Note: Descriptions are shown in the official language in which they were submitted.
CA 022799421999-08-09
Annular chromatogram
The present invention refers to an annular chromatograph with a particle bed
in its
annular gap.
Annular chromatography is a variant of preparative chromatographic separations
that
has been recognised for a number of years and is practised at an ever
increasing pace.
A preferred application of annular chromatography is in the separation of
large
amounts of mixtures of substances, since this type of chromatography can be
operated
continuously while at the same time offering high resolution.
Typical P-CAC equipment ("P-C:AC", preparative continuous annular chromato-
graphy) comprises a particle bed in the shape of a circular ring, that is, an
"annular"
particle bed packed into the space (annular gap) between two concentric
cylinders. At
the upper end the feed solution as well as one or several eluents are fed
continuously
while the particle bed is rotated about its axis. Such operating procedures
are a known
state of the art and widely used (see, e.g., EP-A-371,648).
In preparative chemistry, and particularly so for applications in biochemistry
and
medical chemistry, the purity of chemical reaction products is decisive, which
is why
often extremely demanding procedures of prepurification, work-up and final
purifi-
cation are utilised so as to reduce inevitable impurities to the lowest
possible levels,
ideally below the limits of detection.
Such purification procedures largely occur in separate equipment by batch
operation,
that is, the purification of starting materials and final products occurs for
instance
chromatographically in :pre-colwnns and end-stage separation columns,
respectively,
which prevents a contirnuous operation owing to the need to rinse, regenerate
or re-
equilibrate the separation media after separation of the mixtures of
substances. At best
an intermittent feed stream can be processed semi-continuously. This, however,
limits
CA 02279942 1999-08-09
the capacity of such equipment, and the equipment, time, and financial
requirements
for such processes, consequently also the price for products thus prepared,
are high.
Another problem with which practical chemists are confronted in
chromatographic
separations is the compromise between the time needed for a separation, which
is the
retention time of the substances in the column, and the corresponding
resolution of the
mixtures of substances. Generally, the retention time - and (in most cases)
the reso-
lution as well - decrease;s with increasing eluent flow rate, and vice versa,
so that
relatively low flow rates are preferred for good separation performance.
For chemical reactions i.n flow reactors, to the contrary, high throughputs
and hence
high flow rates normall:~ are desired, which additionally opposes a continuous
reac-
tion and separation process.
It was the aim of the invention, therefore, to provide annular chromatography
equip-
ment for the continuous execution of chemical reactions and prior or
subsequent puri-
fication steps in a continuous, hence more economic operating mode. It was a
further
aim to optimise the retention times of the substances in the individual
sections of the
equipment.
This aim is attained according to the invention by an annular chromatograph
with a
particle bed in its annuhrr gap that is characterised in that at least one
reaction zone for
chemical reactions with at least one associated separation zone for
chromatographic
separation is provided. ~rVith such an arrangement of reaction zone and
separation
zone in a single annular chromatography column (in arbitrary sequence),
successive
reactions) and separations) or prepurification(s) and reactions) can be
conducted
entirely continuously, that is, nol: merely semi-continuously with an
intermittent intro-
duction of the feed stream, because in an annular chromatograph the desired
prod-
ucts) will exit from the column at desired points along the column periphery
or, in
the present case, from the corresponding zone and enter the next zone. It is a
further
advantage of this system that in such a reaction chromatograph, the reaction
products
generated in the reaction zones are continuously withdrawn from the reaction
zone,
which shifts the reaction equilibrium in the direction of the products and
results in
rapid, generally quantitative conversion.
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For the separation of reaction mixtures generated in such a "fixed-bed
reactor" and for
the separation and/or purification of reaction products generated in a
reaction zone,
according to the invention at least one reaction zone is arranged upstream of
at least
one separation zone. Alternatively or additionally, preferably at least one
separation
zone according to the invention can be arranged upstream of at least one
reaction zone
for prepurification of at least one starting material for the chemical
reactions) occurr-
ing in the at least one reaction zone.
Combinations of reaction zones and separation zones in almost any desired
number
and sequence are also possible according to the invention. For instance, a
separation
zone for prepurification can be followed downstream or from top to bottom by
one or
several reaction zones, these in turn can be followed by one or several
separation
zones for the separation of reaction products and side products. In this way
even
multiple-stage reactions and extremely specific and selective separations can
be
carried out in a single reactor chromatograph.
The material used here for the separation zones) can be selected from anion-ex-
change resins, cation-exchange resins, exclusion gels, gel-permeation gels,
affinity
gels, hydrophobic-chronnatography (HIC) gels, displacement resins, reversed-
phase
gels and electrophoresis gels or any other separation media commonly used in
chro-
matographic separations. Depending on the separation task, any combination of
such
separation gels and resins can be utilised. When electrophoretic gels are
utilised,
electrodes will be arranged at the upper and lower edge of the electrophoretic
separa-
tion layer in order to apply a voltage. The electrical connection can for
instance be
accomplished via slip-ring contact to the axis of rotation of the column.
Details con-
cerning this can be found in the co-pending Austrian patent application A
2030/97
submitted on 1 st December 1997 by applicant.
The material for the reacaion zones) can generally be selected from the same
mater-
ials as for the separation zone(s), as well as from materials inert toward the
reactions
occurnng in these zones, for instance glass beads, active carbon, (possibly
modified)
polymers, aluminium oxide, silica gel etc., depending on the reaction type.
Glass
beads and active carbon are preferred according to the invention. However, in
pre-
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ferred embodiments of the invention the material for the reaction zones) can
be im-
pregnated or coated wil:h one or several reaction catalysts such as metals,
metal com-
plexes or enzymes, e.g., Pd/C, Pt/C etc. Proceeding in this way it becomes
possible,
for instance, to feed a number of reaction partners together with a single
feed stream
while the reaction will ~~nly really occur upon contact with one of the
catalysts im-
mobilised in the reaction zone.
As an alternative, according to the invention the material for the at least
one reaction
zone can preferably be coated with at least one reactant, that is, one or
several further
reactants can be introduced together with catalysts) in one feed stream, but
the reac-
tion again will occur, only in the reaction zone within the chromatograph;
according
to the invention, however, even all the reactants can be immobilised on the
particle
material of the reaction zone, and merely the catalysts) required need be
introduced
with the feed, if one or several components of the feed stream will displace
at least
one reactant from the solid phase in order to bring it in contact with the
other reac-
tant(s).
The particle bed in the annular chromatograph according to the invention can
consist
of a single material or o~f different materials for reaction zone and
separation zone,
while the reaction zone material can possibly be impregnated or coated as
described
above, and the change-over between the two particle materials can possibly
occur
continuously. In a preferred embodiment, however, all the zones contained in
the
chromatograph are separated in space by dividing layers in order to prevent a
mixing,
both of the particle materials and of the individual flows between the
corresponding
zones. Such dividing layers can be selected among membranes, nonporous inert
par-
ticle material and - parti.cularly when using electrophoresis - from
electrically non-
conducting material. Here again glass beads which for large part of the
pertinent
reactions are both inert and electrically nonconducting are preferred.
In another preferred embodiment the particle bed is covered with a covering
layer
and/or supported by a foundation layer, where both the covering layer and the
found-
ation layer preferably consist of the same material as the dividing layers,
and partic-
ularly of glass beads. For instance, when the top or bottom zone is designated
to serve
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for an electrophoretic sc;paration, it will always be advisable to provide a
covering or
foundation layer in order to keep the electric field as constant as possible.
In annular chromatographs the particle bed in the cylindrical shell usually
has a uni-
form thickness, which implies that the flow rate of the liquid phase is
essentially con-
stant or varies according to the packing density of the particle bed.
According to the
invention it is proposed to vertically decompose the annular gap in the
annular chro-
matograph into zones o:P different thickness, between which adapting zones may
be
present which provide for a change in flow rate as smooth as possible, and
which as a
rule are curved or conical. In preferred embodiments, however, at least over
part of
the height of the particle: bed the inner and/or outer reactor cylinder is/are
preferably
so designed as to comically approach the opposite cylinder, which serves to
raise the
flow rate within the particle bed and by shortening the pathway of the
solutes, hence
limiting their diffusion ;and migration in the particle bed, results in a
narrowing of the
bands and thus in a concentrating effect.
As an alternative or additional feature, in other embodiments the inner and/or
outer
cylinder of the reactor can be so designed as to recede, preferably comically,
from the
opposite cylinder over at least part of the height of the particle bed. This
serves to
lower the flow rate within the particle bed and results for instance in a
higher resolu-
tion within the separation zones.
Accordingly, constrictions or positions with converging column walls according
to
the invention will preferably be found at the end of reaction zones that are
followed
by a separation zone in order to :introduce a feed at the start of the
separation zone
which is as concentrated as possible, while expansions are preferably found
within the
separation zones where, as mentioned earlier, they serve to improve the
separatory
performance of the column.
By conical design of the; constrictions and expansions a uniform flow can be
provided
in these regions, so that even there undesired congestions, secondary flows or
back-
mixing will hardly occur.
CA 02279942 1999-08-09
In preferred embodiments a temperature control jacket is provided in the
perimeter of
at least one zone at the inner andJor outer cylinder of the chromatograph
according to
the invention in order to~ be able to heat or cool the solutions transported
within the
column. This may be of particular significance in reaction zones where a
particular
reaction temperature must be maintained, but the chromatographic separation in
the
separation zones can also be influenced by temperature, hence temperature
control
jackets both at reaction :cones and at separation zones fall within the scope
of the in-
vention.
In further embodiments., in the perimeter of at least one zone a source of
radiation is
provided at the inner and/or outer cylinder in order to serve as heat source
and/or as
reaction catalyst or reaction initiator. That is, not only a heating of
particular regions
of the chromatograph b~~ IR or microwave radiation can be performed but also,
for
instance, the triggering of photochemical reactions within the column (with
visible or
UV light for instance).
A more detailed description of the invention follows, where reference is made
to the
accompanying drawings where Figures 1 a and lb are schematic views of embodi-
ments of the annular chromatograph of the invention; Figures 2a and 2b are
schematic
views of further embodiments of the annular chromatograph of the invention;
Figure 3
is a schematic sectional view of an embodiment of an annular chromatograph
accord-
ing to the invention corr~prising a source of radiation and a temperature
control jacket;
Figure 4 is a schematic sectional view of an annular chromatograph according
to the
invention having modifications of its flow cross section; and Figures Sa to Sf
are
sketches of possible embodiments of the annular chromatograph according to the
in-
vention comprising con:>trictions or expansions of the liquid stream.
Figure 1 shows schematically two embodiments of the present invention, viz.,
an
annular chromatograph with a raction zone l and one or two separation zones 2,
3 in
an annular column madc: of material inert toward the components of the
reaction
solutions and separation solutions, preferably of glass, the chromatograph
consisting
of an inner cylinder 8 arid an outer cylinder 9 (only the outer cylinder 9 is
shown in
Figure 1 ). The column (driven by a motor that is not shown) is supported so
that it can
rotate around an axis 12, and is continuously supplied via connecting pipes 13
for
CA 02279942 1999-08-09
feed and solvents, a manifold 14, as well as supply channels 15. Channels 1 S
have the
customary design comprising single, multiple, or slit nozzles and the like,
but curved
slit nozzles of variable width fitted to the column perimeter are preferred
for the in-
vention in order to enable the precisest possible tuning of feed and eluent
streams.
At the lower end of the columns, exit channels or pipes 16 for eluate
collection are
provided. These exits 16 can be attached, either to the column (i.e., they
will rotate
together with it around axis 12) or to the axis 12, and for instance be in
contact with
the column rotating relative to this axis via a slip ring, this latter
embodiment being
preferred. The particle :material of the uppermost zone 1 or 2 is each covered
with a
covering layer 6 into which the supply channels 1 S preferably are immersed in
order
to secure a homogeneous feeding. Figure 1 a shows in addition a foundation
layer 7
which (together with a porous bottom plate that is not shown, such as a frit,
mem-
brane disc etc.) serves to prevent an escape of particle material at the
bottom of the
column. The individual reaction zones and separation zones ordinarily are
separated
by dividing layers 5 in .order to prevent a mixing of the particle materials
of the two
zones.
The material for the dividing, covering, and foundation layers 5, 8, 9 is
selected from
membranes as well as nonporous particle material that is inert toward all
components
of the reaction and sep~~ration solutions used in the particular case, and may
be the
same for all three layers or differ, but it must not be electrically
conducting, partic-
ularly in the case of electrophoretic separations. Preferred according to the
invention
are glass beads, which i.n practically all current applications are inert and
readily
packed.
In Figure 1 a single reaction zone l and a separation zone 2 are provided. The
mater-
ial for the reaction zone; can be selected from any particle materials that
are inert to-
ward the reactions taking place in this zone, for instance glass beads,
preferably with
diameters of about 150 to 240 p,m, as well as from materials with separatory
action,
such as ion-exchange rc;sins, exclusion resins, etc., while the particle
material itself
may participate in the reaction (e.g., ion exchanges, H+ catalysis and the
like) or not.
The material of the reaction zone 1 may also be coated with one or several
reactants
CA 02279942 1999-08-09
and/or catalyst (e.g., mc;tal complexes, enzymes, pH modifiers, etc.), so that
the reac-
tion will occur at the solid phase. Theoretically even all reactants can be
immobilised
on the support, if at least one component introduced together with the feed
solvent
(for instance, the solvent itself) will displace at least one of the reaction
partners from
its bond to the solid phase, i.e., strip it from this phase.
During operation of such an annular chromatograph the feed solution containing
at
least one of the reactants and/or catalyst is introduced into the column via
supply
channels 1 S and from there reaches the reaction zone 1, where the desired
chemical
reaction of the reaction partners will occur. At the lower end of zone 1,
these enter the
dividing layer 5 and subsequently the separation zone 2 where a separation and
puri-
fication of the mixture of substances take place.
The components, produ.ct(s), catalyst, starting material, and possible side
products)
thus separated leave the; system at the lower end of the column via ext pipes
16 at a
well-defined position (i.e., a particular angular position) along the
periphery of the
annular chromatograph and possibly are forwarded to tanks or work-up equipment
(for concentration, precipitation, etc.).
The height and diameter of the individual zones will depend on the type of
reaction
and separation, the intended retention time of the substances in the column,
the type
of particle material, the packing density of the corresponding zones, the
desired reso-
lution in the separation and on other factors all familiar to those skilled in
the art. It is
within the capabilities of professionals with average skill in the art to
determine the
dimensions in accordance with t:he specific task, for instance empirically or
by prelim-
tnary runs.
In Figure lb three zones 1, 2, 3 separated from one another by dividing layers
5 are
provided. Of these, zones 2 and 3 are conceived as separation zones, the
intervening
zone 1 is conceived as reaction zone. Thus, one or several components of the
feed
solution introduced via supply pipes 15 can be prepurified in separation zone
2 before
the desired reaction can. take place in zone 1. Subsequently a separation of
the reac-
tion products occurs in zone 3 in a way similar to that described with
reference to
Figure 1 a.
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Figures 2a and 2b are schematic representations of further embodiments of the
in-
vention. In Figure 2a two separation zones 2 and 3 and two reaction zones 1
and 4 are
represented. In such an annular chromatograph a mufti-stage synthesis can be
per-
formed continuously, a first reaction step in reaction zone 1 being followed
by an
intermediate purification in separation zone 2, a second reaction step being
performed
in reaction zone 4 and a.t last the final purification being performed in
separation
zone 3.
Figure 2b shows an embodiment with one reaction zone 1 and two separation
zones 2,
3 where a mixture issuing from :reaction zone 1 can be purified in two stages,
enabling
the collection of highly pure products at the column exit 16.
Figure 3 is a schematic partial sectional view of a particularly preferred
embodiment
of the invention. Here two separation zones and two reaction zones are
provided, that
is, a zone 2 for prepurification similar to Figure lb, two consecutive
reaction zones 1,
4 for a two-step synthesis, and another separation zone 3 for a final
purification step.
Here the first reaction zone is provided with a source of radiation 11 which
is dis-
posed along the inner periphery of inner cylinder 8 and the outer periphery of
outer
cylinder 9 so as to be able to expose the entire volume of the zone as
uniformly as
possible to the radiation. Any kind of electromagnetic radiation can be
considered as
the radiation, for instance visible light and LJV light as reaction catalysts,
IR and
microwave radiation as heat source; preferred are UV and microwave radiation.
This is followed by a further reaction zone 4 for the second reaction step,
which is
provided with a temperature control j acket, i. e., heating or cooling j
acket, which
either serves to bring the reaction mixture to the required reaction
temperature or (as
in the present case) to cool it after inradiation in zone 1 prior to
subsequent separation.
Such temperature control j ackets can of course also be mounted at the
separation
zones in order to directly control the temperature of the mixtures to be
separated, in
most cases to cool therr~.
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Power supply to the source of radiation and to the temperature control jacket
can be
from the interior (via axis 12) or from the exterior.
In each of Figures 1 to 3, a maximum of two reaction and two separation zones
are
shown, but any other practicable number and sequence of such zones falls
within the
scope of the invention.
Provisions to modify the flow cross section can be made in order to adjust the
flow
rate of the mobile phase: in the individual zones. A constriction of this
cross section
for instance causes faster flow in this section and thus - as already
described - a
concentration effect, while an expansion of the flow cross section causes
slower flow
and thus a better interac;tion with the stationary phase. In a reaction zone,
such an
expansion will lead to more complete conversion, in a separation zone it will
lead to
improved separation, that is, a higher resolution.
Figure 4 shows a possible modification at the transition from a reaction zone
1 to a
separation zone 2. At the lower end of zone 1 the cross section of the column
in the
figure is reduced to 1/4 of its original value, which leads to an increase in
flow rate
(here for instance by a j:actor of 16) and hence to a concentration of the
mixture leav-
ing the reaction zone, v~~hich subsequently flows through a dividing layer
consisting,
e.g., of glass beads, which includes a region with narrower cross section but
parallel
cylinder walls designated as concentration zone 17. Finally the mixture enters
the
separation zone 2. In the transition from the dividing layer 5 to the
separation zone 2
the cross section increases (and the flow rate decreases) back to the original
value,
interactions with the solid phase are reinforced, and the separation of the
mixture into
its components is improved. Providing for an even larger cross section than in
zone 1
would produce a further improvement of separation performance. Conical shapes
of
the constrictions and expansions secure uniform flow in these regions, so that
even at
these points congestions, secondary flows and back mixing will hardly occur,
because
flow turbulence is minimised in this way.
In general, however, a compromise between the retention time of the mixture in
the
column, i.e., the throughput of the column, on one hand and the conversion or
reso-
lution on the other hand must be found in order to optimise an annular
chromatograph
CA 02279942 1999-08-09
11
according to the invention for a given reaction/separation system, that is,
there are
upper and lower limits to the flaw cross section.
The ratio of maximum and minimum annular gap width is preferably between 10 :
1
and 1.5 : 1, more particularly between 5 : 1 and 1.5 : 1. The height of the
concen-
trating zones 17 or zones with improved resolution may preferably be between a
value
corresponding to the minimum width of the annular gap and 2/3 of bed height,
with a
value corresponding to maximum width of the annular gap being particularly pre-
ferred.
Figure 5 shows schematically different embodiments of column cross section,
with
Figures Sa and 5e representing a constriction and an expansion, respectively,
which
are obtained by the mere inclination of one cylinder wall (optionally the
inner or outer
cylinder) toward the other or away from the other. Figures Sb and 5 f show the
anal-
ogous cases of a constriction and expansion produced by inclination of both
cylinder
walls, while in Figures Sc and Sd a unilateral and a bilateral constriction
with preced-
ing concentrating zone 17 are shown. Here it can be seen, just as from the
earlier
Figure 4, that even several constrictions and/or expansions can follow one
another in
order to adjust the liquid flow in steps to very high or very low flow rates,
if this is
serving the demands m;~de upon the annular chromatograph of the invention in
each
particular case.
The number of potential applications of the annular chromatograph according to
the
invention is almost unlimited, hence here we merely point by way of suggestion
and
in a general way to the diverse homogeneous and heterogeneous catalytic
processes,
different hydrogenations, dehydrogenations, redox reactions, hydrolyses and
other
solvolyses, enzymatic reactions and many more. A specific example is given by
the
hydrolysis and subsequent separation of oligomeric carbohydrates, e.g., by
acid
catalysis:
H+
Raffinose ~~~ D-Fructose + D-Glucose + D-Galactose
CA 02279942 1999-08-09
12
or by enzymatic cleava~;e:
a-Galactosidase
Raffinose ~--~ D-Fructose + D-Glucose + D-Galactose.
