A system and method for online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled real-time detection and separation of molecular magnets

Abstract

The application discloses a kind of online electron paramagnetic resonance spectrum-high performance liquid chromatography combined system and method for real-time detection and separation molecular magnet, the real-time detection and separation molecular magnet of the system is by sample pump, infusion pump, chromatographic column, six-way valve, UV-Vis detector and EPR detector Composition, integrated continuous sampling, circulating HPLC, EPR tuning and real-time detection function, control operation is simple, with great potential for transformation, easy to promote and expand application.The separation method disclosed in the application can be high-throughput by two separation methods of continuous sampling and circulating HPLC, in combination with EPR detector real-time detection spin signal, real-time detection molecular magnet with EPR signal, with high separation efficiency, significant purification effect, can realize efficient detection and separation of molecular magnet with EPR signal.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing technology, specifically relating to a system and method for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography. Background Technology

[0002] Molecular magnets, as molecules intrinsically possessing electron spin properties, have spin effects that extend to multiple fields, including chemical / biological enzyme catalysis, molecular spin materials, and spin-based therapies. The development of next-generation information processing technologies, such as spintronics and quantum computing, is highly dependent on the research and development of spin materials. Some representative molecular magnets, such as organic free radicals and endohedral fullerenes, are highly promising molecular quantum bit materials; however, their research and applications are limited due to difficulties in separation. On the one hand, apart from magnetism, their structures and properties are extremely similar to their corresponding precursor molecules, resulting in very small differences in their residence time in high-performance liquid chromatography (HPLC), making them difficult to separate. On the other hand, the UV-Vis or IR detectors equipped in commercial HPLC cannot detect electron spin signals, and the yield of endohedral fullerenes is extremely low (usually only 30 ppm to 100 ppm), with very weak UV signals that cannot be detected by UV detectors. Furthermore, some free radicals contain other non-spin-active byproducts that UV detectors cannot distinguish, making it impossible to determine the residence time of the target molecule magnet for collection.

[0003] Early reports indicated the use of highly sensitive electron paramagnetic resonance (EPR) spectroscopy, capable of detecting paramagnetic substances with unpaired electrons, to reconstruct the spin signals of endohedral fullerenes in chromatography. Isr J Chem 2006, 46, 407–412. Analyst 2014, 139, 4519–4524. Fullerene Science and Technology 2001, 9, 329–337.), or using EPR as a detector coupled with HPLC to detect and separate some metal-embedded fullerenes and organic free radicals ( Anal Chem 1994, 66, 2680–2685. Anal Chem 1994, 66, 2675–2679. Applied Superconductivity 1993, 1, 913–923.; Anal Lett(1975, 8, 205–213.). However, these methods failed to achieve high temporal resolution real-time detection, exhibited significant lag, and could not tune the EPR detector or set test conditions before separation began. Under separation conditions with high mobile phase flow rates, this could lead to substantial errors in determining the residence time of the target spin molecule. This is especially true for molecules with extremely narrow spin signal peaks and residence times very similar to other components to be separated, such as P / N@C. 60 Its reactants intrinsic C 60 Separation remains extremely challenging. Furthermore, column conditions may change over long-term use, and the resulting changes in residence time may not be detected promptly. Additionally, crude endogenous fullerene products contain a large amount of unreacted intrinsic fullerenes, meaning that significant amounts of intrinsic fullerenes need to be removed to obtain high-purity endogenous fullerene samples. However, the limited sample loading capacity of the column makes it difficult to process large quantities of samples at once, resulting in low separation efficiency.

[0004] Due to the aforementioned difficulties in detection, a highly efficient separation system and method capable of high-throughput sample processing and real-time detection of molecular magnets has not yet been developed, which greatly hinders further research and application of molecular magnets. Summary of the Invention

[0005] To address the problems of low spin signal detection and low sample throughput in existing technologies for separating molecular magnets, and to further resolve the issues of existing methods using EPR detectors, this invention provides a system and method for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (HPLC). The system is composed of a commercial EPR spectrometer and an HPLC system coupled through a self-designed flow path. The method utilizes flow path design and a six-way valve to achieve pre-separation tuning of the EPR detector, real-time detection mode during separation, and switching between cyclic and continuous injection separation modes. Through ingenious flow path design, this invention achieves a system integrating continuous injection, cyclic separation, and real-time spin signal detection for the real-time detection and separation of molecular magnets with only simple modifications to commercial EPR and HPLC spectrometers. This invention solves the problems of low sample throughput in existing HPLC spectrometers, the inability of UV-Vis / IR detectors to detect electron spin signals, and the detection time lag of EPR detectors used for detecting spin molecules.

[0006] The technical solution of the present invention is as follows: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPC) includes: an injection pump, an infusion pump, a chromatographic column, a UV-Vis detector, an EPR detector, and a six-way valve. The injection pump, infusion pump, chromatographic column, UV-Vis detector, EPR detector, and six-way valve are connected via polytetrafluoroethylene (PTFE) or PEEK tubing. The system features two EPR detection modes and two HPLC separation modes. The two EPR detection modes are a tuned and online detection mode, and the two HPLC separation modes are a continuous injection and a cyclic HPLC mode.

[0007] The objective of this invention is achieved through the following technical solution: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPR-HPLC) includes an HPLC unit and an EPR detection unit. It is based on a commercial HPLC instrument with the addition of an EPR detector and a six-way valve. The HPLC unit includes an injection pump, a liquid pump, a chromatographic column, and a UV-Vis detector. The various parts of the system for real-time detection and separation of molecular magnets are connected by polytetrafluoroethylene or PEEK tubing. The system for real-time detection and separation of molecular magnets has two separation modes: continuous injection and cyclic HPLC, as well as two EPR detection modes: tuning and online detection, which are controlled by six-way valve A and six-way valve B, respectively. The two detection modes are: EPR tuning detection mode and EPR online detection mode; The two separation modes are: continuous injection mode and cyclic HPLC mode; The two detection modes and two separation modes are controlled and switched by two six-way valves. In EPR tuned detection mode, the sample enters the EPR test area after passing through six-way valve B, which is in the dashed line state. In EPR online detection mode, the sample enters the EPR detector after separation by the chromatographic column, and six-way valve B is in the solid line state. In continuous injection mode, the tubing is blue, and six-way valve A is in the solid blue line state, directly connected to the collector. In cyclic HPLC mode, the tubing is orange, and six-way valve A is in the orange dashed line state. The sample returns to the chromatographic column after passing through six-way valve A, and the black line represents the shared tubing.

[0008] Furthermore, the chromatographic column is a normal chromatographic column, a reversed-phase chromatographic column, an ion-exchange chromatographic column, or a gel permeation chromatographic column; even further, it is a reversed-phase chromatographic column.

[0009] Furthermore, the various parts of the system for real-time detection and separation of molecular magnets are connected by polytetrafluoroethylene (PTFE) tubing.

[0010] Furthermore, the portion of the EPR detector area is a polytetrafluoroethylene (PTFE) conduit.

[0011] This invention also provides an online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPLC) coupled with real-time detection and separation method for molecular magnets. This method is performed in the aforementioned online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled with real-time detection and separation system for molecular magnets, and includes the following steps: S1. Dissolve the molecular magnet sample to be separated in the corresponding solvent to prepare a solution with a concentration ≤0.5 mg / mL. The solvent is an organic solvent and should be consistent with the mobile phase in S3. S2. Set the six-way valve B to EPR tuning detection mode, pump the sample to be separated into the EPR test area through the injection pump, tune it, determine the applied microwave frequency, perform continuous wave EPR test at the microwave frequency, determine the resonant magnetic field of the molecular magnet, fix the magnetic field, set the microwave power and modulation amplitude, and detect the change of EPR signal intensity over time under the fixed magnetic field. S3. Switch the six-way valve B to real-time detection mode, and at the same time turn on the infusion pump to deliver the mobile phase for elution. The sample to be separated flows out from the EPR test area, passes through the chromatographic column, UV-Vis detector and EPR detector in sequence, and reaches the six-way valve A. S4. When the separation mode is continuous injection mode, collect the sample and inject it repeatedly at fixed intervals, repeating S3. When the separation mode is cyclic HPLC mode, the sample is returned to the column for the next cycle of separation until the cycle ends. Then, switch the six-way valve A to continuous injection mode and collect the sample.

[0012] Furthermore, the molecular magnet sample to be separated mentioned in S1 is a molecule with an electron paramagnetic resonance signal, preferably a nitrogen-intercalated fullerene, a phosphorus-intercalated fullerene, a metal-intercalated fullerene, or an organic free radical, wherein the content of the crude fullerene product is >50 ppm; the concentration of the prepared solution is preferably 0.1 mg / mL-0.5 mg / mL.

[0013] Furthermore, the organic solvent mentioned in S1 is any mixture of one or more of toluene, dichloromethane, cyclohexane, ethyl acetate, acetone and ethanol.

[0014] Furthermore, as described in S2, the sample to be separated is pumped into the EPR test area by a sample injection pump, with a single injection volume of 1 mL to 6 mL, a fixed microwave frequency of 9 GHz to 10 GHz, a magnetic field range of 50 G to 10000 G, a microwave power attenuation range of 0 dB to 60 dB, and a modulation amplitude of 0.5 G to 5 G.

[0015] Furthermore, as described in S2, the sample to be separated is pumped into the EPR test area by a sample injection pump, with a single injection volume of 3 mL, a fixed microwave frequency of 9.3 GHz-9.6 GHz, a magnetic field range of 3000 G-4000 G, a microwave power attenuation range of 20 dB-30 dB, and a modulation amplitude of 2 G-3 G.

[0016] Furthermore, the elution described in S3 is performed using gradient elution or isocratic elution; the mobile phase is a single organic solvent or a mixture of multiple solvents, such as toluene, dichloromethane, a toluene:dichloromethane = 1:1 solution, or a cyclohexane:ethyl acetate = 9:1 solution; the flow rate of the mobile phase can be 1 mL / min-20 mL / min, preferably 10 mL / min-18 mL / min.

[0017] Furthermore, the UV-Vis detector described in S3 has a wavelength range of 200 nm to 2000 nm, preferably 315 nm and 532 nm.

[0018] Furthermore, the continuous injection interval described in S4 is 4 min-6 min.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPC-HPLC) system for real-time detection and separation of molecular magnets, as described in this invention, integrates continuous injection, cyclic HPLC, and real-time spin signal detection functions with only simple modifications to commercial HPLC and EPR instruments. Furthermore, this system can be further modified to expand its application range by combining other detectors or chromatographic techniques based on the properties of the substances to be separated. It is easy to promote and has universal applicability.

[0020] 2. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPR-HPLC) real-time detection and separation method described in this invention, through ingenious flow path design and six-way valve control, allows for adjustment and setting of conditions before separation. Compared with previously reported spin signal reconstruction methods or EPR detection methods that repeatedly scan the field within a small range, this method is simple to operate, greatly reduces errors caused by detection lag, detects spin signals in real time, accurately measures residence time, and can promptly detect changes in the chromatographic column state, allowing for timely adjustment of the collection strategy. 3. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (HPLC-HPLC) real-time detection and separation method described in this invention separates molecular magnets. Continuous injection mode allows for high-throughput sample processing; cyclic HPLC mode improves resolution; and real-time spin signal detection accurately determines the residence time of target molecules. For molecules with similar residence times and in situations requiring large sample volumes, the separation efficiency is improved, and the purification effect is significant. This method is suitable for separating various molecular magnets with electron paramagnetic resonance signals. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic diagram of the system for real-time detection and separation of molecular magnets according to the present invention (wherein, blue represents the continuous injection mode pipeline, orange represents the circulating HPLC mode pipeline, and black represents the common pipeline). Figure 2 The P@C samples before separation in Examples 1-5 and Comparative Examples 1-3 of this invention. 60 Room temperature continuous wave EPR spectrum of the crude product (approximately 50 ppm); Figure 3 P@C is the embodiment of the present invention 1. 60 Single-injection EPR-HPLC chromatogram in a Buckeyprep-M column (315 nm UV signal corresponds to C) 60 The EPR signal corresponds to P@C 60 ); Figure 4 P@C in Embodiment 2 of the present invention 60 Single-injection EPR-HPLC chromatogram in a 5PBB column (315 nm UV signal corresponds to C) 60 The EPR signal corresponds to P@C 60 ); Figure 5 P@C is the embodiment of the present invention 4. 60 EPR-HPLC chromatogram in continuous injection mode on a Buckeyprep-M column (315 nm UV signal corresponds to C) 60 The EPR signal corresponds to P@C 60 ); Figure 6 P@C in Embodiment 5 of the present invention 60 EPR-HPLC chromatogram in cyclic HPLC mode on a 5PBB column (315 nm UV signal corresponds to C). 60 The EPR signal corresponds to P@C 60 ); Figure 7 The P@C obtained in Example 5 of this invention 60 MALDI-TOF spectrum of pure sample (M=751); Figure 8 The P@C obtained in Example 5 of this invention 60 Room temperature continuous wave EPR spectrum of a pure sample; Figure 9The dual radical of Embodiment 6 of the present invention m EPR-HPLC chromatogram of (TTM)2 in a 5PBB column after a single injection; Figure 10 This is the single-injection EPR-HPLC chromatogram of the triradical TR in a 5PBB column of Example 7 of the present invention; Figure 11 The experimental and simulated spectra of the 100 K continuous wave of the three free radical TR obtained in Example 7 of this invention are shown. Figure 12 P@C is the comparative example 1 of this invention. 60 Spin signal reconstruction spectrum in Buckeyprep column (315nm UV signal corresponds to C) 60 The EPR signal corresponds to P@C 60 ). Detailed Implementation

[0022] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0023] This invention provides an online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPL-HPLC) system for real-time detection and separation of molecular magnets. The system comprises an HPLC unit and an EPR detection unit, which is based on a commercial HPLC instrument with the addition of an EPR detector. The EPR detector is connected in series using polytetrafluoroethylene (PTFE) or PEEK tubing. The HPLC unit includes an injection pump, a dispensing pump, a chromatographic column, and a UV-Vis detector. The system offers two separation modes: continuous injection and cyclic HPLC, as well as two EPR detection modes: tuned and online detection, controlled by six-way valves A and B, respectively.

[0024] like Figure 1 The schematic diagram of the system for real-time detection and separation of molecular magnets is shown. In continuous injection mode, the tubing is blue, with six-way valve A directly connected to the collector. In cyclic HPLC mode, the tubing is orange, and the sample returns to the column after passing through six-way valve A. The black tubing is the common tubing. In EPR tuned detection mode, the sample enters the EPR test area after passing through six-way valve B, which is shown as a dashed line. In EPR online detection mode, the sample is separated by the column before entering the EPR detector, and six-way valve B is shown as a solid line.

[0025] The purpose of this invention is to separate molecular magnets. Experiments have shown that the molecular magnets to be separated have low polarity, and a reverse-phase column is preferable for chromatography.

[0026] The purpose of using PTFE or PEEK tubing is to connect the various parts of the system for real-time detection of separated molecular magnets. PTFE tubing is chosen for the tubing passing through the EPR detector area because experiments have shown that PEEK material has an EPR signal, which can interfere with the detection of the EPR signal of the molecular magnet during separation.

[0027] The present invention provides a real-time detection and separation method using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPLC), performed in the aforementioned online EPC-EPLC real-time detection and separation system, comprising the following steps: S1. Dissolve the molecular magnet sample to be separated in the corresponding solvent, with a concentration not exceeding 0.5 mg / mL. The solvent is a common organic solvent, selected from one or more of toluene, dichloromethane, cyclohexane, ethyl acetate, acetone, and ethanol, and should be consistent with the mobile phase in S3. S2. Set the six-way valve B to EPR tuning detection mode, pump the sample to be separated into the EPR test area through the injection pump, tune it, determine the applied microwave frequency, perform continuous wave EPR test at the microwave frequency, determine the resonant magnetic field of the molecular magnet, fix the magnetic field, set the microwave power and modulation amplitude, and test the change of EPR signal intensity over time under the fixed magnetic field. S3. Switch the six-way valve B to real-time detection mode, and at the same time turn on the infusion pump to deliver the mobile phase for elution. The sample to be separated flows out from the EPR test area, passes through the chromatographic column, UV-Vis detector and EPR detector in sequence, and reaches the six-way valve A. S4. When the separation mode is continuous injection mode, collect the sample and inject it repeatedly at fixed intervals. Repeat S3. When the separation mode is cyclic HPLC mode, return the sample to the column for the next cycle of separation until the cycle ends. Then switch the six-way valve A to continuous injection mode and collect the sample. The molecular magnets to be separated are molecules such as metal-intercalated fullerenes, nitrogen-intercalated fullerenes, phosphorus-intercalated fullerenes, and organic free radicals, which have electron paramagnetic resonance signals that can be detected by EPR. The essence of this invention is to separate molecular magnets with similar residence times from their precursor molecules or byproducts. This invention aims to demonstrate the versatility and effectiveness of systems and methods for real-time detection and separation of molecular magnets. The residence time of atomically embedded fullers differs least from that of intrinsic fullerenes, making them the most difficult to separate. Organic free radicals are extremely common molecular magnets. In the examples, P@C was selected. 60 Triradical TR and Diradical m -(TTM)2 were separated, and their molecular structures are as follows:

[0028] The purpose of S1 is to prepare the sample to be separated into a solution so that it can be injected into a system for real-time detection and separation of molecular magnets via a sample injection pump; wherein P@C 60 The sample spin content is greater than 50 ppm. If the content is too low, the EPR detector will not be able to detect the signal, or the signal-to-noise ratio will be extremely low, making it impossible to identify the signal peak position. The solution concentration is 0.1 mg / mL - 0.5 mg / mL. If the concentration is too high, it will exceed the column capacity, resulting in peak broadening, decreased resolution, and in more serious cases, blockage of the injection pump and column. If the concentration is too low, it will lead to increased experimental time and waste of mobile phase solvent.

[0029] The purpose of S2 is to tune the EPR detector before separation and set experimental conditions in a stable state. The preferred modulation amplitude is 2 G-3 G, and the preferred microwave power attenuation is 20 dB-30 dB. When the microwave power is not saturated, high microwave power and large modulation amplitude can increase the signal peak width and intensity, which helps to resist the interference of magnetic field fluctuations. However, excessive modulation amplitude or oversaturated power will lead to severe signal distortion and affect the stability of EPR test. The preferred single injection volume is 3 mL. If the injection volume is too high, it will exceed the column capacity, resulting in peak broadening and reduced resolution. If the injection volume is too low, it will result in low EPR signal intensity, increase the detection difficulty, and increase the experimental time and waste mobile phase solvent. The purpose of S3 is to initiate separation by detecting the change in EPR signal intensity over time using an EPR detector under a fixed magnetic field to determine the collection time of the target spin molecules. The preferred flow rate of the mobile phase is 10 mL / min-18 mL / min. Too low a flow rate will increase the experimental time, while too high a flow rate will cause excessive pressure on the chromatographic column, leading to its damage. The purpose of S4 is to separate samples using different separation modes. The purpose of continuous injection mode is to process a large number of samples and improve separation efficiency. Cyclic separation only allows single injection and aims to improve the resolution. The injection interval for continuous injection mode is 4-6 minutes, which is determined by the peak width of the sample. If the injection interval is too short, the peaks of the two injections will overlap and cannot be separated; if the injection interval is too long, it will increase the experimental time and waste the mobile phase, reducing the processing efficiency. Therefore, the injection interval should be shortened as much as possible without the sample peaks overlapping.

[0030] The purpose of this invention is to detect and separate molecular magnets. The separation method and conditions are formulated according to the properties of different target molecules. The above conditions are formulated for the molecules in the examples. When separating other molecules, separation conditions can be explored and formulated according to the actual situation and molecular properties.

[0031] The various embodiments will be described in more detail with reference to the following examples and comparative examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the embodiments.

[0032] Raw materials: Phosphorus-intercalated fullerenes, synthesized autonomously Triradical TR, autonomous synthesis Diradical m -(TTM)2 Autonomous Synthesis HPLC grade toluene Thermo Fisher Scientific Testing instruments: EPR detector, Guoyi Quantum EPR200M Infusion pump, six-way valve, sample injection pump, and UV-Vis detector, Ono LC-2000 Buckyprep, 5PBB and Buckyprep-M preparative chromatographic columns (20 mm × 250 mm) Cosmosil.

[0033] Example 1: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography, such as... Figure 1 As shown, 0.5 mg / mL of P@C 60 A crude product (~50 ppm) toluene solution was injected in 3 mL via a sample pump. The six-way valve B was set to tuned mode. After the sample was tuned to the EPR detector, continuous wave EPR testing was performed. The microwave frequency was determined to be 9.8 GHz, the resonant magnetic field to be 3478 G, the microwave power to be 0.5 mW, and the modulation amplitude to be 2 G. The magnetic field was fixed at the resonant magnetic field position. The six-way valve B was then set to real-time detection mode, and the six-way valve A to continuous injection mode. The flow rate was set to 12 mL / min, the mobile phase to toluene, and the UV-Vis detector to 315 nm. Separation began. After separation via a Buckeyprep-M column, the sample passed through the EPR detector and the UV-Vis detector, finally reaching the six-way valve A. The fraction with the EPR signal was collected.

[0034] Figure 2 For P@C 60 Room temperature continuous wave EPR spectrum of the crude product; Figure 3 For the separation of P@C 60 Single-injection EPR-HPLC chromatogram in a Buckeyprep-M column, 315 nm UV signal corresponds to C 60 The EPR signal corresponds to P@C 60 This indicates that real-time detection of the spin signal has been achieved, and the C0 value can be obtained in the Buckeyprep-M column. 60With P@C 60 The dwell time ratio was 1.071.

[0035] Example 2: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography, such as... Figure 1 As shown, 0.5 mg / mL of P@C 60 A crude product (~50 ppm) toluene solution was injected in 3 mL via a sample pump. The six-way valve B was set to tuned mode. After the sample was tuned to the EPR detector, continuous wave EPR testing was performed. The microwave frequency was determined to be 9.8 GHz, the resonant magnetic field to be 3478 G, the microwave power to be 0.5 mW, and the modulation amplitude to be 2 G. The magnetic field was fixed at the resonant magnetic field position. The six-way valve B was then set to real-time detection mode, and the six-way valve A to continuous injection mode. The flow rate was set to 12 mL / min, the mobile phase to toluene, and the UV-Vis detector to 315 nm. Separation began. After separation via a 5PBB column, the sample passed through the EPR detector and the UV-Vis detector, finally reaching the six-way valve A. The fraction with the EPR signal was collected.

[0036] Figure 4 For the separation of P@C 60 A single-injection EPR-HPLC chromatogram in a 5PBB column, with the 315 nm UV signal corresponding to C. 60 The EPR signal corresponds to P@C 60 In a 5PBB column, C can be obtained. 60 With P@C 60 The dwell time ratio was 1.092.

[0037] Example 3: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography, such as... Figure 1 As shown, 0.5 mg / mL of P@C 60A crude product (~50 ppm) toluene solution was injected in 3 mL via a sample pump. The six-way valve B was set to tuned mode. After tuning the EPR detector, continuous wave EPR testing was performed. The microwave frequency was determined to be 9.8 GHz, the resonant magnetic field to be 3478 G, the microwave power to be 0.5 mW, and the modulation amplitude to be 2 G. The magnetic field was fixed at the resonant magnetic field position. The six-way valve B was then set to real-time detection mode, and the six-way valve A to continuous injection mode. The flow rate was set to 12 mL / min, the mobile phase to toluene, and the UV-Vis detector to 315 nm. Separation began. After separation on the Buckyprep column, the sample passed through the EPR and UV-Vis detectors, finally reaching the six-way valve A. The fraction with the EPR signal was collected. In the Buckyprep column, C... 60 With P@C 60 The dwell time ratio was 1.071.

[0038] Examples 1-3 demonstrate that by tuning the EPR and setting experimental conditions before separation, the EPR detector can be brought to its optimal detection state, enabling real-time detection of spin signals in different chromatographic columns without the need to replace the column and reconstruct the spin signal, thus avoiding cumbersome operations.

[0039] Example 4: Systems for real-time detection of separated molecular magnets, such as Figure 1 As shown, the EPR tuning operation and test conditions before separation were set as in Examples 1-3. The six-way valve A was set to continuous injection mode, and 3 mL of P@C was injected every 4 min. 60 The crude product (~50ppm) toluene solution was injected into the Buckeyprep-M column, passed through the EPR detector and UV-Vis detector, and reached the six-way valve A. The fraction with the EPR signal was collected, and the injection was repeated until all the solutions to be separated entered the column.

[0040] Figure 5 For P@C 60 EPR-HPLC chromatogram in continuous injection mode on a Buckeyprep-M column, with the EPR signal being P@C. 60 This indicates that the chromatographic peaks with spin signals do not overlap, allowing for batch sample processing. The EPR detector can achieve real-time detection in continuous injection mode.

[0041] Example 5: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography, such as... Figure 1As shown, the EPR tuning operation and test conditions before separation were set as in Examples 1-3. The six-way valve A was set to cyclic HPLC mode. 3 mL of P@C separated in Example 4 was continuously injected. 60 Toluene solution was injected into a Buckeyprep-M column, passed through an EPR detector and a UV-Vis detector, and reached six-way valve A. The sample was then returned to the column for the next cycle of separation until the cycle ended. Afterward, six-way valve A was switched to continuous injection mode for collection, yielding P@C. 60 The pure sample is a colorless and transparent toluene solution.

[0042] Figure 6 For P@C 60 EPR-HPLC chromatogram in cyclic HPLC mode on a 5PBB column, with the EPR signal being P@C. 60 As the number of cycles increases, the peak gradually broadens, and the EPR detector can achieve real-time detection in cyclic HPLC mode.

[0043] Figure 7 For the separated P@C 60 The MALDI-TOF spectrum of the pure sample (M=751) is the first time that P@C has been obtained. 60 The mass spectrometry results indicate that P@C was successfully separated. 60 ; Figure 8 For the separated P@C 60 The room temperature continuous wave EPR spectrum of the pure sample, and Figure 1 P@C before separation 60 Compared to the continuous wave EPR spectrum of the crude product, it exhibits an extremely high signal-to-noise ratio. This indicates a significant improvement in P@C. 60 Effective purification was carried out.

[0044] Example 6: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography, such as... Figure 1 As shown, the EPR tuning operation and test conditions before separation were set as in Examples 1-3. The six-way valve A was set to continuous injection mode, and 3 mL of 0.2 mg / mL dual radical solution was injected. m -(TTM)2 toluene solution was injected into a 5PBB column, then passed through a UV-Vis detector and an EPR detector, and finally through a six-way valve A to collect the fraction with the EPR signal.

[0045] Figure 9 For separate diradicals mThe single-injection EPR-HPLC chromatogram of (TTM)2 in a 5PBB column shows the simultaneous appearance of UV-Vis and EPR signals, indicating that the free radical can be detected by both detectors, and that only one signal peak appears in each detector, suggesting that the free radical has high purity. The EPR detector can detect free radicals in real time and has versatility.

[0046] Example 7: A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography, such as... Figure 1 As shown, the EPR tuning operation and test conditions before separation were set as in Examples 1-3. The six-way valve A was set to continuous injection mode. 3 mL of 0.2 mg / mL triradical TR toluene solution was injected into the 5PBB column, and then passed through the UV-Vis detector and the EPR detector to the six-way valve A to collect the fraction with the EPR signal.

[0047] Figure 10 The EPR-HPLC chromatogram of the separated triradical TR in a 5PBB column after a single injection shows multiple UV peaks, indicating that the radical may have undergone some degree of decomposition. The UV detector alone cannot determine which component is the target radical. Only the chromatographic peak with a residence time of 4 min has an EPR signal, indicating that this is the triradical TR. The EPR detector can detect the target radical and remove the byproducts when the UV-Vis detector cannot distinguish between byproducts and radicals.

[0048] Figure 11 The 100 K continuous wave EPR experimental and simulated spectra of the isolated triradical TR were used to prove that it is a triradical TR and to effectively purify it.

[0049] Using the methods described in Examples 1-7, the detection of molecules with EPR signals can be achieved using different chromatographic columns and different separation modes, wherein P@C 60 Because the content is too low to be detected by the UV-Vis detector, the free radicals can be detected by both the UV-Vis and EPR detectors, which proves the effectiveness and versatility of the system and method for real-time detection and separation of molecular magnets of the present invention.

[0050] Comparative Example 1: In cyclic HPLC mode, 3 mL of 0.5 mg / L P@C was injected. 60 A toluene solution was used as the mobile phase at a flow rate of 12 mL / min. After multiple cycles through a Buckeyprep column, C was detected by UV chromatography. 60At the start of the chromatographic peak, the eluted fractions were collected every 1 minute, and the continuous wave EPR spectrum of each fraction was tested. The residence time of the phosphorus-intercalated fullerene in the chromatography was reconstructed based on the EPR signal intensity.

[0051] Figure 12 For P@C 60 Spin signal reconstruction spectrum in Buckeyprep column, 315 nm UV signal corresponds to C 60 The EPR signal corresponds to P@C 60 This allows us to obtain P@C in a Buckeyprep column. 60 With C 60 The dwell time ratio was 1.071. This is consistent with the results of real-time detection by the EPR detector in Example 3, mutually confirming the accuracy of the detection results.

[0052] Comparative Example 2: The experimental method was the same as that of Comparative Example 1. Spin signal reconstruction was performed on a Buckeyprep-M column, which yielded the P@C signal in the Buckeyprep-M column. 60 With C 60 The dwell time ratio was 1.068. This is consistent with the real-time detection results of the EPR detector in Example 1, mutually confirming the accuracy of the detection results.

[0053] Comparative Example 3: The experimental method was the same as that of Comparative Example 1. Spin signal reconstruction was performed on a 5PBB column, which yielded the P@C signal in the 5PBB column. 60 With C 60 The dwell time ratio was 1.092. This is consistent with the results of real-time detection by the EPR detector in Example 2, mutually confirming the accuracy of the detection results.

[0054] Results and Discussion: P@C in Examples 1-3 60 With C 60 The residence time ratios correspond to those of Comparative Examples 1-3, mutually confirming the accuracy of the test results. Because P@C 60 With C 60The residence times of the components are very close, requiring multiple cycles to achieve complete separation. In continuous injection mode, the sample undergoes only one separation, resulting in low resolution and narrow peaks. Spin signal reconstruction of eluted components collected at fixed intervals introduces significant errors, limiting its applicability to cyclic HPLC. However, even in cyclic HPLC, the operation is cumbersome, time-consuming, and labor-intensive. Spin signal reconstruction requires re-performing each column change, and changes in column status cannot be detected promptly. In contrast, real-time EPR detection is applicable to various columns and separation modes, offering simple operation and timely, accurate detection.

[0055] Summarize: 1. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPR-HPLC) system for real-time detection and separation of molecular magnets described in this invention can be used as an effective system for real-time detection and separation of molecular magnets. It is suitable for the separation of substances with EPR signals. Through ingenious flow path design, it integrates continuous injection, cyclic HPLC, EPR tuning and real-time detection functions, solving the problems of needing to tune and set test conditions before EPR testing, and is compatible with various chromatographic columns.

[0056] 2. The method for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography (EPR-HPLC) as described in this invention utilizes both continuous injection and cyclic HPLC separation methods, combined with real-time spin signal detection using an EPR detector, to separate molecules containing a large amount of C. 60 P@C 60 Trace amounts of P@C were separated from the crude product. 60 Pure samples; and the separation and detection of byproducts and target radicals that cannot be distinguished by UV-Vis detectors from the complex chemical situations of organic free radicals. Compared with spin signal reconstruction methods, it has high separation efficiency, can process samples in high throughput, accurately detects the residence time of target molecules, has significant purification effect, is simple to test, and is applicable to multiple separation modes.

[0057] 3. The system and method for real-time detection and separation of molecular magnets described in this invention can be achieved by simply modifying commercial HPLC and EPR instruments. Various separation and detection modes can be controlled by switching between two six-way valves. It also has the potential for expansion, can be combined with other detectors or chromatographic techniques to broaden its application scope, is easy to promote, and has universality.

[0058] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0059] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy coupled with high performance liquid chromatography, characterized in that, Including an HPLC unit and an EPR detection unit, it adds an EPR detector and a six-way valve to a commercial HPLC instrument; The HPLC unit includes an injection pump, a liquid pump, a chromatographic column, and a UV-Vis detector. The various parts of the system for real-time detection and separation of molecular magnets are connected by polytetrafluoroethylene or PEEK tubing. The system for real-time detection and separation of molecular magnets has two separation modes: continuous injection and cyclic HPLC, as well as two EPR detection modes: tuning and online detection, which are controlled by six-way valve A and six-way valve B, respectively. The two detection modes are: EPR tuning detection mode and EPR online detection mode; The two separation modes are: continuous injection mode and cyclic HPLC mode; The two detection modes and two separation modes are controlled and switched by two six-way valves. In EPR tuned detection mode, the sample enters the EPR test area after passing through six-way valve B, which is in the dashed line state. In EPR online detection mode, the sample enters the EPR detector after separation by the chromatographic column, and six-way valve B is in the solid line state. In continuous injection mode, the tubing is blue, and six-way valve A is in the solid blue line state, directly connected to the collector. In cyclic HPLC mode, the tubing is orange, and six-way valve A is in the orange dashed line state. The sample returns to the chromatographic column after passing through six-way valve A, and the black line represents the shared tubing.

2. The system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography as described in claim 1, characterized in that, The chromatographic column can be a normal chromatographic column, a reversed-phase chromatographic column, an ion exchange chromatographic column, or a gel permeation chromatographic column.

3. The system for real-time detection and separation of molecular magnets using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography as described in claim 1, characterized in that, The various parts of the system for real-time detection and separation of molecular magnets are connected by polytetrafluoroethylene (PTFE) tubing; the part passing through the EPR detector area is also a PTFE tubing.

4. A real-time detection and separation method using online electron paramagnetic resonance spectroscopy-high performance liquid chromatography, characterized in that, This method is performed in the online electron paramagnetic resonance spectroscopy-high performance liquid chromatography system for real-time detection and separation of molecular magnets as described in any one of claims 1-3, and includes the following steps: S1. Dissolve the molecular magnet sample to be separated in the corresponding solvent to prepare a solution with a concentration ≤0.5 mg / mL. The solvent is an organic solvent and should be consistent with the mobile phase in S3. S2. Set the six-way valve B to EPR tuning detection mode, pump the sample to be separated into the EPR test area through the injection pump, tune it, determine the applied microwave frequency, perform continuous wave EPR test at the microwave frequency, determine the resonant magnetic field of the molecular magnet, fix the magnetic field, set the microwave power and modulation amplitude, and detect the change of EPR signal intensity over time under the fixed magnetic field. S3. Switch the six-way valve B to real-time detection mode, and at the same time turn on the infusion pump to deliver the mobile phase for elution. The sample to be separated flows out from the EPR test area, passes through the chromatographic column, UV-Vis detector and EPR detector in sequence, and reaches the six-way valve A. S4. When the separation mode is continuous injection mode, collect the sample and inject it repeatedly at fixed intervals, repeating S3. When the separation mode is cyclic HPLC mode, the sample is returned to the column for the next cycle of separation until the cycle ends. Then, switch the six-way valve A to continuous injection mode and collect the sample.

5. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled real-time detection and separation method according to claim 4, characterized in that, The molecular magnet sample to be separated described in S1 is a molecule with an electron paramagnetic resonance signal, selected from nitrogen-intercalated fullerenes, phosphorus-intercalated fullerenes, metal-intercalated fullerenes or organic free radicals, wherein the content of crude intercalated fullerene products is >50 ppm; the concentration of the prepared solution is 0.1 mg / mL-0.5 mg / mL.

6. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled real-time detection and separation method according to claim 4, characterized in that, The organic solvent mentioned in S1 is one or a mixture of several of toluene, dichloromethane, cyclohexane, ethyl acetate, acetone and ethanol.

7. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled real-time detection and separation method according to claim 4, characterized in that, As described in S2, the sample to be separated is pumped into the EPR test area by a sample injection pump, with a single injection volume of 1 mL-6 mL, a fixed microwave frequency of 9 GHz-10 GHz, a magnetic field range of 50 G-10000 G, a microwave power attenuation range of 0 dB-60 dB, and a modulation amplitude of 0.5 G-5 G.

8. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled real-time detection and separation method according to claim 7, characterized in that, As described in S2, the sample to be separated is pumped into the EPR test area by a sample injection pump, with a single injection volume of 3 mL, a fixed microwave frequency of 9.3 GHz-9.6 GHz, a magnetic field range of 3000 G-4000 G, a microwave power attenuation range of 20 dB-30 dB, and a modulation amplitude of 2 G-3 G.

9. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled real-time detection and separation method according to claim 4, characterized in that, The elution described in S3 is performed using gradient elution or isocratic elution; the mobile phase is a single organic solvent or a mixture of multiple solvents, such as toluene, dichloromethane, a toluene:dichloromethane = 1:1 solution, or a cyclohexane:ethyl acetate = 9:1 solution; the flow rate of the mobile phase can be 1 mL / min-20 mL / min, preferably 10 mL / min-18 mL / min; the wavelength range of the UV-Vis detector described in S3 is 200 nm-2000 nm.

10. The online electron paramagnetic resonance spectroscopy-high performance liquid chromatography coupled real-time detection and separation method according to claim 4, characterized in that, The continuous injection interval described in S4 is 4 min-6 min.

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