GB2362338A

GB2362338A - Multilevel support for parallel chemical processes

Info

Publication number: GB2362338A
Application number: GB0012172A
Authority: GB
Inventors: Martyn Deal; Kevin Hobbs
Original assignee: Radleys Discovery Technologies Ltd
Current assignee: Radleys Discovery Technologies Ltd
Priority date: 2000-05-20
Filing date: 2000-05-20
Publication date: 2001-11-21
Anticipated expiration: 2020-05-20
Also published as: GB0012172D0; GB2362338B

Abstract

The support has legs 5 attached to a base 2 that is adapted to hold a microtitre plate 3. The legs 5, 6 support two support plates 7, 8. Each plate 7, 8 has holes 9 for supporting reaction vessels or syringe barrels 10. The syringes are positioned to cooperate with cells 4 in the microtitre plate 3. Each hole 9 has a luer fitting 11 into which the syringe 10 may be plugged. The luer fitting 11 of one layer enters the reaction vessel 10 or cell 4 immediately beneath it. Each support plate 7, 8 has a marginal groove 12 filled with a sealing gasket. The liquid in a vessel 10 on the upper plate 8 is forced down by a plenum box 13.

Description

2362338 1 IMPROVEMENTS IN AND RELATING TO APPARATUS FOR THE PARALLEL

CHEMICAL PROCESSING OF MATERIALS This invention relates to apparatus for the parallel chemical processing of materials. Such parallel processing includes filtration, liquid separation, solid phase extraction, parallel synthesis and combinatorial chemistry generally.

Parallel reactions are typically carried out in a number of reaction vessels placed in a suitable array which is conveniently arranged to correspond with the arrangement of a standard microtitre plate which commonly has 12, 24, 48 or 96 cells. The reaction vessels may be of glass, or plastics material such as polypropylene or polytetrafluoroethylene and they may be individual vessels or they may be combined in a single block. Individual vessels may be closed tubes or open tubes such as syringe barrels.

Following a reaction step in which a desired chemical reaction takes place it is usually required to perform some purification process to separate a required product of the reaction from other materials that may be present in the reaction mixture. These materials which constitute impurities are either left over from the synthesis or created as a result of. it. There are various ways in such purification may be achieved. These include:

Filtration involving passing the reaction mixture through a filter membrane which will separate any solid material from any liquid material.

A phase separation in which the mixture is partitioned between an aqueous phase and a non-miscible organic phase whereby any water 2 soluble material may be separated from material that is not water soluble. An aqueous phase may be separated from an organic phase by the use of a suitable hydrophobic membrane for example 5 pm PTFE.

A chromatographic separation in which the reaction mixture is allowed to interact with a stationary phase in such a way that differentiation may be achieved between the components of the reaction mixture. The stationary phase is typically a micro-granular silica coated with an organo-functional silane. The stationary phase is conveniently held in a reaction vessel formed as a cartridge constituted as a syringe barrel containing a filter membrane which allows the passage of liquids through it. As an alternative the stationary phase may be adapted to participate in an ionic exchange interaction with the material in the column.

It may be desirable to use one or more of these methods in sequence in order to obtain the desired level of purification.

When such parallel chemical reactions are being employed to synthesise or analyse or separate more than one compound or sample at the same time it is desirable that these compounds or samples are also purified at the same time, and it is desirable to perform any subsequent processing such as purification using an apparatus that maintains the same footprint as the synthesis or analysis apparatus. For example if 24 samples are synthesised in 24 reaction vessels arranged in a 6 x 4 array, it is desirable to perform any subsequent processes in the same 6 x 4 array. This allows easy transfer of the reaction mixture from one process stage to the next and minimises a risk of error by cross contamination of the different reaction mixtures or samples.

3 It is an object of the present invention to provide an apparatus which facilitates this transfer.

According to the present invention there is provided apparatus for the parallel chemical processing of materials which comprises a frame having a base portion adapted to receive a microtitre plate comprising a plurality of cells arranged in a predetermined array of locations, first and second sets of support legs and first and second support plates each having support means for supporting a plurality of reaction vessels in an array of locations, said support plates being adapted to be supported by said respective sets of legs so that each location in the array of locations associated with one said support plate is in vertical registration with a location in the array of locations associated with the other said support plate and with a location in said predetermined array of locations.

is Such an apparatus allows the various separation techniques previously referred to be performed conveniently with minimal risk of confusion between the different reaction species.

It is convenient for the base and legs to be formed of stainless steel and for the support plates to be formed of a thermoplastics material. A set of support legs may be constituted by a set of pillars, or the sets may be constituted by sets of spacer sleeves which surround continuous pillars.

In preferred embodiments of the invention the two support plates are identical in form.

Typically the reaction vessels will be constituted as the barrels of syringes which may be packed with an appropriate chromatograph 4 stationary phase, and the invention includes apparatus as aforesaid provided with a plurality of such vessels.

The invention also includes apparatus as aforesaid further comprising a 5 microtitre plate.

In the most preferred embodiments of the invention the cells of the microtitre plate are arranged in an array of 6 x 4 locations.

Such microtitre plate is preferably provided with liners for its cells.

In the most preferred embodiments of the invention the support means are constituted by holes. These holes may be arranged to accommodate the reaction vessels directly, or they may each be arranged to accommodate an intermediary fitting which in turn accommodates such a reaction vessel.

Preferably the lengths of the support legs are arranged so that a fluid outlet from a said reaction vessel, or intermediary fitting where present, is located beneath the level of the mouth of a vessel in vertical register beneath it.

Preferably at least one said support plate comprises a marginal groove in one face thereof which is at least partly filled with a sealing gasket. It is preferred to provide a plenum box having an open base whose edges are registrable with said marginal groove and having a gas inlet whereby gas under pressure can be introduced into the plenum box in order to assist passage of reagent from or through vessels supported by the plate to which the plenum box is applied.

A preferred embodiment of the invention will now be described with reference to the accompanying drawings in which:

Figure 1 is a schematic perspective view of an embodiment of apparatus according to the invention.

Figures 2 to 4 are perspective views of further embodiments of apparatus in accordance with the invention.

In the drawings, apparatus for the parallel chemical processing of materials comprises a frame generally indicated at 1 having a base portion 2 which is adapted to received a microtitre plate 3. Two different forms of microtitre plate are shown in Figures 2 and 4 and in Figure 3 respectively. In Figures 2 and 4 the microtitre plate is of transparent plastics material and it is illustrated with a few reaction/collection vessels which are placed in an array of cells 4 in that plate 3. The microtitre plate of Figure 3 is shown as a stainless steel sub-frame defining an array of locations 4 for containing reaction/collection vessels for receiving material. In each case, the microtitre plate is arranged in a 6 x 4 array.

The frame comprises a first set of legs 5 which are attached to the rectangular base portion 2 at each of its corners and are arranged to support a first support plate 7, together with a second set of support legs 6 arranged to support a second support plate 8. The legs 5 and 6 at each corner of the frame 1 may be separate pillars, or they may be constituted as support sleeves slid over a common pillar.

The support plates 7, 8 each have support means 9 constituted as holes for supporting a plurality of reaction vessels 10 in an array of locations which corresponds with the array of the microtitre plate 3. The reaction 6 vessels 10 are each constituted as a syringe barrel as shown in Figures 2 to 4.

As will be seen in Figure 1, alternate rows of locating holes 9A are recessed. The effect of that is to allow luer fittings 11 (Figure 2) associated with holes 9A and associated syringe vessels 10 to be located at a slightly different height from those associated with the rows of holes 9. In this way, the flanges 10A at the mouths of the syringe vessels 10 associated with those different rows of locating holes do not interfere with one another. This permits a closer spacing of the syringe vessels 10 allowing registration with the microtitre plate matrix to be maintained.

In the embodiments illustrated in Figures 2 to 4 each of the holes 9 in the support plates contains a luer fitting 11 into which a syringe vessel 10 may be plugged. The lengths of the luer fittings 11 and the spacing of the support plates due to the sets of legs 5 and 6 is such that the luer fitting 11 of one layer enters the reaction vessel or cell immediately beneath it as shown in Figure 2. This is of great assistance in preventing cross contamination of the reactions proceeding in the various vessels.

Each of the support plates 7, 8 is provided with a marginal groove 12 as most clearly shown in Figure 1. This groove may be filled with a sealing gasket not shown. In order to force the passage of liquid contained in any reaction vessel 10 supported by an upper support plate a plenum box 13 is provided. The plenum box 13 has an open base and its side walls fit into the groove 12 and may be manually pressed into engagement with the sealing gasket. The plenum box may be connected to a source of gas for example nitrogen and thus pressurised. Since the box surrounds all the reaction vessels supported by the plate against which it is pressed gas 7 pressure within the plenum box will force the contents of those reaction vessels 10 downwardly into the next level of vessels.

Where parallel synthesislcombinatorial chemistry is being employed to synthesise more than 1 compound at the same time, it is desirable that these compounds are also purified at the same time. It is also desirable to perform the purification method(s) using purification apparatus that maintains the same footprint as the synthesis apparatus. For example, if 24 samples are synthesised in 24 reaction vessels arranged in a 6 x 4 matrix, it is desirable to perform the purification in the same 6 x 4 matrix. This allows easy transfer of the reaction mixture from one process to the other, and minimises the risk of error in mixing the reaction mixtures up.

The apparatus of the invention as illustrated in the accompanying drawings allows the three purification techniques previously listed to be conveniently used in a 6 x 4 arrangement, using a cartridge 10 (typically polypropylene) of an appropriate dimension (typically 15mm). This cartridge 10 may contain either a filter membrane to allow filtration, a hydrophobic membrane (e.g. 5 jum PTFE) to allow separation of an aqueous phase from an organic phase, or a stationary phase (e.g. silica) held above a filter membrane, for chromatography.

The cartridges 10 can be located into female luer push fittings 11 which are positioned in a support plate 7, 8 formed from a polymer such as polypropylene at an appropriate spacing (18mm between each cartridge, and 18mm between each row of cartridges).

The polypropylene plate containing the luer fittings 11 may be located, 30 via four support legs, above a collection rack constituted by a microtitre 8 plate containing 24 collection vessels also in a 6 x 4 format. The support plate is located at a suitable height so that the bottom of the spout of the luer fitting is located below the level of the top of the collection tube, to minimise the risk of cross contamination. This height may be conveniently adjusted to allow for different heights of collection tube by choice of a set of spacing cylinders or support legs of appropriate height.

A further feature of the apparatus is that a second, preferably identical, plate containing further luer fittings in an identical 6 x 4 format can be stacked above the first plate. The bottom of the spout of the luer fitting in the upper polypropylene plate is located below the level of the top of the lower layer of cartridges 10, to minimise the risk of cross contamination. The height of the top polypropylene plate can also be adjusted relative to the height of the lower polypropylene plate by choice of a set of spacing cylinders or support legs of appropriate height.

This feature allows the separated solution (filtrate) from the top layer of cartridges to pass directly into the second (lower) layer of cartridges, thereby allowing two of the purification techniques to be "stacked" together. This is a very convenient feature, as it removes a manual transfer step (typically performed with a pipette) if the two techniques were performed individually.

In some circumstances it may be necessary to apply a pressure differential across a reaction vessel or cartridge to promote flow of material through the system. Hitherto, this has usually been achieved by applying a partial vacuum to the lower end of a reaction vessel to suck the material through.

When using the apparatus of the invention, we prefer to apply an 30 overpressure to the top of the cartridge after loading the reaction mixture 9 to force the solution to pass through the filter membrane, -or stationary phase that it contains. In this case, it is desirable to apply the positive pressure to all reaction vessels or cartridges at the same time. This may be conveniently achieved by the use of a plenum box as referred to which can be connected to a positive pressure gas supply (typically air or nitrogen). This plenum box is ideally made from a clear material such that it is possible to view inside it, and is of a size such that it fits over all 24 cartridges when located in the polypropylene plate. It locates on a sealing ring recessed into the upper support plate, such that a fully enclosed working area is obtained. The positive pressure gas supply thus provides a positive pressure to the top of all twenty four cartridges at the same time. The positive pressure may be maintained simply by pressing the top of the plenum box by hand, or by securing the box to the support plate with clips. The plenum box may be used to promote flow through the vessels carried by the lower support plate after removal of the top support plate. In Figure 1, the plenum box 13 is shown mounted between the two support plates 7 and 8 for convenience in the drawing rather than as an indication of its intended use.

Apparatus of the invention may be used in the performance of any of the following analytical techniques:

Filtration This is by far the simplest separation method. Using a membrane or frit, particulate matter is removed from the sample.

Liquid-Liquid Extraction This is traditionally performed using a separating funnel. An aqueous phase is added to a non-miscible organic reaction mixture (or vice versa).

The two phases are mixed together and allowed to separate. The different impurities within the sample typically have an affinity (are dissolved in or attracted to) the aqueous phase. Furthermore different types of aqueous phase; acid, neutral and basic will remove different types of impurities. The two phases are then separated; for example by passing the sample through a hydrophobic membrane.

Flash Chromatography The columns are traditionally made of glass with a glass sinter or frit at the bottom. Silica is added to the column as stationary phase and covered with a bed of sand, then the reaction solvent is added. The repeated washing of the column with solvents (the mobile phase) will elute off specific components of the sample at different intervals. These fractions are collected as desired. Using Thin Layer Chromatography, prior to and after the flash chromatography, it is possible to identify when any target component is likely to elute off and to collect that fraction. Thin layer chromatography may be used to optimise the separation.

Solid Phase Extraction This process is not dissimilar to Flash Chromatography. Solid phase extraction cartridges or columns vary from a 2mI to 50 or 10OmI in volume. They are typically plastic syringe barrels packed with different types of silica.

After wetting or conditioning the column, the sample is added. This is conventionally pulled through the column by a vacuum (using a 12 or 24 position vacuum manifold) or allowed to drip through under gravity. The components of the sample are attracted to the silica allowing the carrier solvent or mobile phase to pass through. The target component is then extracted by washing the column with other appropriate solvents. Different solvents will elute different components from the silica depending upon their polarity. The more polar the sample, the stronger 11 the bond to the silica and therefore the more polar the washing solvent should be. Typical solvents and their polarity are:- 3.4 5 4.3 6.2 Cyclohexane DCM Ethyl Acetate Acetonitrile 6.6 - Methanol This is a very precise and accurate method of separation for smaller volumes.

Ion Exchange Columns are identical in appearance to SPE columns. Ion exchange columns are either Cationic (e.g. SCX) or Anionic (e.g. SAX or NI12). Cationic columns have an acid group on the stationary phase, again usually silica-based, and they thus attract basis materials. Anionic columns have a basic group on the silica, so attract acidic components.

These columns allow you to wash off in one fraction everything that does not stick.

12

Claims

Apparatus for the parallel chemical processing of materials which comprises a frame having a base portion adapted to receive a microtitre plate comprising a plurality of cells arranged in a predetermined array of locations, first and second sets of support legs and first and second support plates each having support means for supporting a plurality of reaction vessels in an array of locations, said support plates being adapted to be supported by said respective sets of legs so that each location in the array of locations associated with one said support plate is in vertical registration with a location in the array of locations associated with the other said support plate and with a location in said predetermined array of locations.
2. Apparatus according to claim 1, wherein the base and legs are formed of stainless steel.
3. Apparatus according to claim 1 or 2, wherein the support plates are formed of a thermoplastics material.
4. Apparatus according to any preceding claim, wherein said sets of support legs are each constituted by a set of pillars.
5. Apparatus according to any of claims I to 3, wherein the sets of support legs are constituted by sets of spacer sleeves which surround 20 continuous pillars.
6. Apparatus according to any preceding claim, wherein the two support plates are identical in form.
7. Apparatus according to any preceding claim, provided with a plurality of reaction vessels constituted as the barrels of syringes optionally 25 packed with an appropriate chromatograph stationary phase.
8. Apparatus according to any preceding claim further comprising a microtitre plate.

13
9. Apparatus according to claim 8, wherein the microtitre plate arranged in an array of 6 x 4 locations.
10. Apparatus according to claim 8 or 9, wherein the microtitre plate comprises cells and liners for its cells.
11. Apparatus according to any preceding claim, wherein the support means are constituted by walls of holes leading through the support plates.
12. Apparatus according to claim 11, wherein the holes are each arranged to accommodate an intermediary fitting which in turn accommodates a reaction vessel.
13. Apparatus according to any preceding claim and provided with a plurality of reaction vessels, wherein the reaction vessels and the lengths of the support legs are arranged so that a fluid outlet from a said reaction vessel, or intermediary fitting where present, is located beneath the level of the mouth of a vessel in vertical register beneath it.
14 Apparatus according to any preceding claim, wherein at least one said support plate comprises a marginal groove in one face thereof which is at least partly filled with a sealing gasket.
Apparatus according to claim 15, wherein there is further provided a plenum box having an open base whose edges are registrable with said marginal groove and having a gas inlet whereby gas under pressure can be introduced into the plenum box in order to assist passage of reagent from or through vessels supported by the plate to which the plenum box is applied.
16. Apparatus substantially as herein described with reference to the accompanying drawings.