Nmr tube

Abstract

A nuclear magnetic resonance tube (100) is disclosed, sized for fitting within an NMR machine (101). It comprises a semi-permeable membrane (105) located within the NMR tube (100) for providing a semi-permeable boundary between first a sample region (103) within the NMR tube (100) and a second sample region (104) within the NMR tube (100). The first sample region (103) is configured for holding a first sample. The second sample region (104) is configured for holding a second sample.

Description

[0001] NMR Tube

[0002] TECHNICAL FIELD

[0003] This invention relates to nuclear magnetic resonance (NMR) tubes for NMR spectroscopy.

[0004] BACKGROUND

[0005] Nuclear magnetic resonance (NMR) spectroscopy is a spectroscopic technique which measures nuclear spin coherences created by radio frequency pulses in a magnetic field. In typical NMR experiments, a sample is placed in a magnetic field to align (quantize) its magnetic nuclear spins. This alignment of the nuclear spins is subsequently perturbed by a radio-frequency pulse transmitted to the sample via a coil and, in response, electromagnetic waves are emitted by the nuclei of the sample which are detected and analysed.

[0006] A liquid sample to be studied by NMR is typically placed within a substantially cylindrical glass NMR tube, which is then inserted into the probe of an NMR machine. Typical NMR tubes are made from borosilicate glass or quartz, are around 15-20 cm in length, and have an outer diameter of between 3 mm - 10 mm (commonly 5 mm) with a wall thickness of less than 1 mm.

[0007] This approach works well for studying single, homogenous, liquid samples. However, the present invention seeks to provide a nuclear magnetic resonance (NMR) tube apparatus that is suitable for performing more complex NMR spectroscopy analysis.

[0008] SUMMARY OF THE INVENTION

[0009] From a first aspect, the invention provides a nuclear magnetic resonance (NMR) tube sized for fitting within an NMR machine, wherein the NMR tube comprises a semi-permeable membrane located within the NMR tube for providing a semi- permeable boundary between a first sample region within the NMR tube and a second sample region within the NMR tube, the first sample region being configured for holding a first sample and the second sample region being configured for holding a second sample.

[0010] Thus it will be seen that, in accordance with embodiments of the present invention, the semi-permeable membrane provides a semi-permeable boundary between two sample regions arranged within the NMR tube. This can allow for NMR spectroscopy reaction-monitoring or other multiple-chamber monitoring experiments to be performed. By providing a semi-permeable boundary between the sample regions, this apparatus can be used even when the first and second samples comprise two closely-related solvents that would not form a smooth liquid-liquid interface if placed one above the other in a standard NMR tube. Embodiments disclosed herein may be used to study transport phenomena, or to control an experiment (i.e. a diffusion controlled titration) by using diffusion across the boundary. For example, the two samples may be allowed to mix slowly over time, due to diffusion across the semi-permeable membrane, while using NMR spectroscopy to monitor change in the amounts, e.g. concentrations, of certain compounds.

[0011] In particular, the semi-permeable membrane can allow multiple-chamber monitoring of miscible samples. This means that two aqueous phases can be used - making embodiments of the invention useful for many biological experiments. Embodiments of the invention may enable two closely-related solvent samples to be studied, which have similar properties. The slow mixing of such samples may be analysed spectroscopically over time.

[0012] The semi-permeable membrane may be arranged perpendicularly to an axis of the NMR tube. It may be arranged to be horizontal in use — i.e. to separate the first and second sample regions vertically.

[0013] The semi-permeable membrane may be a synthetic or natural polymer membrane (e.g. a cellulose filter), or a porous glass membrane, or any other type of semi- permeable membrane. It may be permeable to a first set of one or more molecules present in the first and / or second sample, and impermeable to a second set of one or more molecules present in the first and / or second sample. The NMR tube may comprise a hollow cylindrical (preferably circular-cylindrical) tube body. The NMR tube or tube body may be open at both ends or may be closed at one end.

[0014] The NMR tube or tube body may have a circular outer cross-section, which may have a diameter of between 3 mm and 10 mm — e.g. 5 mm. It may have an outer wall of a glass material, e.g. borosilicate glass. The glass material may have a magnetic susceptibility that is tuned to match the magnetic susceptibility of an NMR solvent, such as water, dimethyl sulfoxide, methanol, or chloroform.

[0015] In a first set of embodiments, the semi-permeable membrane is permanently fixed within the NMR tube. In some embodiments, the NMR tube may comprise a hollow cylindrical tube body, wherein the semi-permeable membrane is fixed at a position within the tube body and extends across a transverse section of the tube body. The position may be spaced away from both ends of the tube body — e.g. by 10 mm, 20 mm, or more.

[0016] However, in a second set of embodiments, the semi-permeable membrane is movably positioned within the NMR tube. In some embodiments, the NMR tube comprises a hollow cylindrical tube body and a nuclear magnetic resonance (NMR) tube-insert at least partly within the NMR tube, wherein the first sample region is within the NMR tube-insert, and wherein the NMR tube-insert comprises: a first end for receiving the first sample the NMR tube-insert; and a semi-permeable membrane adjacent a second end of the NMR tube-insert for providing the semi-permeable boundary between the first sample region and the second sample region within the NMR tube.

[0017] The NMR tube-insert may comprise a hollow cylindrical (preferably circular- cylindrical) tube-insert body. The first end of the tube-insert may be a first end of the tube-insert body, and the second end may be a second end of the tube-insert body.

[0018] At least some such embodiments allow the tube-insert to be used with a standard NMR tube, and may allow the NMR tube to be re-used once the NMR tube-insert is removed. Such a tube-insert for fitting at least partly within an NMR tube (e.g. for at least a proportion, or all, of a length of the tube-insert) forms a further aspect of the invention. Therefore, from a second aspect, the invention provides a nuclear magnetic resonance (NMR) tube-insert sized for fitting at least partly within an NMR tube that is sized for fitting within an NMR machine, the NMR tube-insert comprising: a first end for receiving a first sample into a first sample region within the NMR tube-insert; and a semi-permeable membrane adjacent a second end of the NMR tube-insert for providing a semi-permeable boundary between the first sample region within the NMR tube and a second sample region within the NMR tube.

[0019] The semi-permeable membrane may be mechanically fastened, or chemically- bonded or heat-bonded, to a body of the NMR tube-insert. It may cover some or all of the second end of the tube-insert body. In some embodiments the semi- permeable membrane and the tube-insert body may be monolithically integrated — for example, the semi-permeable membrane may be provided by a membrane of porous glass, and the tube-insert body may be formed of porous or non-porous glass, integrally formed with the semi-permeable membrane such that the semi- permeable membrane and the tube-insert body together are a single piece of glass.

[0020] From a third aspect, the invention provides a method of fabricating a nuclear magnetic resonance (NMR) tube, the method comprising inserting the NMR tubeinsert into an NMR tube so that the semi-permeable membrane of the NMR tubeinsert is located within the NMR tube and provides a semi-permeable boundary between the first sample region within the NMR tube-insert and a second sample region within the NMR tube.

[0021] The method may further comprise fabricating the NMR tube-insert by affixing the semi-permeable membrane to an end of an open tube, the open tube being sized for fitting at least partly within the NMR tube.

[0022] In any of the embodiments disclosed herein, the semi-permeable membrane may be positioned within the NMR tube so that, when the NMR tube is placed in an NMR machine, a probe of the NMR machine extends over the first sample region and over the second sample region. This may allow both sample regions to be spectroscopically monitored at the same time, e.g. using z-selection.

[0023] In a set of embodiments, the first and second sample regions contain a first and second sample, respectively. Preferably, the first sample region is configured for holding a first powder of fluid sample, and the second sample region is configured for holding a second powder or fluid sample. In some embodiments, the first and second samples may both be liquids. They may both be aqueous solutions. The second sample may be different from the first sample.

[0024] The invention extends to a kit for assembling a nuclear magnetic resonance (NMR) tube-insert, the kit comprising: an open tube sized for fitting at least partly within an NMR tube that is sized for fitting within an NMR machine, the open tube comprising: a first end for receiving a first sample into a first sample region within the open tube; and a second end comprising a fixing surface; and a semi-permeable membrane for affixing to the fixing surface, wherein, when fixed to the fixing surface, the semi-permeable membrane is arranged for providing a semi-permeable boundary between the first sample region and a second sample region within the NMR tube.

[0025] The kit may further comprise the NMR tube. The insert may be single-use, e.g. disposable. The kit may be used in a method of fabricating a nuclear magnetic resonance (NMR) tube as disclosed herein.

[0026] Affixing the semi-permeable membrane to the fixing surface may be done in any suitable way. The fixing may be permanent or releasable. For example, the semi- permeable membrane may be mechanically fastened to the fixing surface, e.g. by a clip, or using a collar so that the membrane ‘caps’ one end of the open tube. In one set of embodiments, the membrane is a circular disc that is secured between an end of a tube-insert body (providing the fixing surface) and a cap member that caps the end of the tube-insert body. The cap member may be toroidal, i.e. defining a circular opening for the passage of fluid through the cap. It may be secured by glue or by tension. This can allow use with membranes that are incompatible with gluing or heat-bonding of the membrane itself. In other embodiments, the semi-permeable membrane may be affixed to the fixing surface by chemical-bonding (e.g. using an adhesive) or by heat-bonding, e.g. thermoplastic bonding. For example, the fixing surface may be partially melted and the membrane then applied to the fixing surface so that, when set, the membrane is bonded to the fixing surface.

[0027] In a set of embodiments, the NMR tube and / or open tube and / or NMR tube-insert comprises or is formed of a non-magnetic material that is transparent to radio waves. It may also be optically transparent. The open tube may comprise a hollow (e.g. borosilicate) glass or plastic tube. It may have a magnetic susceptibility that is tuned to match the magnetic susceptibility of an NMR solvent, such as water, dimethyl sulfoxide, methanol, or chloroform.

[0028] In a set of embodiments, the NMR tube-insert or open tube comprises a ring, e.g. a plastic ring, to which the semi-permeable membrane is or may be attached. The ring may be at the second end of the open tube, and may provide the fixing surface for the semi-permeable membrane. In an example embodiment, the NMR tubeinsert comprises a glass open tube with a plastic fixing surface, the fixing surface being provided by a plastic ring. In another example embodiment, the entire NMR tube-insert is made from a uniform material, such as a polymer, (e.g. including both the open tube and the fixing surface); this may help to allow the membrane to be affixed to the NMR tube-insert without needing to provide a separate plastic ring or other attachment for providing the fixing surface.

[0029] In some embodiments, the NMR tube and / or NMR tube-insert may comprise a support member, such as a coarse mesh, arranged to support the semi-permeable membrane. The support member may touch the semi-permeable membrane. It may be arranged parallel to the semi-permeable membrane. It may comprise a set of holes each having a diameter that is 10, 100, 1000 or more times greater than the maximum pore diameter of the semi-permeable membrane. In this way, the support member will not significantly impede the passage of fluid through the membrane.

[0030] The NMR tube-insert (e.g. a tube-insert body thereof) may be sized and shaped for a close fit within an NMR tube, e.g. a friction fit. Preferably, everywhere around a circumference of an outer wall of the tube-insert, a distance between an outer wall of the tube-insert and an inner wall of the NMR tube is less than 1 mm, e.g. less than 100 pm, or less than 10 pm. As will be appreciated by the skilled person, the smaller the gap, the closer the fit between the NMR tube-insert and the NMR tube; having only a small gap may help to avoid relative movement between the two parts in use. However, a gap (e.g. of at least 1 pm or 10 pm) may usefully be provided around at least part, or all, of a circumference of an outer wall of the tube-insert, e.g. in order to allow air to escape the NMR tube as the tube-insert is pushed down into the top of the NMR tube so as to prevent the formation of air bubbles adjacent the semi-permeable membrane.

[0031] In some embodiments, the NMR tube-insert comprises one or more seals (e.g. an O-ring) arranged to form a fluid (e.g. liquid) seal between the NMR tube-insert and the second sample region. The seal may help prevent leakage of the second sample into the first sample region and / or into a void between the NMR tube-insert and the NMR tube body.

[0032] In a set of embodiments, the NMR tube (e.g. the tube body of the NMR tube) comprises at least one feature for holding the semi-permeable membrane of the tube-insert at a predetermined position within the NMR tube or tube body, at least when the NMR tube is assembled for use. In some embodiments, the NMR tube may comprise one or more grooves on an inner wall of the NMR tube body for holding the semi-permeable membrane and / or tube-insert in position within the NMR tube. Such a groove may be arranged to interface (e.g. mate) with a protruding element of the tube-insert, such as a plastic ring to which the membrane is attached. In some embodiments, the NMR tube comprises one or more protrusions (e.g. dimples) extending radially inwards for holding the semi- permeable membrane in position. In some embodiments, the NMR tube has a non- uniform inner diameter that is smallest at a waist of the NMR tube. The waist may be provided uniformly by a toroidal protrusion or it may be provided at one or more discrete diameters only, e.g. by one, two or more discrete inwardly-extending protrusions (e.g. dimples). The waist may be sized for retaining the tube-insert at a predetermined position within the NMR tube. The waist has a diameter that is narrower than an outer diameter of the tube-insert, at least in a vicinity of the semi- permeable membrane. The semi-permeable membrane may be positioned at or adjacent the waist, at least when the NMR tube is in use. The tube-insert may comprise one or more features for engagement with a positioning tool for moving the tube-insert axially within the NMR tube body. Each feature may comprise a groove, plate, hole or patterned element. It may be configured for engagement by a hooked tool. In some embodiments, the tube-insert may comprise a pair of diametrically-opposed engagement features (e.g. a pair of blind holes or through holes in a side wall of the tube-insert). They may be arranged for engagement by respective parts of a positioning tool. They may facilitate more precise positioning than having just a single feature.

[0033] Not all embodiments comprise a tube-insert, but instead have a semi-permeable membrane fixed (e.g. bonded) within the tube body and extending across a transverse section of the tube body. Such embodiments, without an insert, may desirably provide a uniform wall thickness adjacent both sample regions of the NMR tube.

[0034] The invention extends to a method for performing nuclear magnetic resonance (NMR) spectroscopy, comprising using an NMR tube as disclosed herein in an NMR machine to perform NMR spectroscopy.

[0035] The method may comprise spectral acquisition over a predetermined period of time, e.g. over 1 to 60 seconds. In a set of embodiments, the method comprises generating a time series of NMR data, e.g. over minutes, or hours (possibly 12 hours or more). Such NMR monitoring over time may be able to generate data with a higher temporal resolution, on multiple components, compared to existing techniques such as phospholipid vesicle permeation assays (PVPA).

[0036] In a set of embodiments, the method comprises performing NMR spectroscopy on a first sample in the first sample region and / or a second sample in the second sample region. It may comprise determining or monitoring a change in concentration of a component of the first sample or of the second sample caused by diffusion through the semi-permeable membrane. It may comprise studying a change in concentration of a component in a sample region on one or both sides of the semi- permeable membrane. In some embodiments, it may comprise determining or monitoring a change in a structure or state of a component. In some embodiments, it may comprise imaging the first and / or second sample, and using the imaging to monitor one or more diffusion phenomena.

[0037] In some embodiments, the method comprises simultaneously performing NMR spectroscopy on the first sample region and the second sample region in order to study a change in concentration, structure or state of one or more components on both sides of a semi-permeable membrane. The method may comprise performing more than one type of NMR spectroscopy at once - e.g. performing proton 1 D1H NMR, deuterium2H NMR and / or 2D15N HSQC (Hetereonuclear Single Quantum Coherence) at the same time. This allows a higher number of characteristics to be monitored within the samples.

[0038] In a set of embodiments, the method comprises monitoring the transport of components across the semi-permeable membrane and / or changes in the transport of components across the semi-permeable membrane. Therefore, the membrane may be selected as a membrane to be studied for its transport phenomena.

[0039] In a set of embodiments, the method comprises applying a magnetic field gradient to the NMR tube and monitoring the change of concentration of one or more components at a first position in the first sample region and a second position in the second sample region using spatial selection. This method may use a Pulsed Field Gradients (PFG) - NMR technique. Thus, in a set of embodiments, the magnetic field gradient is pulsed. This provides an increased sensitivity of measurement compared to non-pulsed experiments and has been found to be particularly useful in diffusion experiments.

[0040] This may be especially useful for studying bacterial-like membranes. Transport phenomena is of great interest in a number of scientific fields. Having the ability to study transport phenomena across the membrane allows the composition and construction of the membrane to be tested and thus adjusted to fit any number of biological and non-biological use cases where movement of material across a barrier is of interest. Experiments making use of the invention may include, for example, experiments on: the blood brain barrier; gastrointestinal barriers; biofilms; drug release; chemical reaction monitoring and / or battery cells. The opportunity to study biological membranes in this way, in an NMR machine rather than in-vivo, may also reduce the need for animal models in the testing of pharmaceuticals, while offering monitoring of transport of certain components over time.

[0041] Embodiments exploiting diffusion to introduce chemical entities from a first position in the first sample region to a second position in the second sample region may be especially useful for performing diffusion-controlled titration experiments on biomolecules — for example the titration of an interaction, pH dependence or denaturation / unfolding. The opportunity to perform titrations without the need for manual sample handling may save instrument time and operator time compared with traditional approaches using conventional NMR tubes without any insert.

[0042] Some embodiments of the NMR tube may comprise a further semi-permeable membrane located within the NMR tube, for providing a semi-permeable boundary between the first or second sample region and a third sample region within the NMR tube, the third sample region being configured for holding a third sample. The further membrane may be provided by a tube-insert, which may be separate from a tube-insert providing the first semi-permeable membrane. The tube-insert may be arranged to support the further membrane at a different predetermined position within the NMR tube compared with a position of the first membrane.

[0043] In some embodiments, the NMR tube may comprise two, three or more semi- permeable membranes and may define three, four or more sample regions.

[0044] Features of any aspect or embodiment described herein may, wherever appropriate, be applied to any other aspect or embodiment described herein. Where reference is made to different embodiments or sets of embodiments, it should be understood that these are not necessarily distinct but may overlap.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Certain preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0047] FIG. 1 is a schematic diagram of a nuclear magnetic resonance (NMR) tube embodying the invention in an NMR spectroscopy machine;

[0048] FIG. 2 is a schematic diagram of an NMR tube-insert embodying the invention within an NMR tube, thus forming an NMR tube embodying the invention; FIG. 3 is a schematic diagram of an alternative NMR tube embodying the invention, having a fixed semi-permeable membrane within the NMR tube;

[0049] FIG. 4A is a schematic diagram of the NMR tube of FIG. 2 placed within an NMR coil for monitoring two sample regions;

[0050] FIG. 4B is a schematic diagram of the NMR tube of FIG. 2 with the NMR tube-insert positioned above the NMR coil for monitoring only one sample region;

[0051] FIG. 5 is a schematic diagram of an NMR tube embodying the invention alongside example data obtained from simultaneous monitoring of the change of concentration of certain components in both sample regions of the NMR tube;

[0052] FIG. 6 is a schematic diagram showing an NMR tube embodying the invention arranged for tracking multiple components simultaneously;

[0053] FIG. 7 is a schematic diagram showing an NMR tube embodying the invention having protrusions for holding a tube-insert in a desired position;

[0054] FIG. 8A is a schematic diagram of components for assembling an NMR tube-insert embodying the invention;

[0055] FIG. 8B is a schematic diagram of the assembled NMR tube-insert of FIG. 8A;

[0056] FIG. 9 is a schematic diagram of an NMR tube-insert embodying the invention and a positioning tool for adjusting the position of the NMR tube-insert within an NMR tube body; and

[0057] FIG. 10 is a schematic diagram of a further NMR tube-insert embodying the invention and a different positioning tool hook for adjusting the position of the NMR tube-insert within an NMR tube body.

[0058] DETAILED DESCRIPTION OF EMBODIMENTS

[0059] FIG. 1 shows a nuclear magnetic resonance (NMR) system comprising an NMR spectroscopy machine 101 having inserted therein an NMR tube 100 according to an embodiment of the invention. The NMR machine 101 has RF coils 106 surrounding the portion of the sample region(s) for exciting the sample. Not shown is a magnet, probe, vacuum chamber and nitrogen / helium ports within the NMR machine 101.

[0060] The NMR tube 100 has a hollow cylindrical tube body 107 made from borosilicate glass and having a 5 mm (outer) diameter with walls that are approximately 0.3 mm thick. In some examples its magnetic susceptibility matches that of water or some other NMR solvent.

[0061] The NMR tube 100 comprises a semi-permeable membrane 105, which, in this embodiment, is provided by an NMR tube-insert 102 located within the tube body 107. The NMR tube 100 of FIG. 1 is elongate and extends along a longitudinal axis which coincides with the central vertical axis (i.e. the bore) of the NMR machine 101. The longitudinal axis of the NMR tube 100 lies normal to the membrane 105. The membrane 105 provides a semi-permeable boundary between a first sample region 103 and a second sample region 104. Having both sample regions 103, 104 measured by the NMR machine 101 allows for multiple-chamber NMR experiments to be done, e.g. even using miscible samples.

[0062] The NMR tube 100 of FIG. 1, according to a first embodiment, comprises an NMR tube-insert 102 for inserting within a standard NMR tube 107.

[0063] FIG. 2 shows another, similar NMR tube 200 in more detail. This also comprises a tube insert 202 within a tube body 207, with the tube insert 202 comprising a semi- permeable membrane 205.

[0064] In both FIG. 1 & 2, the tube-insert 102, 202 comprises an open cylindrical tubeinsert body sized for fitting at least partly within the standard, e.g. 5 mm, NMR tube 107, 207 (i.e. for all or at least a proportion of its length). The tube-insert 102, 202 has a first end for receiving a first sample 103, 203 and a second end having a fixing surface 202a to which the semi-permeable membrane 105, 205 is affixed, e.g. by heat-bonding or gluing. In FIG. 2, a downwards arrow shows the direction of insertion, i.e. the direction in which the tube-insert 202 should enter the NMR tube 207.

[0065] The insert 102, 202 acts as a cylindrical compartment for the first sample region 203 with a porous filter 105, 205 fused to one end. In this particular example, the semi-permeable membrane 105, 205 is a cellulose filter. The NMR tube-insert may be designed to fit a range of NMR tube sizes. The cellulose filter 105, 205 may be coated with phospholipids to mimic the membrane properties of eukaryotic and bacterial cells. The tube-insert 102, 202 is sized and shaped to fit closely within a conventional NMR tube body 107, 207; so as to suspend the membrane 105, 205 within the NMR tube 100, 200 at a distance above the base of the NMR tube 100, 200. The membrane 105, 205 may be joined or bonded to the tube-insert 102, 202 by a ring of plastic or rubber or by fusing to a fixing surface (e.g. through thermoplastic bonding). The fixing surface may be formed of polylactic acid (PLA), polycaprolactone (PCL), polyethylene terephthalate (PET), or any other material that can be partially melted at a temperature that does not damage the membrane 105, 205. In other embodiments, the membrane may be an integral part of a monolithic tube-insert, e.g. with the membrane being of porous glass and with the cylindrical body of tube-insert being of the same porous glass or of a different glass.

[0066] In use, the membrane-covered tube-insert 102, 202 is filled with a first liquid sample in the first sample region 103, 203. The first liquid sample is a ‘donor’ solution containing a compound of interest. The NMR tube 100, 200 is partially pre-filled with a second liquid sample, e.g. an ‘acceptor’ solution. The NMR tube-insert 102,

[0067] 202 is placed within the NMR tube 100, 200 such that the first sample region 103,

[0068] 203 lies on top of the second sample region 104, 204. The membrane 105, 205 may rest on top of the second sample region

[0069] The contents of the ‘donor’ region 103, 203 and the ‘acceptor’ region 104, 204 can be monitored independently over time. A time-series of acquisitions may be made over a period of time. These may be obtained alternately for the first and second regions, resulting in two interleaved time series of data. The time period could be seconds, minutes or hours. For some experiments, it may be between 12 hours or more.

[0070] The NMR method according to embodiments of the invention may make use of z- gradient spatially selective pulse programs to track the movement of multiple components across the membrane barrier 105, 205 over time. This may enable the characterization of the effect of membrane disrupting compounds in detail.

[0071] Embodiments may thus make it possible to distinguish and quantify multiple components from one another spectroscopically. For example,1H,2H and19F z- selected pulse programs may be used to detect ion, water and small molecules, however other common nuclei such as13C and15N may be used. 2D experiments like1H,15N-HSQC or1H,13C-HSQC may be used to monitor changes in larger molecules, and imaging experiments may be used to map diffusion phenomena.

[0072] FIG.3 shows a second embodiment of the NMR tube according to the invention. According to the second embodiment, the NMR tube 300 comprises a hollow cylindrical tube body 307, open at both ends, and having a semi-permeable membrane 305 fixed within the tube body 307, and extending across a transverse section of the tube body 307, so as to separate it into two chambers, i.e. the first sample region 303 and the second sample region 304.

[0073] In use, the bottom-most sample region 304 is completely filled with a liquid sample and sealed or capped at the end, e.g. with a bung 308, before flipping the tube 300 over so that the other sample region 303 can be filled with another liquid sample.

[0074] This NMR tube 300 is dimensioned similarly to a conventional NMR tube and can be directly loaded into the NMR machine 101, like the one shown in FIG.1 , e.g. for analysis of movement of components through the semipermeable membrane 305 over time.

[0075] FIGS. 4A and 4B show two ways to use the NMR tube according to embodiments the invention.

[0076] Both drawings show an NMR tube-insert 402a, 402b capped with a semi-permeable membrane 405a, 405b. Each tube-insert 402a, 402b is filled with a liquid sample in a first sample region 403a, 403b. The tube-inserts 402a, 402b are inserted within NMR tubes 400a, 400b that are pre-filled with a second liquid sample in the second sample region 404a, 404b. The NMR tubes 400a, 400b are inserted into a probe comprising coils 406a, 406b.

[0077] FIG. 4A shows the tube-insert 402a positioned within the coil 406a so that z- gradients can be used to detect radio waves from nuclei in both the first sample region 403a and the second sample region 404a to allow monitoring of the donor and acceptor chambers simultaneously. FIG. 4B shows an alternative mode, where the tube-insert 402b is positioned so that the first sample region 403a lies outside of the coils 406b such that only the second sample region 404b or the ‘acceptor’ chamber, is monitored, allowing for the use of non-gradient selective pulse-programs. This allows the tube-insert 402b to be used with NMR instruments that are not capable of gradient selection. In such a case, only signals from the sample in the second sample region 404b are detected by the coils 406b. This may still be useful for two-chamber experiments involving monitoring chemical reactions or transport of components across the membrane 405b as the changes of component concentrations in the second sample region 404b could provide information on how the samples in the first and second sample regions 403b, 404b behave when placed either side of a semi- permeable membrane 405b.

[0078] FIG. 5 shows how the technique shown in FIG.4A results in data for both sample regions 403a, 403b as a result of applying a magnetic field gradient and using z- selection to detect component concentrations at different heights. FIG. 5 shows an NMR tube 500 embodying the invention, with a tube-insert 502 comprising the membrane 505 positioned within the coil 506 to allow monitoring of both regions 503, 504 simultaneously.

[0079] Applying a magnetic field gradient that varies along the z-axis (see vertical arrow indicating the direction of the z-axis). When acquisition is in the presence of a magnetic field gradient, a position-dependent change of resonance frequency gives a vertical profile of the frequency, i.e. also known as spatial encoding. The magnetic field gradient may be pulsed - i.e. pulse field gradient NMR (PFG-NMR).

[0080] The graph in FIG.5 shows the amount of ‘guest’ molecule detected in two different regions over 1080 minutes -1H NMR measuring guest concentration at a z-position in the first sample region 503, i.e. shown by line 510, and1H NMR measuring guest concentration at a z-position in the second sample region 504, i.e. shown by line 511. The graph shows an increase in concentration in the guest molecule in the second sample region 504 and a decrease in concentration in the guest molecule in the first sample region 503. FIG. 6 shows that embodiments of the invention can also allow a plurality of components to be monitored. FIG. 6 shows an NMR tube 600 embodying the invention, with a tube-insert 602 comprising the membrane 605 positioned within the coil 606 to allow monitoring of both regions 603, 604 simultaneously. With this arrangement, it is possible for z-selected1H NMR and2H NMR to be performed simultaneously.

[0081] FIG. 7 shows an exemplary NMR tube 700 that is very similar to the NMR tube 200 shown in FIG. 2 apart from two differences. First, the tube-insert 702 is shorter in height than the tube-insert 202 of FIG. 2. This illustrates that tube-inserts may be produced in any of a variety of heights, and in use may protrude from a top of the NMR tube body 707, or may fit wholly within the height of the tube body 707. Secondly, and independently from this first difference, the NMR tube body 707 in this example has two inwardly-extending, diametrically-opposed protrusions or dimples 708a, 708b, positioned at approximately one third of the height of the tube body 707 up from the base of the tube body 707. These dimples 708a, 708b provide a narrow waist to the tube body 707 at this height, the waist having a diameter that is narrower than an outer diameter of the tube-insert 702 at the semi- permeable membrane 705. The dimples 708a, 708b help to retain the tube-insert 702 at a predetermined position within the tube body 707, by resisting the force of gravity on the tube-insert 702.

[0082] In some variant embodiments, there may be more three or more dimples, e.g. uniformly spaced around the inner circumference of the tube body 707. In a further variant, instead of discrete dimples 708a, 708b, the tube body 707 may have a toroidal protrusion that extends inwardly uniformly around the circumference of the tube body 707, thereby defining a continuous waist.

[0083] The provision of a waist or other feature for holding the semi-permeable membrane of an NMR tube-insert at a predetermined position can reduce or eliminate a reliance on friction to hold the tube-insert in position, which may lead to more consistent positioning and / or results.

[0084] Any of the tube-inserts disclosed herein may optionally be used with NMR tube bodies that have one or more such protrusions. FIG. 8A shows parts for assembling an NMR tube-insert 802 in which a semi- permeable membrane 805 is sandwiched (i.e. clamped) between an end face 809a of a cylindrical tube-insert body 809 and a toroidal end cap 810. The tube-insert 802 can be automatically or manually assembled by placing the membrane 805 against the open lower end of the tube-insert body 809, abutting the fixing surface provided by the end face 809a, and then pressing the cap 810 over the outer walls at the lower end of the tube-insert body 809 so as to secure the membrane 805 against the fixing surface 809a. The cap 810 may be glued in place or may be stretched and held by tension within the cap 810. FIG. 8B shows the assembled NMR tubeinsert 802. This approach allows the tube-insert 802 to use membranes that may not withstand heat-bonding or direct gluing.

[0085] In other variant embodiments of an NMR tube-insert, the semi-permeable membrane may be held in place by one or more additional components such as a cap 810 or an O-ring, or may be held in place mechanically, or by heat-bonding, or chemical bonding, or gluing, or any combination of these methods.

[0086] FIG. 9 shows a cylindrical NMR tube-insert 902 having a semi-permeable membrane 905 at its lower end. The side wall of the tube-insert 902 has a horizontal, circular through-hole 911 , close to the upper end of the tube-insert 902. This hole 911 is sized to receive the hooked tip of a positioning tool 912 (e.g. made of metal). When the tool 912 is engaged in the hole 911, it can be used by a human or robotic operator to raise or lower the tube-insert 902 to a desired position within an NMR tube body.

[0087] FIG. 10 shows a variant NMR tube-insert 1002 that has a pair of diametrically- opposed through holes 1011 , 1013 in its cylindrical side wall, arranged for engagement by different respective hooked legs of a sprung positioning tool 1012. This may provide a more secure engagement for manipulating the tube-insert 1002 to a desired position within an NMR tube body.

[0088] More generally, a tube-insert may comprise a groove or pattern or blind-hole or through-hole or other feature to enable a tool to apply a force to the tube-insert to remove and / or reposition the NMR tube-insert within an NMR tube body. In some embodiments, a plurality of NMR tube-inserts 102, 202, 402, 502, 702, 802, 902 (e.g. two or more) may be stacked vertically within a single NMR tube body 107, 207, 407, 707 to form an NMR tube having three or more distinct sample regions, separated by semi-permeable membranes. Alternatively, one or more NMR tube-inserts may be placed within an NMR tube 307 that already has a permanently fastened membrane 305 to define three or more sample regions.

[0089] It will be appreciated by those skilled in the art that the invention has been illustrated by describing one or more specific embodiments thereof, but is not limited to these embodiments; many variations and modifications are possible, within the scope of the accompanying claims.

Claims

Claims1. A nuclear magnetic resonance (NMR) tube sized for fitting within an NMR machine, wherein the NMR tube comprises a semi-permeable membrane located within the NMR tube for providing a semi-permeable boundary between a first sample region within the NMR tube and a second sample region within the NMR tube, the first sample region being configured for holding a first sample and the second sample region being configured for holding a second sample.

2. The NMR tube of claim 1, wherein the first sample region is configured for holding a first powder or fluid sample, and the second sample region is configured for holding a second powder or fluid sample.

3. The NMR tube of claim 1 or 2, comprising a hollow cylindrical tube body and a nuclear magnetic resonance (NMR) tube-insert at least partly within the tube body, wherein the first sample region is within the NMR tube-insert, and wherein the NMR tube-insert comprises: a first end for receiving the first sample into the NMR tube-insert; and a semi-permeable membrane adjacent a second end of the NMR tube-insert for providing the semi-permeable boundary between the first sample region and the second sample region within the NMR tube.

4. The NMR tube of claim 3, wherein, everywhere around a circumference of the outer wall of the NMR tube-insert, a distance between the outer wall of the NMR tube-insert and an inner wall of the NMR tube is less than 1 mm.

5. The NMR tube of claim 3 or 4, wherein the tube body comprises at least one feature for holding the semi-permeable membrane of the NMR tube-insert at a predetermined position within the NMR tube.

6. The NMR tube of claim 5, wherein the tube body comprises one or more inwardly-extending protrusions on an inner wall of the NMR tube body for holding the tube-insert with the semi-permeable membrane at the predetermined position.

7. The NMR tube of claim 5 or 6, wherein the NMR tube has a non-uniform inner diameter that is smallest at a waist of the NMR tube and wherein the waist is sized for retaining the tube-insert with the semi-permeable membrane at the predetermined position.

8. The NMR tube of claim 1 or 2, wherein the NMR tube comprises a hollow cylindrical tube body, wherein the semi-permeable membrane is bonded to the tube body at a position within the tube body and extends across a transverse section of the tube body.

9. The NMR tube of any preceding claim, wherein the semi-permeable membrane is positioned within the NMR tube so that, when the NMR tube is placed in an NMR machine, a probe of the NMR machine extends over the first sample region and over the second sample region.

10. A nuclear magnetic resonance (NMR) tube-insert sized for fitting at least partly within an NMR tube that is sized for fitting within an NMR machine, the NMR tube-insert comprising: a first end for receiving a first sample into a first sample region within the NMR tube-insert; and a semi-permeable membrane adjacent a second end of the NMR tube-insert for providing a semi-permeable boundary between the first sample region and a second sample region within the NMR tube.

11. The NMR tube-insert of claim 10, wherein the semi-permeable membrane is mechanically fastened to a body of the NMR tube-insert.

12. The NMR tube-insert of claim 11 , wherein the semi-permeable membrane is clamped between a fixing surface at the second end of the NMR tube-insert and a cap member.

13. The NMR tube-insert of claim 10, wherein the semi-permeable membrane is chemically-bonded or heat-bonded to a body of the NMR tube-insert.

14. The NMR tube-insert of any preceding claim, comprising a feature for engagement with a positioning tool for moving the NMR tube-insert axially within the NMR tube body.

15. A kit for assembling a nuclear magnetic resonance (NMR) tube-insert, the kit comprising: an open tube sized for fitting at least partly within an NMR tube that is sized for fitting within an NMR machine, the open tube comprising: a first end for receiving a first sample into a first sample region within the open tube; and a second end comprising a fixing surface; and a semi-permeable membrane for affixing to the fixing surface, wherein, when fixed to the fixing surface, the semi-permeable membrane is arranged for providing a semi-permeable boundary between the first sample region and a second sample region within the NMR tube16. The kit of claim 15, wherein the open tube is formed of a non-magnetic material that is transparent to radio waves.

17. The kit of claim 15 or 16, wherein the open tube comprises a plastic ring at the second end of the open tube, the plastic ring providing the fixing surface for the semi-permeable membrane.

18. The kit of any of claims 15 to 17, further comprising the NMR tube.

19. A method of fabricating a nuclear magnetic resonance (NMR) tube, the method comprising inserting an NMR tube-insert according to any of claims 10 to14 into an NMR tube so that the semi-permeable membrane of the NMR tube-insert is located within the NMR tube and provides a semi-permeable boundary between the first sample region within the NMR tube-insert and a second sample region within the NMR tube.

20. The method of claim 19, further comprising fabricating the NMR tube-insert by affixing the semi-permeable membrane to an end of an open tube, the open tube being sized for fitting at least partly within the NMR tube.

21. A method of performing nuclear magnetic resonance (NMR) spectroscopy, comprising using an NMR tube according to any of claims 1 to 9 in an NMR machine to perform NMR spectroscopy.

22. The method of claim 21 , comprising performing NMR spectroscopy on a first sample in the first sample region and / or on a second sample in the second sample region, and determining a change in concentration of a component of the first sample or of the second sample caused by diffusion through the semi-permeable membrane.

23. The method of claim 21 or 22, comprising performing NMR spectroscopy on a first sample in the first sample region and / or on a second sample in the second sample region, and monitoring a change in state or structure of a component of the first or second sample.

24. The method of any of claims 21 to 23, comprising performing NMR spectroscopy on a first sample in the first sample region and / or on a second sample in the second sample region; imaging the first and / or second sample; and using the imaging to monitor one or more diffusion phenomena.

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