AU740246B2

AU740246B2 - Annular chromatograph

Info

Publication number: AU740246B2
Application number: AU14747/99A
Authority: AU
Inventors: Adalbert Prior
Original assignee: Prior Separation Technology GmbH
Current assignee: Prior Separation Technology GmbH
Priority date: 1997-12-09
Filing date: 1998-12-09
Publication date: 2001-11-01
Anticipated expiration: 2018-12-09
Also published as: CN1252009A; EA199900732A1; ATA207697A; AT405026B; EA001580B1; JP2001512370A; EP0963227A1; CA2279942A1; BR9807203A; AU1474799A; WO1999029388A1

Description

I.

Annular chromatograph 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 preparative continuous annular chromatography) 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, 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 ofprepurification, work-up and final purification 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-columns and end-stage separation columns, respectively, which prevents a continuous operation owing to the need to rinse, regenerate or reequilibrate the separation media after separation of the mixtures of substances. At best an intermittent feed stream can be processed semi-continuously. This, however, limits 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 resolution as well decreases with increasing eluent flow rate, and vice versa, so that relatively low flow rates are preferred for good separation performance.

For chemical reactions in flow reactors, to the contrary, high throughputs and hence high flow rates normally are desired, which additionally opposes a continuous reaction and separation process.

It was the aim of the invention, therefore, to provide annular chromatography equipment for the continuous execution of chemical reactions and prior or subsequent purification 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.

In accordance with one aspect of the invention there is provided an annular chromatograph with a particle bed that is arranged in an annular gap between two concentric cylinders and rotates around a vertical axis, wherein the particle bed comprises two or more zones vertically arranged one on top of the other and wherein the zones comprise at least one reaction zone for chemical reactions and at least one separation zone for the chromatographic separation. With such an arrangement of reaction zone and separation zone in a single annular chromatography column (in arbitrary sequence), successive reaction(s) and separation(s) or prepurification(s) and reaction(s) can be conducted entirely continuously, that is, not merely semi-continuously with an intermittent S0": introduction of the feed stream, because in an annular chromatograph the desired 30 product(s) will exit from the column at desired points along the column periphery Melbourne\003876555 Printed 30 August 2001 (16:19) 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.

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 reaction(s) occurring 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.

20 The material used here for the separation zone(s) can be selected from anionex-change resins, cation-exchange resins, exclusion gels, gel-permeation gels, -affinity gels, hydrophobic-chromatography (HIC) gels, displacement resins, reversed-phase gels and electrophoresis gels or any other separation media commonly used in chromatographic separations. Depending on the separation task, 25 any combination of such separation gels and resins can be utilised. When electrophoresis gels are utilised, electrodes will be arranged at the upper and lower edge of the electrophoretic separation 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 concerning this can be found in the co- S 30 pending Austrian patent application A 2030/97 submitted on 1st December 1997 by Melbourne\003876555 Printed 30 August 2001 (16:19) applicant.

The material for the reaction zone(s) can generally be selected from the same materials as for the separation zone(s), as well as from materials inert toward the reactions occurring 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 preferred embodiments of the invention the material for the reaction zone(s) can be impregnated or coated with one or several reaction catalysts such as metals, metal complexes or enzymes, 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 only really occur upon contact with one of the catalysts immobilised 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 catalyst(s) in one feed stream, but the reaction 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 catalyst(s) 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 reactant(s) The particle bed in the annular chromatograph according to the invention can consist of a single material or of different materials for reaction zone and separation zone, while the reaction zone material can possibly be impregnated or coated as 25 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 oo* Printed 30 August 2001 (16:19) the corresponding zones. Such dividing layers can be selected among membranes, nonporous inert particle material and particularly 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 foundation layer preferably consist of the same material as the dividing layer(s), and particularly of glass beads. For instance, when the top or bottom zone is designated to serve

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*o*ooo* Melboume\003876555 Printed 30 August 2001 (16:19) for an electrophoretic separation, 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 uniform thickness, which implies that the flow rate of the liquid phase is essentially constant 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 chromatograph into zones of 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 conically 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 conically, 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 resolution 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 backmixing will hardly occur.

In preferred embodiments a temperature control jacket is provided in the perimeter of at least one zone at the inner and/or 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 zones and at separation zones fall within the scope of the invention.

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 by 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 embodiments 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 according to the invention comprising 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 5a to 5f are sketches of possible embodiments of the annular chromatograph according to the invention comprising constrictions or expansions of the liquid stream.

Figure 1 shows schematically two embodiments of the present invention, viz., an annular chromatograph with a raction zone 1 and one or two separation zones 2, 3 in an annular column made of material inert toward the components of the reaction solutions and separation solutions, preferably of glass, the chromatograph consisting of an inner cylinder 8 and an outer cylinder 9 (only the outer cylinder 9 is shown in Figure 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 feed and solvents, a manifold 14, as well as supply channels 15. Channels 15 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 invention 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 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 15 preferably are immersed in order to secure a homogeneous feeding. Figure la shows in addition a foundation layer 7 which (together with a porous bottom plate that is not shown, such as a frit, membrane 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 separation solutions used in the particular case, and may be the same for all three layers or differ, but it must not be electrically conducting, particularly in the case of electrophoretic separations. Preferred according to the invention are glass beads, which in practically all current applications are inert and readily packed.

25 In Figure l a a single reaction zone 1 and a separation zone 2 are provided.

The material 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 [tm, as well as from materials with separatory action, such as ion-exchange resins, exclusion resins, etc., while the *o 30 particle material itself may participate in the reaction ion exchangers,

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Melbourne\003876555 Printed 30 August 2001 (16:19) catalysis and the like) or not. The material of the reaction zone 1 may also be coated with one or several reactants and/or catalyst metal complexes, enzymes, pH modifiers, etc.), so that the reaction 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 15 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 purification of the mixture of substances take place.

The components, product(s), catalyst, starting material, and possible side product(s) thus separated leave the system at the lower end of the column via exit pipes 16 at a well-defined position a particular angular position) along the periphery of the annular chromatograph and are collected there, 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 20 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 resolution 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 the specific task, for instance 25 empirically or by preliminary 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 S 30 zone 2 before the desired reaction can take place in zone 1. Subsequently a Melbourne\003876555 Printed 30 August 2001 (16:19) separation of the reaction products occurs in zone 3 in a way similar to that described with reference to Figure 1 a.

Figures 2a and 2b are schematic representations of further embodiments of the invention. In Figure 2a two separation zones 2 and 3 and two reaction zones 1 and 4 are represented. In such an annular chromatograph a multi-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 at 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 1 is provided with a source of radiation 11 which is disposed along the inner periphery of inner cylinder 8 and the outer periphery of 20 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 V considered as the radiation, for instance visible light and UV light as reaction catalysts, IR and microwave radiation as heat source; preferred are UV and microwave radiation.

25 This is followed by a further reaction zone 4 for the second reaction step, which is provided with a temperature control jacket, heating or cooling jacket, which either serves to bring the reaction mixture to the required reaction temperature or (as in the present case) to cool it after irradiation in zone 1 prior to O• O subsequent separation. Such temperature control jackets can of course also be S 30 mounted at the separation zones in order to directly control the temperature of the Melbourne\003876555 Printed 30 August 2001 (16:19) mixtures to be separated, in most cases to cool them.

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 interaction 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 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 factor of 16) and hence to a concentration of the mixture leaving the reaction zone, which subsequently flows through a dividing layer consisting, 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 Melbourne\003876555 Printed 30 August 2001 (16:19) in the column, the throughput of the column, on one hand and the conversion or resolution on the other hand must be found in order to optimise an annular chromatograph *S o* *o *e Printed 30 August 2001 (16:19) according to the invention for a given reaction/separation system, that is, there are upper and lower limits to the flow 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 concentrating 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 preferred.

Figure 5 shows schematically different embodiments of column cross section, with Figures 5a 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 5b and 5f show the analogous cases of a constriction and expansion produced by inclination of both cylinder walls, while in Figures 5c and 5d a unilateral and a bilateral constriction with preceding 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 made 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, by acid catalysis: H Raffinose D-Fructose D-Glucose D-Galactose 12 or by enzymatic cleavage: ct-Galactosidase Raffinose D-Fructose D-Glucose D-Galactose.

Claims

1. An annular chromatograph with a particle bed that is arranged in an annular gap between two concentric cylinders and rotates around a vertical axis, wherein the particle bed comprises two or more zones vertically arranged one on top of the other and wherein the zones comprise at least one reaction zone for chemical reactions and at least one separation zone for the chromatographic separation.

2. An annular chromatograph according to claim 1, wherein at least one reaction zone is arranged upstream of at least one separation zone for the separation and/or purification of at least one of the reaction products generated in the at least one reaction zone.

3. An annular chromatograph according to claim 1 or 2, wherein at least one separation zone is arranged upstream of at least one reaction zone for prepurification of at least one of the starting materials for the chemical reaction(s) taking place in the at least one reaction zone.

4. An annular chromatograph according to any one of the claims 1 to 3, wherein 20 the material for the separation zone(s) is selected from anion-exchange resins, cation-exchange resins, exclusion gels, gel-permeation gels, affinity gels, hydrophobic chromatography (HIC) gels, displacement resins, reversed-phase gels, o:ooo and electrophoresis gels. •oo.oi 25

5. An annular chromatograph according to any one of the preceding claims, wherein the material for the reaction zones is selected from the same materials as the separation zone(s) as well as from material inert toward the reactions taking place in the reaction zones, glass beads or active carbon.

6. An annular chromatograph according to any one of the preceding claims, 14 wherein the material for the at least one reaction zone is impregnated or coated with reaction catalyst as for instance metals, metal complexes, or enzymes.

7. An annular chromatograph according to any one of the preceding claims, wherein the material for the at least one reaction zone is coated with at least one reactant.

8. An annular chromatograph according to any one of the preceding claims, wherein all zones are separated in space by dividing layers.

9. An annular chromatograph according to any one of the preceding claims, wherein at least one dividing layer is selected from membranes, nonporous inert particle material, and electrically nonconducting material, preferably glass beads.

10. An annular chromatograph according to any one of the preceding claims, wherein the particle bed is covered with a covering layer and/or supported by a foundation layer, where the covering layer and foundation layer preferably consist of the same material as the dividing layer(s).

11. An annular chromatograph according to any one of the preceding claims, wherein over at least part of the height of the particle bed the inner cylinder and/or the outer cylinder of the reactor is/are so designed to approach the other, preferably in a conical or curved shape, in order to raise the flow rate in the particle bed.

12. An annular chromatograph according to any one of the preceding claims, wherein over at least part of the height of the particle bed the inner cylinder and/or :i the outer cylinder of the reactor is/are so designed to depart from the other, preferably in conical or curved shape, in order to lower the flow rate in the particle bed. Printed 30 August 2001 (16:19)

13. An annular chromatograph according to claim 11 or 12, wherein the inner cylinder and/or the outer cylinder of the reactor in the lower terminal section of at least one reaction zone is/are so designed as to approach the other, preferably conically.

14. An annular chromatograph according to any one of the claims 11 to 13, wherein the inner cylinder and/or the outer cylinder of the reactor in the region of at least one separation zone is/are so designed to recede from the other, preferably conically.

An annular chromatograph according to any one of the preceding claims, wherein in the perimeter of at least one zone at the inner and/or outer cylinder a temperature control jacket is provided.

16. An annular chromatograph according to any one of the preceding claims, characterised in that in the perimeter of at least one zone at the inner and/or outer cylinder a source of radiation is provided as heat source and/or as reaction catalyst or initiator.

17. An annular chromatograph substantially as herein before described with reference to the accompanying drawings. Dated 30 August 2001 25Freehills Carter Smith Beadle Patent Attorneys for the Applicant PRIOR SEPARATION TECHNOLOGY GMBH 16555 Printed 30 August 2001 (16:19)