will form between the.-outlet adaptor (which is normally used as -
the inlet adaptor for the desorption process) and the non-
expanded bed.
Washing and desorption may be effected in accordance with
Figures 3c and 4c.
The embodiments illustrated in Figures 2, 3 and 4 are the
embodiments most preferred on the date from which priority is
claimed, with the embodiment according to Figure 3 being the
main preference.
