WO2021176393A1

WO2021176393A1 - An attachment for benchtop nuclear magnetic resonance (nmr) spectrometer and a method for conducting a measurement

Info

Publication number: WO2021176393A1
Application number: PCT/IB2021/051822
Authority: WO
Inventors: Dariusz GOŁOWICZ; Javier Augustin ROMERO
Original assignee: Uniwersytet Warszawski
Priority date: 2020-03-04
Filing date: 2021-03-04
Publication date: 2021-09-10
Also published as: PL433143A1

Abstract

The subject of the invention is an attachment for benchtop nuclear magnetic resonance (NMR) spectrometer having a motor (14) equipped with a gear wheel (10) and a tube holder (3) equipped with a gear rack (1), wherein gears (1, 10) form a mechanism enabling the tube (4) displacement inside a permanent magnet of the spectrometer. Also, the subject of the invention is a method for conducting a measurement using benchtop nuclear magnetic resonance (NMR) spectrometer, in which between subsequent scans the tube (4) with the sample being tested is displaced vertically inside the permanent magnet cavity of the spectrometer, within the region of the high magnetic field.

Description

An attachment for benchtop nuclear magnetic resonance (NMR) spectrometer and a method for conducting a measurement

Description The invention relates to an attachment for benchtop nuclear magnetic resonance spectrometer (Benchtop NMR) equipped with a permanent magnet, making it possible to improve the sensitivity of the accumulated measurement on said spectrometer due to the optimalisation of the nuclear relaxation during the measurement. The invention relates also to a method of conducting a measurement using benchtop nuclear magnetic resonance (NMR) spectrometer.

In contrary to the high field NMR spectrometers characterized by larger sizes and necessity of cooling to temperatures below the critical values in the range of single kelvins (K), benchtop NMR spectrometers are equipped with permanent magnets and thus they have small sizes, are portable and are cheap to maintain, The magnetic field strength generated by the employed permanent magnets is however much lower then the strength generated by the superconducting magnets (even several dozen times), which translates into relatively low sensitivity and resolution of measurements carried out using benchtop NMR spectrometers. This is one of the most important limitations in the applicability of these devices.

The subject of research using benchtop NMR spectrometers are liquid samples, most often prepared for measurement in the form of a several centimeter column of liquid in a glass tube placed in the magnetic field of a permanent magnet inside the spectrometer. During the measurement, the tube is static, i.e. it does not change its position inside the spectrometer in any way. The signal in each of the recorded scans comes from the same volume of the sample, which is only a fraction of the total volume of the liquid column in the tube, which results directly from the size of the transmit-receive coil of the spectrometer.

The basic one-scan ¹D NMR experiment consists in applying a strong radio pulse of a given frequency to the sample and then recording the signal of decay of free induction of the nuclear spins. Both activities take place using the aforementioned transmit-receive coil. In order to be able to record another scan during accumulated measurements, some of the nuclei in the sample, and in the case of quantitative measurements even 99.3% of them, must return to the state of thermodynamic equilibrium. The primary cause of the transition of atomic nuclei from excited state to ground state is the process of nuclear relaxation.

The basic relaxation processes are transverse and longitudinal relaxation. Transverse relaxation, of spin-spin type, i.e. relaxation of the transverse component of the macroscopic magnetization vector of the sample, is related to the return of spins to equilibrium, mainly due to fluctuations in the magnetic field created by the surrounding of the nucleus and the interaction of the spins of adjacent atomic nuclei. The longitudinal relaxation, of the spin-lattice type, is the relaxation of the longitudinal component of magnetization. The magnetic field fluctuations are mainly responsible for the decay of magnetization in this direction but only under the condition that the frequency of the local magnetic field caused by atomic nuclei is approximately equal to the frequency of the Larmor precession. For this reason, inter alia, the longitudinal relaxation time T 1 is longer than the transverse relaxation time T2, and for small and medium-sized chemical molecules, it may be equal from several to several dozen seconds.

Due to the long time of nuclear spin-lattice relaxation with each successive scan there may be a significant reduction in the intensity of the collected signal which results in lowering signal-to-noise ratio in the obtained NMR spectrum.

In order to avoid the reduction of the signal intensity in subsequent scans, it is necessary to properly select the scan repetition time, i.e. the time between consecutive scans. Due to the mentioned relaxation it should be sufficiently long. This causes a significant extension of the measurement time or, in the case of inadequate adjustment of the pause length between scans (when the pause is too short), a reduction in the signal-to-noise ratio in subsequent scans and, consequently, a reduction in the sensitivity of the measurement.

The aim of the invention is to solve the above-described problem by constructing an attachment for a benchtop NMR spectrometer that allows to improve the sensitivity of the accumulated measurement or shorten its duration by optimizing the nuclear relaxation during the measurement.

It is also an object of the invention to propose a new method for carrying out measurements using a benchtop nuclear magnetic resonance spectrometer. The strong magnetic field generated by the magnet of a benchtop NMR spectrometer is present in a larger volume than the standard measuring volume used defined by the dimensions of the transmit/receive coil. As a result, in conventional measurement a large part of the sample outside the coil is polarized (magnetized) but is not used. The problem of slow spin-lattice nuclear relaxation can therefore be solved by mechanically displacing the sample in order use the portion of the sample that is normally outside the coil. For this purpose, an attachment was developed, the operation of which is based on displacing the test sample inside the spectrometer, so as to make it possible to perform a scan for the sample portion coming from the region of the magnetic field located outside the measuring coil inside the permanent magnet, i.e. one that had previously also been kept in a high magnetic field and is therefore magnetically polarized. Thus, the unexploited region of the magnetic field outside the measuring coil is used as a place to pre-polarize a new sample portion and relax the sample portion that had previously excited. Another scan is thus performed on a "fresh" sample portion and the volume that has left the measuring coil returns to thermodynamic equilibrium during this time.

Subsequent "fresh" volumes are measured thanks to the attachment bearing in mind that the sample volume is limited and that the sample should not stay outside the high magnetic field region of the spectrometer as it will lose its polarization. This necessitates certain sample displacement patterns based on a sequence of vertical displacements, preferably preceded by system calibration.

The attachment for benchtop NMR spectrometers, which is the subject of the invention, allows for fully controlled, mechanical displacement of the sample between successive scans of the NMR spectrum. The proposed device is cheap to implement, does not require any modification of the original construction of the spectrometer and, depending on the relaxation parameters of the tested sample, it allows for even a 10-fold increase in measurement sensitivity or for a several-fold reduction of the measurement time, without any negative impact on the quality of the results.

The design of the attachment is based on an electric motor which, with the help of gears, displaces the glass tube mounted in the holder, with high precision. The tube moves inside the permanent magnet cavity of the benchtop NMR spectrometer within the high magnetic field region.

The motor operation is synchronized with the sequence of radio pulses generated by the spectrometer, i.e. with recording of subsequent scans of the NMR signal. As a result, each subsequent spectrum scan is performed using "fresh", polarized sample volume.

The attachment is placed outside the spectrometer, and with its side surface, or optionally legs, it rests on the upper surface of the spectrometer. It is made from a non-magnetic material and it has openings in the side walls (in the case of a closed surface) that allow air to circulate in the space below it. An electric motor with a gear wheel is mounted on the upper surface of the attachment in the mounting bracket.

The tube holder is also made of a non-magnetic material. Its lower part is in the form of a rod with a diameter corresponding to the inner diameter of the NMR glass tube. In its upper part, the holder has a flat part for mounting a gear rack.

The gear wheel of the electric motor has a tooth spacing compatible with the gear rack mounted in the upper part of the tube holder and engages it.

The top surface of the base of the attachment has an opening in the center to allow the tube holder with the rack to move vertically through the surface. A non magnetic support element perpendicular to the upper surface of the attachment base is adjacent to said opening and from the opening side it has a profiled groove acting as a guide. The groove diameter corresponds to the diameter of the tube holder, which allows for a stable, vertical sliding motion. The gear rack of the electric motor together with the gear rack of the tube holder, adjacent to the support element at the groove location, form a mechanism that enables controlled vertical displacement of the tube inside the permanent magnet of the spectrometer. Synchronization of the sample displacement with the recorded NMR signal scans is ensured by a programmed microcontroller and electric motor controller system, which is connected, wired or wireless, with a computer controlling the benchtop NMR spectrometer, and connected in a wired manner with the electric motor.

The subject of the invention is an attachment for benchtop nuclear magnetic resonance (NMR) spectrometer having a motor equipped with gear wheel and tube holder equipped with gear rack, wherein gears form a mechanism enabling the tube displacement inside a permanent magnet of the spectrometer when the attachment is placed on the spectrometer near the upper opening of the spectrometer cavity. Preferably, the motor is a stepper motor.

Preferably, the attachment is equipped with a base with an opening wherein the motor is mounted on the base.

Preferably, the base is made from non-magnetic material.

The side surface of the base is in form of legs or is closed and preferably equipped with side openings.

Preferably a support element, adjacent to the opening, is mounted on the upper surface of the base.

Preferably, the attachment on opening side it has a profiled groove having shape corresponding to the shape of the holder. Preferably, the motor is connected with the motor controller system and microcontroller.

The subject of the invention is also a method for conducting a measurement using benchtop nuclear magnetic resonance (NMR) spectrometer wherein between subsequent scans the tube with the sample being tested is displaced vertically inside the permanent magnet cavity of the spectrometer, within the region of the high magnetic field, wherein the tube is moved using the attachment for benchtop nuclear magnetic resonance (NMR) spectrometer having the motor equipped with gear wheel and tube holder equipped with gear rack, wherein gears form a mechanism enabling the tube displacement inside a permanent magnet of the spectrometer.

Preferably, the motor operation is synchronized with the radio impulse sequence generated by the benchtop NMR spectrometer.

Preferably, before the measurement is conducted, an optimal sample displacement distance is determined by: a) measuring short NMR signal, b) displacing the sample as fast as possible by a distance as short as possible (up or down) and repeating the NMR signal measurement, c) comparing amplitudes of the signals recorded during the first and second scan d) repeating steps a), b) and c) for increasing distances of the sample displacement, e) selecting the shortest distance for which the ratio of signal amplitudes of both scans is 1 , within the measurement error, as the optimal sample displacement distance.

Preferably, before the measurement is conducted, an approximate profile of the magnetic field strength in the magnet cavity of the benchtop NMR spectrometer is determined using a series of measurements using the attachment, in which short NMR signal is measured, the sample is displaced as fast as possible by a distance corresponding to the optimal displacement distance and another short NMR signal is recorded, wherein the measurement is conducted for various distances corresponding to the multiple of the optimal displacement distance, and then a region in which the amplitude ratio of the signals measured before and after the sample is displaced is 1 , within the measurement error, is selected as a sample displacement range. Below, the invention will be described in greater detail with reference to drawings on which:

Fig. 1 is a schematic view of the attachment placed on the spectrometer.

Fig. 2 shows schematically the construction of the attachment of the invention where:

1 - gear rack,

2 - flat cutout for mounting the gear rack,

3 - tube holder,

4 - glass tube, 5 - upper surface of the attachment base,

6 - side surface of the attachment base,

7 - side openings in side surface of the attachment base

8 - profiled groove,

9 - opening in the upper surface of the attachment base, 10 - gear wheel,

11 - electric motor bracket,

12 - support element

13 - longitudinal openings of the lower base of the motor bracket,

14 - electric motor, 15 - mounting openings,

16 - microcontroller and electric motor controller system,

17 - computer.

The holder (3) made of non-magnetic material has the shape of a rod, preferably circular in cross-section. In the upper part of the holder (3), on one side, there is a flat cutout (2) on which a gear rack (1 ) is mounted, also made of non-magnetic material. Any other suitably durable connection of the gear rack (1) with the handle (3) is also possible. In the lower part, the holder (3) ends with a short section of embossing with a diameter corresponding to the internal diameter of the NMR glass tube (4). Connecting the tube (4) with the holder (3) is accomplished in a compression manner, because the shape of the tip of the holder (3) fits the upper opening of the tube (4). Other connections of the holder and test tube are allowed, but magnetic elements cannot be used in the connection.

From about 1 to about 1 .5 ml of the liquid substance to be tested are placed in a glass tube (4).

During the operation of the device, the gear rack (1 ) is in contact with the gear wheel (10) of the electric drive (14), creating a mechanism that allows the tube (4) and its holder (3) to move vertically (the entire element 1 ,2,3,4 is moved).

The side surface of the attachment base (6) made of non-magnetic material prevents the electric motor (14) from being directly placed on the spectrometer surface. It is sufficiently high so the range of vertical displacement of the sample in the spectrometer is not limited (too low setting would block the linear rack (1 ) against the upper cover of the NMR spectrometer). Moreover, the appropriate height of the side surface of the base makes it possible to eliminate the disadvantageous influence of the heat and the magnetic field generated by the electric drive (14) on the stability of the measurement.

The side surface of the base (6) is preferably closed and provided with openings (7) for air circulation under the attachment. The shape of the openings and their number are not limited. The side surface of the attachment base can also have the form of legs allowing it to be raised on the top surface of the spectrometer. In this case, no openings are required.

The upper surface of the base of the attachment (5), made of non-magnetic material, has an opening (9) in its central part, the size and shape of which allow the test tube (4) to pass along with the holder (3) and the gear rack (1 ) through this surface.

The device has an electric motor (14) equipped with a gear wheel (10) constituting the drive transmission element. The tooth spacing of the gear wheel (10) corresponds to the tooth spacing of the gear rack (1 ). In an embodiment, the motor (14) has four mounting opening (15) in its bracket (11 ) for its mounting. The motor bracket (11 ) is preferably L-shaped and is mounted on the upper surface of the attachment base (5) by means of bolts and nuts (not shown) through the longitudinal openings (13) in its lower base, allowing the position of the motor (14) on the upper surface of the base (5) to be adjusted.

However, bracket/engine mount (14) may be of any type as long as it ensures a stable and stationary position. The possibility of adjusting its position is not obligatory.

There is a support element (12) perpendicular to the upper surface of the attachment base (5), and adjacent to the hole (9). The support element (12) has a profiled semicircular groove (8) on the opening (9) side

The groove (8) diameter corresponds to the diameter of the NMR glass tube (4) holder (3).

During the operation of the device the upper part of the holder (3) with the gear rack (1 ) is in the groove (8) which acts as a guide and the linear rack (1 ) engages with the gear wheel (10) of the electric drive (14), creating a mechanism controlling the position of the tube (4). In the measuring configuration, the gear wheel (10) exerts a slight pressure on the gear rack (1 ) and on the holder (3) which is located in the semicircular groove (8) of the support element (12). The movement of the gear wheel (10) causes the movement of the gear rack (1 ) and consequently the entire holder (3) and the tube (4).

The support element (12) with the groove (8) ensures a straight, vertical displacement of the tube (4) and the holder (3) during the measurement. Moreover, the presence of the support element (12) allows the tube (4) to be stopped in different positions without collapsing. The gear wheel (10) of the electric drive (14) gently presses the linear rack (1 ) against the groove (8) of the support element (12) so that the tube (4) may be kept in any position within the range corresponding to the length of the gear rack (1 ).

In other embodiments, the groove (8) may be rectangular rather than semicircular if the tube holder (3) also has a rectangular cross section. The motor (14) is connected by a wire to the motor controller system and the programmed microcontroller (16), and to a computer (17) controlling the benchtop NMR spectrometer and the operation of the electric motor (14).

The electric motor controller system and the programmed microcontroller (16) are connected with the computer (17) in a wired or wireless manner.

During the measurement, after each successive scan of the NMR signal the computer (17) sends a command to the microcontroller system and controller (16) to rotate the gear wheel (10) by a given angle, in a selected direction and at a given speed. The procedure for carrying out the measurement with the attachment according to the invention is described below.

1. The NMR glass tube (4) with the tested liquid sample is placed in the holder (3).

2. The tube (4) with the holder (3) and the gear rack (1 ) located on the holder is manually inserted through the central opening (9) in the upper surface of the attachment base (5) until the gear rack (1) and the gear wheel are in contact. (10). At this point, the tube (4) is already partially in the magnet cavity of the benchtop NMR spectrometer. The whole thing (elements 1 ,2,3,4) rests on the teeth of a gear wheel.

3. By means of the electric motor (14) the gear wheel (10) is rotated by a predetermined angle so as to center the position of the gear rack (1) in relation to the gear wheel (10). At this point, the holder (3) behind the linear rack (1 ) fits tightly in the groove (8) that acts as a guide. In this setting, the tube (4) and the sample placed inside are within the high magnetic field of a permanent magnet.

4. In this moment the attachment and the benchtop NMR spectrometer are ready for carrying out the measurement.

5. The multi-scan NMR measurement is started from the computer (17) controlling the benchtop NMR spectrometer. After each scan made by the spectrometer, the computer (17) sends a signal to the microcontroller and controller system (16) ordering the rotation of the gear wheel (10) by a predetermined angle at a predetermined speed and indicates the direction of rotation. Rotation of the gear wheel (10) forces the gear rack (1 ) to move vertically up or down. Since the gear rack (1 ), the holder (3) and the tube (4) are connected to each other, the liquid sample in the tube (4) also changes its position in the permanent magnet inside the benchtop NMR spectrometer, similar to the gear rack (1 ). In this way, a "fresh" (non- excited) sample volume appears inside the measuring coil of the benchtop NMR spectrometer (4).

The tube displacement pattern (4), the time interval between movements, the displacement distance and the displacement speed are a matter of proper calibration and may depend on the type of measurement being carried out.

Due to the relatively small region of high magnetic field in the benchtop NMR spectrometer, a calibration is preferably performed which allows for the optimal use of this region in the measurement with the attachment. Omitting the calibration in case of benchtop NMR spectrometer may result in the lack of the expected results, i.e. improved sensitivity or reduced measurement time. The purpose of the calibration is to determine the minimum distance of the tube displacement for which the signal in the next scan will come from the "fresh" volume, and to estimate the range of sample displacement, so that during the measurement there is no significant loss of polarization due to the sample being outside the region of high magnetic field. Calibration consists in performing a series of short experiments using the attachment as follows: a) the basic experiment to determine the minimum sample displacement distance is performed by measuring a short NMR signal, displacing the sample as fast as possible by a distance as short as possible (up or down) and measuring the NMR signal again. Then the amplitudes of the signal collected in the first and second scans are compared. The calibration experiment is repeated for increasing sample displacement distances. If the signal in the second scan comes only from the “fresh” sample volume, the amplitude ratio of the signals in both scans will be equal to 1 , within the measurement error. The shortest displacement distance for which this ratio is achieved corresponds to the optimum sample displacement distance and should be used in real measurements with the attachment. b) the estimation of the sample displacement range requires knowing the magnetic field strength profile in the magnet cavity of a benchtop NMR spectrometer.

A sufficient approximation of the profile can be obtained again through a series of two-scan experiments using the attachment. The tube is placed in the holder and then in the spectrometer in a position in which the gear rack is centered with respect to gear wheel. In the first scan, a short NMR signal is recorded for the starting position of the tube, then the sample is displaced up as quickly as possible by the distance corresponding to the optimal displacement distance and another short NMR signal is recorded. The signal amplitude ratio of both scans will be equal to 1 , within the measurement error, as long as the sample volume in the second scan has not been previously outside the high magnetic field. The measurement is repeated several times for different distances corresponding to the multiple of the optimal displacement distance. Next, an analogous measurement is performed but the sample is moved down. The obtained approximate profile of the magnetic field strength makes it possible to assess how many up and down movements relative to the central position can be made by the sample without a clear loss of polarization.

The prototype of the attachment built by the inventors was tested using the Magritek Carbon 43MHz NMR benchtop spectrometer. Taking into account the design of the available models of Magritek FT-NMR benchtop spectrometers, the attachment should be compatible with each of them.

Claims

1. An attachment for benchtop nuclear magnetic resonance (NMR) spectrometer characterized in that it comprises a motor (14) equipped with gear wheel (10) and tube holder (3) equipped with gear rack (1), wherein gears (1 , 10) form a mechanism enabling the tube (4) displacement inside a permanent magnet of the spectrometer when the attachment is placed on the spectrometer near the upper opening of the spectrometer cavity.

2. The attachment according to claim 1 , characterized in that the motor (14) is a stepper motor.

3. The attachment according to claim 1 or 2, characterized in that it is equipped with a base (5, 6) with opening (9), wherein the motor (14) is mounted on the base.

4. The attachment according to claim 3, characterized in that the base (5,6) is made from non-magnetic material.

5. The attachment according to claim 3 or 4, characterized in that the side surface of the base (6) is in form of legs.

6. The attachment according to claim 3 or 4, characterized in that the side surface of the base (6) is closed.

7. The attachment according to claim 6, characterized in that the side surface of the base (6) is equipped with side openings (7).

8. The attachment according to any of the claims 3 to 6, characterized in that a support element (12), adjacent to the opening (9), is mounted on the upper surface of the base (6).

9. The attachment according to claim 8, characterized in that on the opening (9) side it has a profiled groove having shape corresponding to the shape of the holder (3).

10. The attachment according to any of the claims 1 to 8, characterized in that the motor is connected with a motor controller system and a microcontroller (16).

11. A method for conducting a measurement using benchtop nuclear magnetic resonance (NMR) spectrometer including introduction of the tube with sample into the spectrometer and making at least two scans, characterized in that between subsequent scans the tube (4) with the sample being tested is displaced vertically inside the permanent magnet cavity of the spectrometer, within the region of the high magnetic field, wherein the tube is displaced using the attachment for nuclear magnetic resonance (NMR) spectrometer having a motor (14) equipped with a gear wheel (10) and a tube holder (3) equipped with a gear rack (1), wherein gears (1 , 10) form a mechanism enabling the tube (4) displacement inside a permanent magnet of the spectrometer.

12. The method according to claim 11 , characterized in that the motor (14) operation is synchronized with the radio impulse sequence generated by the benchtop NMR spectrometer.

13. The method according to any of the preceding claims, in which before the measurement is conducted, an optimal sample displacement distance is determined by: a) measuring short NMR signal, b) moving the sample as fast as possible by a distance as short as possible (up or down) and repeating the NMR signal measurement, c) comparing amplitudes of the signals recorded during the first and second scan, d) repeating steps a), b) and c) for increasing distances of the sample displacement, e) selecting the shortest distance for which the ratio of signal amplitudes of both scans is 1 , within the measurement error, as the optimal sample displacement distance.

14. The method according to claim 13, in which before the measurement is conducted, an approximate profile of the magnetic field strength in the magnet cavity of the benchtop NMR spectrometer is determined using a series of measurements using the attachment, in which short NMR signal is measured, the sample is displaced as fast as possible by a distance corresponding to the optimal displacement distance and another short NMR signal is recorded, wherein the measurement is conducted for various distances corresponding to the multiple of the optimal displacement distance, and then a region in which the amplitude ratio of the signals measured before and after the sample is displaced is 1 within the measurement error, is selected as a sample displacement range.