WO2014187655A1

WO2014187655A1 - Device and method for separating components of a solution

Info

Publication number: WO2014187655A1
Application number: PCT/EP2014/058957
Authority: WO
Inventors: Harald RITCHIE; Ken MEADOWS
Original assignee: Thermo Electron Manufacturing Limited
Priority date: 2013-05-21
Filing date: 2014-05-01
Publication date: 2014-11-27
Also published as: CN205413065U; DE212014000128U1

Abstract

A device for separating components of a solution comprising: a well for receiving the solution;an exit pipe connected to the well for receiving the solution from the well wherein the exit pipe is of smaller diameter than the well; and a porous composite sorbent within the exit pipe for retaining one or more components from the solution, wherein the composite sorbent is in the form of a sintered solid composition comprising at least porous polymer particles and sorbent particles sintered together that is push fitted into the exit pipe so as to form a close fit within the exit pipe,and wherein the exit pipe has a restriction therein that acts to stop travel of the composite sorbent in the exit pipe beyond the location of the restriction. The device may be a concentrator for liquids, e.g. prior to LC or LC/MS analysis.

Description

Device and method for separating components of a solution

Field of the invention

This invention relates to separating components of a liquid solution and thereby to purification and/or concentration of the solution, especially, but not exclusively, as part of sample preparation for liquid chromatography and/or mass spectrometry.

Background of the invention

Liquid chromatography (LC), especially when coupled to mass spectrometry (MS), is an important technique for the analysis of pharmaceutical drugs and biological samples for example. Peptides are just one type of molecule that nowadays routinely requires analysis by LC/MS. Traditional sample preparation before analysis by LC/MS requires a purification and/or concentration step. However, a concentration step can lead to evaporative loss of up to 95% of the components of interest, e.g. peptides, due to irreversible binding onto the walls of the concentration vessel. The evaporation and concentration step is thus a time consuming and costly activity in terms of productivity.

Solid phase extraction (SPE) is a technique for preparing samples prior to LC. It comprises using a sorbent typically held in some type of column or cartridge to remove one or more chemical components from a liquid solution typically containing a plurality of components whilst leaving other components in the liquid. In this way, unwanted components can be removed prior to analysis. In one type of embodiment, the component of interest is removed using the sorbent and subsequently recovered from the sorbent in another liquid. The component of interest can be purified and/or concentrated in this way. In another type of embodiment, one or more unwanted components are removed from the liquid using the sorbent. In a conventional SPE, the sorbent is in the form of a powder, i.e. loose particles. The sorbent particles are then held between two fixed porous frits and the liquid to be concentrated is flowed through the first frit, followed by the sorbent particles and then the second frit. However, the use of loose sorbent particles has numerous disadvantages. In addition to the simple drawback that two frits are required to be provided and fitted together with the sorbent, the presence of the frits increases the volume occupied which can increase the width of the sample band in a chromatographic analysis. Each time a SPE column is prepared, the amount of sorbent particles must be measured and the amount may vary slightly from column to column, as well as packing density and other factors, which can affect the separation properties from column to column. Furthermore, the use of sorbent particles can affect the flow characteristics in terms of causing reduced flow resistance and liquid channeling among the particles.

GB 2441528 discloses a co-sintered porous polymer comprising a porous polymer substrate and a molecule specific powder immobilized therein. The co- sintered polymer is described for use as the frits in SPE such that a separate sorbent powder is not required. However, the configuration described in that document are not suitable for concentrating the very small quantities of liquid sample as often required for the analysis of biological samples nowadays. In US 6048457 and its continuations is described a housing used for separating samples wherein the housing is provided with a structure comprising a plurality of sorptive particles entrapped in a porous polymer matrix. The matrix is formed by a casting method within the housing and the matrix is thereby adhered to the housing. A drawback is that the amounts of casting solution deployed and the efficiency of casting may vary from one housing to another leading to variations in separation efficiencies between housings. Similarly, in EP 1533015) is disclosed a sintered mix of polymer particles and sorptive beads, which is sintered directly into a housing of a liquid container such as a pipette. However, similar disadvantages also apply to that device.

In another approach, US 6,723,236 describes an extraction device having a reservoir, a well and an exit spout, wherein the well is tapered and contains a first filter (frit) and a second filter sealed between the tapered well walls, wherein an amount of sorbent particles partially fills the volume between the filters to provide a void volume between the sorbent and the first filter above it. As well as having the problems associated with using loose sorbent particles and separate porous frits as described above, both the frits and media are prone to variation in position when manufactured and movement post manufacture. For example the position of frits can be different due to variations in tolerances of the frits and the well and the position of the media can be different due to inconsistencies in packing. Moreover, the tapered well shape will adversely affect the uniformity of liquid flow velocity as the liquid progresses along the well.

Against this background the present invention has been made.

Summary of the invention

According to an aspect of the present invention there is provided a device for separating components of a solution comprising: a well for receiving the solution; an exit pipe connected to the well for receiving the solution from the well; and a porous composite sorbent located within the exit pipe for retaining one or more components from the solution. The present invention preferably provides a device for separating components of a solution comprising: a well for receiving the solution; an exit pipe connected to the well for receiving the solution from the well wherein the exit pipe is of smaller diameter than the well; and a porous composite sorbent within the exit pipe for retaining one or more components from the solution, wherein the composite sorbent is in the form of a sintered solid composition comprising at least porous polymer particles and sorbent particles sintered together that is push fitted into the exit pipe so as to form a close fit within the exit pipe, and wherein the exit pipe has a restriction therein that acts to stop travel of the composite sorbent in the exit pipe beyond the location of the restriction.

The device is useful for solid phase extraction. The invention relates in one aspect to separating components (also termed analytes) of a solution, i.e. separating components from each other and/or separating one or more components from the solvent of the solution. The device may be for concentrating and/or purifying components of a solution. The invention may be utilized for extracting one or more components from a solution and subsequently re-dissolving or recovering them in the same or another solution for subsequent analysis, for example by LC/MS. The invention thus may be used for concentrating one or more analytes of interest in a solution. The sorbent can retain the one or more components so that they can be subsequently re-dissolved into solution (the same or different solution from which they were retained) in a more concentrated state than they originally entered the device in. A purified and/or concentrated solution obtained may be utilized for analysis. The device may therefore be a concentrator for liquids, especially a concentrator for preparing solutions prior to LC or LC/MS analysis. A purified or concentrated solution that elutes from the device, i.e. after passing through the sorbent, may be injectable into an LC system, especially an LC/MS system. The solution may be a solution of biological or biologically active molecules or pharmaceutical drugs for example. The device is especially suitable for processing (concentrating) small amounts of solution, such as concentrating solutions of, for example, 0.2 to 2.0 ml (milliliter) or 1 .0 to 2.0 ml, into solution of, for example, 0.01 to 0.5 ml or less (or 0.02 to 0.5 ml, or less). The device may concentrate a solution of more than 0.1 ml (100 μΙ) into a solution of 0.1 ml or less (e.g. 0.05 ml or less). The device thus may be suitable for microelution of samples (i.e. it may be a microelution device for concentration from a larger solution (e.g. 1 ml or more) into a smaller solution wherein the smaller solution has volume of 500 μΙ or less, preferably 200 μΙ or less, more preferably 100 μΙ or less, 50 μΙ or less, or 25 μΙ or less, or even 10 μΙ or less.

The invention removes the need for evaporation and avoids the sample loss associated with evaporative adsorption on well walls. The invention thus addresses the need in pharmaceutical and clinical laboratories to concentrate biological samples for LC/MS without an evaporative concentration step. Thus, operational efficiency is improved by reducing workflow steps and in turn reducing operational cost and improving productivity. In addition, more efficient use can be made of the available sample solution.

The invention dispenses with the need in the prior art for frits to retain loose sorbent particles. The invention is suitable for concentrating small volumes of sample especially of biological or pharmaceutical origin. The sorbent is a porous composite material located in the exit pipe that is smaller in diameter than the main solution well. A smaller amount of composite sorbent is therefore required and it is suitable for concentrating smaller volumes of solutions and smaller quantities of analytes than prior art designs. Furthermore, by virtue of the geometry of the device and the configuration of the composite sorbent therein, such as the respective aspect ratios, there may be an increased residence time of solution in contact with the sorbent which leads to improvement in separation efficiency and subsequent recovery.

Numerous preferred features and additional advantages of the invention will now be summarised.

Preferred embodiments

The well of the device is a part of the device which acts as a reservoir for an amount of a solution to be processed. The solution, which has been obtained in a separate process and may be a biological sample for example, may be dispensed to the well by a liquid delivery device, e.g. pipette. In use, the device is arranged with the well above the exit pipe so that under gravity or other force (such as suction), fluid flows from the well into the exit pipe.

The well is typically cylindrical in shape or has a cylindrical portion. The well preferably has a vertical wall (when the exit pipe is positioned vertical), preferably wherein the vertical wall has parallel sides (i.e. not like a funnel or tapered shape) along at least a portion of its length. The well may have circular or square cross sectional shape (in transverse section). The well may have a portion (e.g. an upper portion in use) which is of square cross sectional shape and another portion (e.g. a lower portion in use) which is of circular cross sectional shape (such as a cylindrical or preferably conical portion). The well may have an internal volume of between 0.5 and 5.0 ml, or between 0.5 and 2.5 ml, or between 0.75 and 2.0 ml. The well may have a volume of 0.75, 1 .5 or 2.0 ml for example. Preferably, the well has a volume of at least 1 .5 ml, especially at least 2ml thereby giving the ability to dilute a sample of volume 1 ml 1 :1 which is larger than existing approaches. The well feeds the solution to the exit pipe wherein there is located the composite sorbent. In some embodiments, a wall, e.g. bottom wall, of the well that connects to the exit pipe is preferably configured to be at a defined angle to the horizontal when the exit pipe is vertical to aid the exit of fluid, such as at an angle of from 1 to 85 degrees or 1 to 60 degrees, preferred being 15 to 60 degrees and especially 30 to 60 degrees (e.g. 45 degrees), or in other cases 1 to 15 degrees to the horizontal plane. This assists in preventing eddys in the fluid flow exiting from the well and reduces collection of fluid in corners of the well.

The device has an exit pipe in fluid communication with the well. The exit pipe is preferably of smaller diameter and especially smaller volume than the well (i.e. internal diameter and internal volume). The internal diameter of the exit pipe is typically about half or less than half the diameter of the well. Where the well and/or exit pipe has more than one value of diameter, it is the average diameter of the exit pipe that is preferably smaller than the average diameter of the well. Where the well and/or exit pipe is not circular, it is the diameter or width at the widest point of the exit pipe and/or well that is considered. The exit pipe is typically cylindrical in shape. Preferably, the exit pipe is elongate and is substantially straight in the direction of a longitudinal axis of the device. In use, the device is designed to be used with the exit pipe in a vertical orientation, i.e. with the exit pipe longitudinal axis vertical, whereby the well is located above the exit pipe such that solution flows from the well to the exit pipe. Preferably, the exit pipe has a circular transverse section. Preferably, the exit pipe has a substantially constant internal diameter (i.e. is not tapered) along at least a portion of its length, in some embodiments along at least a major portion of its length, and in some embodiments substantially along its length. Preferably, the diameter of the narrowest diameter of the exit pipe is at least 60%, more preferably at least 70% and most preferably at least 80% of the diameter of the widest diameter of the exit pipe. The composite sorbent is preferably located in this constant internal diameter section. Due to a constant internal diameter of the exit pipe, the liquid flow velocity of the solution is uniform, especially through the sorbent. By providing the composite sorbent in the narrow exit pipe, the volume occupied by the sorbent is reduced compared to providing sorbent in the well which enhances separation efficiency, especially for small amounts of sample (as is the case for many biological samples) and also elution efficiency when the separated components are re- dissolved into solution.

The internal wall of the exit pipe preferably has a restriction therein that acts to stop the travel of the composite sorbent in the exit pipe beyond the location of the restriction. The restriction may be of various forms, such as an internal step or reduction in internal diameter of the exit pipe or one or more protrusions into the internal volume of the exit pipe.

The composite sorbent preferably fits closely against the internal wall of the exit pipe but allows push fitting of the sorbent into the exit pipe during assembly. The composite sorbent preferably fits closely so that no significant amount of liquid solution can flow around the edges of the sorbent, i.e. between the edges of the sorbent and the adjacent internal wall of the exit pipe. The solution thus flows effectively through the sorbent, which increases the efficiency of separation. The weight of sorbent located in the exit pipe is preferably not more than 10mg (milligram), more preferably not more than 5mg for example it may be between 1 and 10mg, for example 2mg, 5mg or 10mg. Preferably, the composite sorbent is not positioned contiguously with the outlet of the exit pipe but rather there is a length of the exit pipe between the composite sorbent and the outlet of the exit pipe. This may be a length of exit pipe of reduced internal diameter compared to the length of the exit pipe in which the composite sorbent is located.

The aspect ratio of the exit pipe (length(L)/internal diameter(D)) is preferably between 2 and 10 (more preferably between 2 and 8, still more preferably between 2 and 6, and most preferably between 3 and 5), especially about 4. Where the internal diameter of the exit pipe has more than one value, the aspect ratio is to be calculated from the average diameter. The internal diameter (D) of the exit pipe (average diameter where the internal diameter has more than one value) is preferably between 1 .0 and 3.0 mm, preferably between 1 .0 and 2.5 mm, especially between 1 .0 and 2.0 mm. The length (L) of the exit pipe is preferably at least 6mm, more preferably at least 8mm. Solution or liquid that has passed through the sorbent emerges from the outlet of the exit pipe, which is configured so that the solution or liquid leaves typically as droplets. The exit pipe is preferably dimensioned and shaped for optimized liquid droplet formation at its outlet. It has been found, for example, that droplets with minimal spraying can be formed. The outlet of the exit pipe is preferably flared, i.e. opening outward, on its internal surface to provide focusing on the droplet size. The outlet of the exit pipe should also preferably be designed to reduce creep back of fluid up the outside of the pipe. The exit pipe has a rim at its outlet (between its internal and external walls). For a circular section pipe the rim will likewise be circular. Preferably, the width of the rim should be at least 0.1 , or at least 0.2, or at least 0.3, or at least 0.4, or at least 0.5 mm. Moreover, the outer surface of the exit pipe at its outlet end is preferably angled to the horizontal plane (when the exit pipe is vertical), preferably at 5 to 85 degrees (especially 15 to 85 degrees, 45 to 85 degrees, 60 to 85 degrees, and 75 to 85 degrees).

The term sorbent herein means a material having the capability to adsorb or absorb components from a liquid. The sorbent used in the invention is a composite sorbent and is a fixed solid composition (not loose or powdered). The composite sorbent thereby removes the need for providing two separate filters or frits to retain loose sorbent particles. Indeed, additional filters (frits) are preferably not present in the exit pipe to retain the composite sorbent.

Preferably, the composite sorbent is a sintered composite, wherein two or more materials (preferably as powders) have been sintered together. The composite sorbent is accordingly mechanically robust. Its sintered form is in contrast to the loose powdered sorbent forms found in the prior art. Most preferably, the two or materials are mixed together and pressure is applied to the mixture, for example by means of a tablet press, to form a composite material that is then subjected to heat treatment (e.g. by passing through an oven between 100-200 deg C) to sinter the composite material. The composite sorbent may thus be formed as a solid plug or tablet. At least one of the two or more materials sintered together is a sorbent. There may be one, two or more sorbent materials included in the composite sorbent. One material is preferably a porous polymer and another material is preferably a sorbent. The sintered composite may thus comprise at least porous polymer particles and sorbent particles sintered together. Among suitable composite sorbents are those sintered sorbent compositions described in GB 2441528 (Porvair) and EP 1533015 (Eppendorf). Examples of preferred porous polymers for the composite sorbent are polyethylene (preferably HDPE) or polypropylene (for example HDPP) (preferably polyethylene). Examples of preferred sorbents for the composite sorbent are poly(styrene / divinyl benzene) (PS/DVB), functionalized PS/DVB, silica, polymer coated silica, carbon, methacrylate polymers, PTFE, and polypropylene, with functionalized PS/DVB being more preferred among these. In general, sorbents should be porous or at least superficially porous if not bulk porous.

Preferably, the composite sorbent is pre-formed, for example as described above, and pushed into the exit pipe of the device and as such is not cast or sintered directly into the device (i.e. not sintered "in-situ") with the problems that that entails. This enables sorbent composite plugs to be formed in a separate process with uniform dimensions and properties across multiple plugs, which improves reproducibility of performance between devices.

The aspect ratio of the composite sorbent (length(L)/ outer diameter(OD)) is preferably about 0.5 or more, preferably about 1 or more, e.g. between 0.5 and 10 (or between 1 and 10), more preferably between 0.5 and 8 (or between 1 and 8), still more preferably between 0.5 and 6 (or between 1 and 6).

Preferably, the sorbent is a rationally designed sorbent wherein the property of extraction (extraction efficiency) is selective towards a particular analyte or group of analytes. Preferably, the sorbent has a high enrichment capacity for a particular analyte or group of analytes (e.g. analytes of a particular chemical class). Examples of particular analytes or groups of analytes include steroids, hormones, opiates, environmental pollutants and food additives.

The composite sorbent can be manufactured with uniform separation properties. In this way, each device that is fitted with the composite sorbent in accordance with the invention will have uniform separation properties, unlike prior art devices employing a dispensed amount of loose sorbent into a separation column. In addition, frits are not required in the device and so are preferably not present.

Typically, the composite sorbent is manufactured as a solid plug, preferably of defined size and shape, which fits within the exit pipe, especially with a close fit. As the exit pipe is typically cylindrical, in such embodiments, the solid plug of composite sorbent is desirably of cylindrical shape. The weight of the solid plug is preferably not more than 10mg, for example it may be between 1 and 10mg, for example 2mg, 5mg or 10mg. The width of the solid plug is preferably 2 to 3 mm. The height (length) of the solid plug is preferably 2 to 6 mm.

The solution may be one of numerous different solution types requiring separation of its components or their purification or concentration. The solution may be aqueous or non-aqueous. The device is suitable for use with different solution matrices, for example such as blood plasma, or urine. A component or components of the solution requiring separation and/or concentration may comprise chemical a component or components belonging to one or more of the following types: small organic molecules, macronnolecules, pharmaceutical drugs, peptides, therapeutic peptides, proteins, biological molecules, biological macronnolecules, DNA, RNA, etc.

A plurality of devices according to the present invention may be utilized in a multi-well plate, e.g. a standard 96-well elution plate. Such a plate can be used with robotics systems, e.g.: Tecan and TomTec robotics.

Description of the drawings

Figure 1 shows schematically embodiments of a device according to the invention, wherein the sorbent bed has different bed weights and positions (Figures 1A-1 D);

Figure 2 shows schematically a part of another embodiment of a device according to the invention comprising a preferred exit pipe design;

Figure 3A shows schematically a further embodiment of a device according to the invention based on a solid phase extraction (SPE) cartridge design.

Figures 3B and 3C show schematically a model of another design similar to the design of SPE cartridge shown in Figure 3A.

Figure 3D shows schematically a further design of SPE cartridge similar to that shown in Figures 3B and 3C.

Figure 3E shows the Figure 3D embodiment with the composite sorbent plug in position. Figure 3F shows schematically a further embodiment of a device according to the invention.

Figure 3G shows schematically yet another embodiment of a device according to the present invention.

Figure 4 shows schematically a plan view of a 96-well plate for receiving devices according to the invention.

Figure 5A shows schematically a side view of a long side of the plate of Figure 4 and Figure 5B shows schematically a side view of a short side of the plate.

Figure 6 shows experimental results of the microelution of two analytes procainamide and amitryptiline using a device according to the invention and a comparative device.

Description of Embodiments of the Invention

In order to enable further understanding of the invention, but without limiting the scope thereof, various exemplary embodiments of the invention are now described with reference to the accompanying drawings.

Referring to Figure 1A-D, there is shown schematically a device that is useful as a concentrator for biological and other fluids, especially for concentrating such fluids prior to LC and LC/MS analysis. The device as a whole can be considered as a cartridge, which in some embodiments may be disposable (i.e. single use). The device 1 comprises a well 2 of defined size and shape that acts as a reservoir to receive a solution that is to be concentrated, such as blood or urine for example. The well has an open inlet 4 at one end (the upper end in use) via which the solution can be admitted into the well, for example by pipette or other injector. Such solution injection may be automated, e.g. using a robotic system. The well has a generally cylindrical shape (circular cross section) and is of defined internal volume, preferably at least 1 .5 ml, for example 2ml. The well may be of any desired length (height) depending on the internal volume required. Optionally, a syringe piston could be positioned in the well to force solution from the well and through the sorbent. Otherwise, gravity or suction can feed the solution from the well through the sorbent.

The well 2 has an exit pipe 6 that depends from the opposite end of the well to the inlet of the well and which is in fluid communication with the internal volume of the well. The end of the well from which the exit pipe depends is the lower end in use as shown in the figure. The well thereby feeds the solution to the exit pipe wherein there is located a composite sorbent 8. The wall or bottom 10 of the well that connects to the exit pipe is configured at a defined angle 12 to the horizontal when the exit pipe is positioned vertically. The angle is from 1 to 15 degrees to the horizontal plane. Thus, the wall or bottom 10 of the well that connects to the exit pipe is of a shallow conical configuration. The material used for construction of the device is preferably of low extractable content to allow samples to be prepared for LCMS without contamination, e.g. made of a polyolefin such as polypropylene for example.

The exit pipe 6 is straight sided and generally cylindrical shape (circular cross section). The diameter of the exit pipe is significantly smaller than the diameter of the well however and the exit pipe has a smaller internal volume than the well. In this embodiment, the internal diameter d of the exit pipe is 2.5mm. The well and exit pipe have a central axis (i.e. longitudinal axis) 14. In use, the device is designed to be used with the exit pipe in a vertical orientation, i.e. with the longitudinal axis vertical, whereby the well is located above the exit pipe such that solution flows from the well to the exit pipe. The exit pipe shown has a substantially constant internal diameter (i.e. is not tapered) along its length. The composite sorbent is located in the internal diameter of the exit pipe. Due to a constant internal diameter of the exit pipe, the liquid flow velocity of the solution is uniform, especially through the sorbent. By providing the composite sorbent in the narrow exit pipe, the volume occupied by the sorbent is reduced compared to providing sorbent in the well which enhances separation efficiency and also elution efficiency when the separated components are re-dissolved into solution. The exit pipe has an outlet 16 via which the solution leaves after passing through the sorbent. Thus, components concentrated by the sorbent are eluted via the outlet 16. The sorbent 8 located in the exit pipe is porous to allow through solution that flows from the well. The sorbent is a composite sorbent in the form of a fixed solid plug (i.e. not loose or powdered). The composite sorbent thereby is not required to be retained in its position by separate frits. The composite sorbent is preferably a sintered composite, comprising two or more materials (e.g. particulate materials) that have been sintered together to form a solid plug. At least one of the two or more materials sintered together is a sorbent. One material is preferably a porous polymer and another material is preferably a sorbent. The sintered composite may thus comprise porous polymer particles and sorbent particles sintered together. Preferably, the composite sorbent 8 has a retention or extraction property that is selective towards a particular analyte or group of analytes. Preferably, the sorbent has a high enrichment capacity for a particular analyte or group of analytes (e.g. analytes of a particular chemical class). The chemistry of the sorbent and hence its selective retention property is chosen based on the needs of the application. Similarly, the porosity of the sorbent is chosen based on the needs of the application.

The width of the composite sorbent 8 is such that it fits closely against the internal wall of the exit pipe, but allows push fitting of the sorbent into the exit pipe during assembly, so that no significant amount of solution can flow around the edges of the sorbent, i.e. between the edges of the sorbent and the adjacent internal wall of the exit pipe. The solution thus flows effectively through the sorbent, not around it, which increases the efficiency of separation. The weight of sorbent located in the exit pipe is preferably not more than 10mg, for example it may be between 1 and 10mg, for example 2mg, 5mg or 10mg. The weight of the sorbent may be chosen based on the needs of the application, e.g. the expected loading. Figures 1A and to 1 D show various embodiments of the device having different sorbent dimensions (i.e. length (h) of the sorbent plug) and thus sorbent weight and/or positions of the sorbent in the exit pipe. Figure 1A shows a short sorbent plug (low weight) located high in the exit pipe. Figure 1 B shows a short sorbent plug located low in the exit pipe (nearer the outlet). Figure 1 C shows a medium sized sorbent plug located high in the exit pipe. Figure 1 D shows a long sorbent plug that extends most of the length of the exit pipe, e.g. for increased retentive capacity and/or use with higher loadings. Typically, the length (h) of the composite sorbent is not greater than 10 mm and may commonly be not greater than 5 mm. Typically, the exit pipe extends for a length s below the sorbent. Examples of the length s are between 2 and 10 mm, preferably 4 to 8mm, e.g. 4mm, 6mm or 8mm.

Referring to Figure 2 there is shown schematically a part of a device according to the present invention comprising a preferred design of the exit pipe 6. The end of the exit pipe adjacent its outlet 16 is shown in detail. The exit pipe again has an internal diameter of 2.5mm in its upper portion (closest to the well). This is a wider internal diameter portion of the exit pipe. Below this wider portion of the exit pipe is a portion 18 of narrower internal diameter, n. The reduced internal diameter portion provides a lip or rim 20 that is 0.2mm thick around the internal surface of the exit pipe and is located approximately 4mm from the outlet 16 of the exit pipe. The narrower diameter portion of the pipe is only slightly narrower than the wider diameter portion so that the solution flow velocity is not adversely affected between the portions. With the rim thickness of 0.2mm in this embodiment, the narrower diameter portion 18 is only 0.4mm less than the wider diameter portion (figure not drawn to scale). Preferably, the diameter of the narrower diameter portion is at least 60%, more preferably at least 70% and most preferably at least 80% of the diameter of the wider portion. The purpose of the reduced diameter portion is to provide the rim 20 against which the composite sorbent plug 8 can be located and thereby retained in the exit pipe. In other words, the internal diameter of the narrow diameter portion of the pipe is less than the outer diameter of the sorbent plug so that the sorbent is stopped against the rim 20 of the narrow diameter portion of the pipe. In Figure 2A the sorbent plug is shown out of the exit pipe, i.e. prior to assembly, to enable a clear view of the rim 20. In Figure 2B, the sorbent plug is shown in its operational position inserted in the exit pipe against the rim 20. The design shown in Figure 2 also exhibits a further preferred feature in the form of an internal surface 22 of the end portion of the exit pipe outwardly flared towards outlet 16. This flared end portion of the pipe assists in reducing droplet creep up the outside of the pipe at the outlet and thus improves droplet formation.

Alternatively, instead of the reduced internal diameter portion and its associated rim, another means of locating the composite sorbent could be used, such as one or more protrusions or projections at a longitudinal location on the internal surface of the exit pipe that act to stop the longitudinal travel of the sorbent plug in the exit pipe beyond that longitudinal location.

The present invention may also be applied in certain embodiments in the form of a cartridge similar to a conventional solid phase extraction (SPC) cartridge, as shown in schematically in Figure 3A. Referring to Figure 3A, with dimensions (if not stated) in millimeters (mm), there is shown a part of an embodiment of cartridge design according to the invention, which is adapted for elution of small quantities of analyte, wherein a housing 32 provides a well for receiving the solution to be processed. The housing 32 is a cylindrical shape having a circular cross section (internal diameter 5.60mm). The housing has a bottom wall 42 that is angled to the horizontal plane (sloping downwards toward the centre of the housing) and is connected to and in fluid communication with an 8mm long exit pipe 36 having a first (upper) portion with internal diameter 2.25mm. An exit pipe not longer than about 10 or 12 mm is typically employed. A second (lower) portion 48 of the exit pipe has a narrower internal diameter of only 1 .6mm. The upper lip or rim provided by the narrower internal diameter portion 48 of the exit pipe serves to locate a composite sorbent plug 38 and prevent it from being push too far down the exit pipe. The composite sorbent plug 38 is 4mm in length (height) and is 2.2mm wide so that it fits tightly inside the upper (wider) portion of the exit pipe but is stopped by the lower (narrower) portion 48 of the exit pipe. Finally, the exit pipe 36 has an outwardly flared inner surface 52 at its outlet end to promote proper droplet formation, wherein the flared surface makes an angle of 18 degrees to the horizontal plane. In other embodiments, this angle could be larger, e.g. 25 degrees, 30 degrees, 60 degrees or 75 degrees. A flared surface that makes an angle of 15 to 75 degrees to the horizontal plane is preferred.

Figures 3B and 3C show a solid model of a design similar to the design of SPE cartridge shown in Figure 3A where like parts are denoted by like reference numerals. The sorbent plug is not shown inserted in Figure 3B and 3C. Figure 3C shows close-up detail of the portion of the cartridge circled in Figure 3B (in the vicinity of the exit pipe). In this embodiment, the outer surface of the exit pipe at its outlet end is angled to the horizontal plane, e.g. at 80 degrees to the horizontal plane as shown. Other angles of the outer surface of the exit pipe at its outlet end, e.g. 60 or 75 degrees, are also possible. Angles between 15 and 85 degrees are preferred, with 45 to 85 degrees more preferred). This may assist reduction of liquid creep up the outside of the exit pipe. The exit pipe 36 in this case again has a first (upper) portion with internal diameter 2.25mm but the second (lower) portion 48 of the exit pipe in this case has a internal diameter of 2.00 mm, very similar to the diameter of the upper portion. The lower, narrower portion 48 of the exit pipe in this case is 3.6 mm long.

Figure 3D shows another embodiment analogous to the design shown in Figures 3B and 3C. However, in this case, the exit pipe 36 has its outwardly flared inner surface 52 at its outlet end making a larger angle to the horizontal of 75 degrees. Other angles of flared inner surface 52, e.g. 60 degrees, are also possible. Moreover, the flared portion in this case has a longer length of over 1 .0 mm (an actual length of 1 .12 mm) allowing greater focusing on droplet formation. Figure 3E shows the Figure 3D embodiment with the composite sorbent plug 38 (4mm in length (height) and 2.2mm wide) fitted inside the upper (wider) portion of the exit pipe and stopped by the rim of the lower (narrower) portion 48 of the exit pipe.

Figure 3F shows still another embodiment of a device according to the present invention. All measurements refer to internal dimensions in mm. The embodiment comprises an upper well portion 62 of square cross section (in plan or transverse section) and having a width A of 7.5 mm and a height 1 of 35 mm. The bottom of the well portion has curved corners 60 to reduce the collection of fluid in the corners of the well. A first tapered or conical section 64 joins the bottom of the well portion to an upper part 66 of the exit pipe. The diameter C of the top end of the tapered section 64 is 5.0 mm and the diameter D of the bottom end of the tapered section is 2.8 mm. The first tapered section 64 has a length 3 of 5.0 mm. The upper part 66 of the exit pipe is of circular section and is straight and has the diameter D at its top end and a diameter E at its bottom end both of 2.8 mm. The upper part 66 of the exit pipe has a length 4 of 3.4 mm. A second tapered or conical section 68 joins the upper part 66 of the exit pipe to a lower part 70 of the exit pipe, which is also of circular section. The diameter E of the top end of the second tapered section 68 is 2.8 mm and the diameter F of the bottom end of the second tapered section is 1 .2 mm. The second tapered section 68 has a length 5 of 2.0 mm. The lower part 70 of the exit pipe is straight and has the diameter F at its top end and a diameter G at its bottom end both of 1 .2 mm. The length 6 of the lower part 70 of the exit pipe is 4.8 mm. The composite sorbent plug (not shown) fits inside the upper part 66 of the exit pipe. The sections 62, 64, 66, 68 and 70 are in fluid communication with their respective adjacent sections such that a solution with components requiring concentration and/or purification can be initially received in the well 62 to flow through the device, through the composite sorbent plug (not shown) inside the upper part 66 and out of the exit pipe 70.

Figure 3G shows schematically yet another embodiment of a device according to the present invention. The device has a well comprising a wall with an upper (in use) portion 102 and a lower portion 104. The well upper portion 102 is of square transverse cross section and the lower portion 104 of circular transverse cross section. The lower section is also tapered so that it is of conical shape (narrowing toward the exit pipe). The total volume of the well (upper and lower portions) is more than 2ml to accommodate sample solutions of e.g. 1 -2 ml.

A composite sorbent 106 is push fitted into the top of an exit pipe 108 that is connected to the bottom of the well lower portion 104 to receive solution from the well. The composite sorbent comprises a polymeric powder and PTFE powder combined together (by pressing and sintering) to form a 2mg solid which is packed into the device. No movement of the composite sorbent occurs in the device during normal use after push fitting of the sorbent. It is held in place by virtue of tight control of product dimensions during manufacture. It should be noted that no frits are used to hold the sorbent in place. The internal diameter of the portion of exit pipe in which the sorbent 106 is located is substantially constant providing a uniform fluid flow velocity. The exit pipe has a reduction in internal diameter in the form of an internal step 114 which acts as a stop for the sorbent. A reduced internal diameter portion 116 of the exit pipe extends from the step 114 to the outlet 118 of the pipe from which eluate flows out of the device. The reduced internal diameter portion 116 of the exit pipe is also of substantially constant internal diameter providing a uniform fluid flow velocity.

The design shown in Fig. 3G also has a feature of a skirt or wing 110 depending downwardly (in use) from the bottom of the well portion 104. The skirt 110 thereby surrounds an upper part of the exit pipe, particularly the part of the exit pipe in which is located the composite sorbent, with a gap 112 between. This skirt permits the outer dimension of the device at the location of the exit pipe to be of sufficient diameter e.g. so as to fit the dimensions of existing standard size well plates. The skirt allows having such outer dimension whilst using a moulding process that is designed to provide a uniform plastic wall thickness throughout the whole device. The device is generally moulded from a polymer such as polypropylene. Without the skirt, it would require a thicker wall of the device at that point, which is more difficult to mould, so the solution is to provide the skirt in the device by having a mould with an 'annulus" that produces the 'gap' 112 in the wall at that point and thus all wall portions have substantially the same thickness.

The device can be used for the extraction and thereby concentration of compound(s) from a matrix solution. The device allows, for example, for the loading of 1 -2ml of matrix into the well and subsequent elution in a smaller solution volume < 100 μΙ or <25μΙ, thereby allowing for a significant increase in sensitivity in detection (e.g. by LC/MS).

The device thus provides an improvement in sensitivity in the recovery of samples since it uses a small volume (weight) composite positioned in the narrow exit pipe so that the sample is thereby retained in a smaller volume which hence allows minimizing of the volume of solvent to release it. Thus a more concentrated sample can be obtained giving higher sensitivity detection.

The device differs from existing microelution formats, which are comprised of a sorbent material being positioned between 2 spherical frits, whereby significant deviation in reproducibility of recovery results as there are wide tolerances in positioning the sorbent and frit material.

The composite sorbent in accordance with the invention that is packed into the device as described provides a format which minimizes these issues and as a result provides improved reproducibility of recovery at low elution volumes (especially <1 ΟΟμΙ) and also allows higher recoveries to be achieved at <10μΙ than with a sorbent material being positioned between 2 spherical frits. The invention is applicable to being used in a multi-well plate, e.g. a standard 96-well elution plate. Such a plate can be used with robotics systems, e.g.: Tecan and TomTec robotics. Referring to Figure 4, there is shown schematically in a plan view an array of 96 wells 72 (8x12) in a plate 70 that can each receive one of the concentration devices according to the invention. In Figure 5A is shown a schematic side view of a long side (12 wells) of the plate wherein devices 1 according to the invention are shown positioned in the wells 72. Depth guides 76 in the plate define how far the exit pipes 6 of the devices protrude from the bottom of the plate and thus how far into a receiving vessel (not shown) they extend to reduce positive pressure strain on the fluid. Each device 1 may elute into its own receiving vessel. In Figure 5B is shown a schematic side view of a short side (8 wells) of the plate.

It can be seen that in embodiments the invention provides a device that directly outputs an injectable sample for LC or LC/MS. It achieves this through enrichment or concentration on a sorbent composite plug which is porous, of defined adsorbent capacity (i.e. surface area/mass ratio) and has no independent frit or screen arrangement that would classically be used to retain adsorbents. The plug has small dimensions and is housed in a device in such a manner, i.e. within a narrow exit pipe, as to permit concentration of small quantities of solution. The device is dimensioned and shaped to optimize droplet formation with small solution quantities and reduce creep back of solution along the outside of the outlet of the device.

An example of results from the use of a device as depicted in Fig. 3G is shown in Figure 6 and in Table 1 below. A 100 μΙ solution which was 0.1 % formic acid in water containing analytes including procainamide and amitryptiline was introduced into the well and subsequently the analytes were eluted from the sorbent using 5% ammonium hydroxide in methanol with volumes of 100 μΙ, 50 μΙ, 25 μΙ and 10 μΙ. The same procedure was conducted using a comparative device (Waters MCX microelution well product). Table 1 and Figure 6 show the % recovery (% REC) of the analytes as determined by LC/MS detection. It can be seen that the device according to the present invention exhibited significantly improved recovery performance for microelution. Table 1

It will be appreciated the composite sorbent could also be employed by fitting it into the tip of a pipette to act as separation device or into a centrifuge tube as another application.

The foregoing described embodiments are merely examples of devices according to the invention. It should be understood that various modifications may be made to the shown embodiments whilst still falling within the scope of the invention.

As used herein, including in the claims, unless the context indicates otherwise, singular forms of the terms herein are to be construed as including the plural form and vice versa. For instance, unless the context indicates otherwise, a singular reference, such as "a" or "an" means "one or more".

Throughout the description and claims of this specification, the words "comprise", "including", "having" and "contain" and variations of the words, for example "comprising" and "comprises" etc, mean "including but not limited to", and are not intended to (and do not) exclude other components.

It will be appreciated that variations to the foregoing embodiments of the invention can be made while still falling within the scope of the invention. Each feature disclosed in this specification, unless stated otherwise, may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The use of any and all examples, or exemplary language ("for instance", "such as", "for example", "e.g." and like language) provided herein, is intended merely to better illustrate the invention and does not indicate a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Any steps described in this specification may be performed in any order or simultaneously unless stated or the context requires otherwise.

All of the features disclosed in this specification may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. Likewise, features described in non-essential combinations may be used separately (not in combination).

Claims

1 . A device for separating components of a solution comprising: a well for receiving the solution; an exit pipe connected to the well for receiving the solution from the well wherein the exit pipe is of smaller diameter than the well; and a porous composite sorbent within the exit pipe for retaining one or more components from the solution, wherein the composite sorbent is in the form of a sintered solid composition comprising at least porous polymer particles and sorbent particles sintered together that is push fitted into the exit pipe so as to form a close fit within the exit pipe, and wherein the exit pipe has a restriction therein that acts to stop travel of the composite sorbent in the exit pipe beyond the location of the restriction.

2. A device as claimed in claim 1 wherein the well is cylindrical in shape.

3. A device as claimed in claim 1 or 2 wherein the well has a bottom that connects to the exit pipe and the bottom is configured to be curved or at a defined angle of from 1 to 85 degrees to the horizontal when the exit pipe is vertical.

4. A device as claimed in any preceding claim wherein the exit pipe is cylindrical in shape.

5. A device as claimed in any preceding claim wherein the exit pipe has a substantially constant internal diameter along at least a portion of its length.

6. A device as claimed in claim 5 wherein the composite sorbent is located in the substantially constant internal diameter section.

7. A device as claimed in any preceding claim wherein the exit pipe is at least 8 mm in length.

8. A device as claimed in any preceding claim wherein the exit pipe has more than one value of diameter and the narrowest diameter of the exit pipe is at least 60% of the widest diameter of the exit pipe.

9. A device as claimed in any preceding claim wherein the restriction is in the form of an internal step or reduction in internal diameter of the exit pipe or in the form of one or more protrusions into the internal volume of the exit pipe.

10. A device as claimed in claim 9 wherein the restriction is in the form of an internal step or reduction in internal diameter of the exit pipe that is provided by the exit pipe comprising a wider diameter portion closest to the well into which the composite sorbent is push fitted and a narrower diameter portion connected to the wider narrow portion that acts to stop the travel of the composite sorbent into the narrower diameter portion.

1 1 . A device as claimed in claim 10 wherein the wider portion of the exit pipe has a substantially constant internal diameter along its length.

12. A device as claimed in claim 10 or 1 1 wherein the narrower diameter portion of the exit pipe is at least 60% of the wider diameter portion of the exit pipe.

13. A device as claimed in any preceding claim wherein the aspect ratio of the exit pipe (length/internal diameter) is between 2 and 10.

14. A device as claimed in any preceding claim wherein the aspect ratio of the composite sorbent (length(L)/outer diameter(OD)) is between 0.5 and 10, or between 1 and 10, or between 0.5 and 8, or between 1 and 8, or between 0.5 and 6, or between 1 and 6.

15. A device as claimed in any preceding claim wherein frits are not present in the exit pipe to retain the composite sorbent therein.

16. A device as claimed in any preceding claim wherein the composite sorbent has a weight of not more than 10mg or not more than 5mg.

17. A device as claimed in any preceding claim wherein the composite sorbent has an extraction property that is selective towards a particular analyte or group of analytes.

18. A device as claimed in any preceding claim wherein the outlet of the exit pipe is flared on its internal surface.

19. A device as claimed in any preceding claim wherein the outer surface of the exit pipe at its outlet end is angled to the horizontal plane at 60 to 85 degrees.

20. A device as claimed in any preceding claim wherein the device is for use in a multi-well plate.

21 . A device as claimed in any preceding claim wherein the device is for concentrating a solution for injection into an LC or LC/MS system.

22. A method of using the device as claimed in any preceding claim comprising concentrating a solution by retaining one or more components from the solution with the sorbent and eluting a concentrated solution from the device for injection into an LC or LC/MS system.