PH measuring method based on multi-core J coupling constant

Abstract

The invention discloses a multi-core J coupling constant-based pH measurement method, which comprises the following steps of: 1, preparing a series of compound samples with different pH values, and measuring J coupling constants of compounds in the samples; 2, drawing a coupling constant and pH relation curve of the compound J, and deducing a pH calculation formula; and 3, measuring the J coupling constant of the compound-containing sample to be measured, and determining the pH value of the sample to be measured. According to the method, the J coupling constant is measured through a nuclear magnetic resonance spectrum method or a zero field-near zero field J coupling spectrum method, and the pH value of the surrounding environment of the compound can be measured.

Description

Technical Field

[0001] This invention belongs to the fields of magnetic resonance technology and analytical measurement, and specifically relates to a pH measurement method based on multinucleus J coupling constant. This method is applicable to measuring the pH value of the environment surrounding a compound by measuring the J coupling constant of the compound. Background Technology

[0002] pH measurement holds significant importance in scientific research, industrial production, and the field of life and health. pH is not only a core indicator for measuring the acidity or alkalinity of a solution, but also a crucial parameter for understanding chemical equilibrium, regulating biological metabolism, and ensuring environmental safety. In biomedicine, accurate pH measurement directly impacts disease diagnosis (such as acidosis and tumor microenvironment assessment) and drug efficacy. In industrial production, pH control determines reaction efficiency and product quality (such as food fermentation and wastewater treatment). From high-precision electrodes in the laboratory to magnetic resonance imaging of living tissues, advancements in pH measurement technology continuously drive interdisciplinary innovation in chemistry, biology, and medicine, providing indispensable support for human health, industrial optimization, and environmental protection.

[0003] Existing pH detection methods include: electrochemical methods (such as pH electrodes) offer high precision and fast response, but require sample contact and are susceptible to contamination; optical methods (such as fluorescent probes) offer high sensitivity and can achieve micron-level spatial resolution, but suffer from photobleaching issues and limited penetration depth; semiconductor methods (such as pH-FETs) are easy to miniaturize and integrate, making them suitable for wearable devices, but are susceptible to ion interference and lack stability; colorimetric methods (such as pH test strips) are simple to operate and low in cost, but have lower precision and rely on subjective judgment; and conventional nuclear magnetic resonance methods (such as those based on chemical shift changes) are also available. 31 P NMR and CEST MRI (based on chemical exchange saturation transfer) are non-invasive and have unlimited penetration depth, making them particularly suitable for biomedical research. However, they are susceptible to field inhomogeneities and have complex data processing requirements. Existing pH detection methods each have their own characteristics and are applicable to different scenarios, but they also have certain limitations. Therefore, there is a need to develop new methods for quantitative pH detection. Summary of the Invention

[0004] To address the aforementioned problems and shortcomings of existing pH detection technologies, this invention provides a pH measurement method based on the multi-nuclear J coupling constant. This invention is based on the following approach:

[0005] The J-coupling constant is a physical quantity characterizing the strength of spin-spin coupling between atomic nuclei. Its value is mainly determined by factors such as the bonding structure, spatial configuration, electron cloud distribution, and electronegativity of neighboring groups between the coupled nuclei. A key characteristic is that the J-coupling constant is independent of the strength and homogeneity of the external magnetic field. This characteristic gives the use of the J-coupling constant a unique advantage in pH measurement: changes in pH can alter the molecular structure or electronic environment of pH-sensitive compounds through mechanisms such as protonation / deprotonation, conformational changes, or metal coordination. This leads to the coexistence of compounds in solution in two or more different forms (e.g., protonated and deprotonated states) until acid dissociation equilibrium is reached. Importantly, these different forms typically possess their own characteristic J-coupling constants. When the rate of interconversion between different forms is faster than the timescale of nuclear magnetic resonance (NMR), the experimentally observed J-coupling constant is a concentration-weighted value of the J-coupling constants of the different existing forms. This concentration fraction is directly related to the pH of the solution and the acid dissociation constant (pKa) of the compound. Therefore, by measuring the J coupling constant of the target compound in the sample using techniques such as nuclear magnetic resonance spectroscopy or zero-field-near-zero-field J coupling spectroscopy, and combining this with a pre-established curve showing the relationship between the J coupling constant of the compound and pH value, the pH of the sample environment can be quantitatively determined.

[0006] A pH measurement method based on multi-nuclear J coupling constant includes the following steps:

[0007] Substituting the J-coupling constants of solutions containing the target compound at multiple different pH values ​​into the equation set, we obtain the J-coupling constants of all dissociated substances after acid dissociation of the target compound and the acid dissociation constant for each dissociation step. Substituting these J-coupling constants and the acid dissociation constants for each dissociation step into the equation set, we obtain the pH value of the solution containing the target compound. The equation set includes:

[0008]

[0009] f i = f(pKa1, ..., pKa) j pH)

[0010] Where N is the number of dissociated substances after acid dissociation of the target compound, f i and J i These represent the molar concentration fraction of the i-th intermediate dissociation product and the coupling constant J, respectively. PH pKa is the measured J coupling constant. j f is the acid dissociation constant for the j-th dissociation step. i = f(pKa1, ..., pKa) j , where pH) is a function of the molar concentration fraction of the first to i dissociated substances, j = N-1.

[0011] The steps for obtaining the J-coupling constant of a solution containing the target compound include:

[0012] Obtain solutions of the same target compound at different pH values.

[0013] The J-coupling constant of the solution of the target compound was determined by either of the following two techniques:

[0014] (a) Nuclear magnetic resonance spectroscopy (NMR); (b) Zero-field-near-zero-field J-coupled spectroscopy.

[0015] The target compound contains chemical groups, and the J-coupling constant of the chemical groups changes with pH value.

[0016] The J coupling constant of the solution of the target compound is either the phosphorus-hydrogen internuclear coupling constant or the phosphorus-fluorine internuclear coupling constant.

[0017] A pH measurement system based on multi-nuclear J coupling constant, comprising:

[0018] The data acquisition module is used to obtain the J coupling constant J of the target compound solution and the test solution containing the target compound at different pH values. PH ,

[0019] The data processing module stores and executes equation-solving programs to obtain the coupling constants J of all intermediate dissociated substances after the acid dissociation of the target compound, as well as the acid dissociation constant for each dissociation step. Each equation-solving program establishes a set of equations based on a single target compound.

[0020]

[0021] f i = f(pKa1, ..., pKa) j pH)

[0022] Where N is the number of dissociated substances after acid dissociation of the target compound, f i and J i These represent the molar concentration fraction of the i-th intermediate dissociation product and the coupling constant J, respectively. PH pKa is the measured J coupling constant. j f is the acid dissociation constant for the j-th dissociation step. i = f(pKa1, ..., pKa) j , where pH) is a function of the molar concentration fraction of the first to i intermediate dissociated substances, j = N-1;

[0023] The calculation output module takes the J coupling constant of the solution containing the target compound obtained by the data acquisition module and the output data of the data processing module, substitutes them into the equation system corresponding to the target compound, and outputs the pH value calculation result of the solution to be tested.

[0024] A computer-readable storage medium storing a computer program that, when executed by a processor, implements the steps of the pH measurement method based on the multi-core J coupling constant.

[0025] Compared with existing technologies, this invention has the following advantages: ① Compared with conventional NMR methods, the J coupling constant is not affected by magnetic field strength and homogeneity, overcoming the inherent defects of conventional NMR methods and exhibiting strong anti-interference capabilities; ② Compared with other existing methods besides conventional NMR methods, this invention does not require contact with the sample, has unlimited penetration depth, and is suitable for pH detection in living biological tissues and complex systems; ③ It has a wider range of applications and is easier to operate. This invention is particularly suitable for zero-field to near-zero-field J coupling spectroscopy methods where chemical shift information cannot be obtained, expanding application scenarios and avoiding complex data processing, thus lowering the implementation threshold. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 J is the phosphorus-hydrogen coupling constant J in 50 mmol / L Na2HPO3 and phosphate solutions. PH Relationship curve with pH;

[0028] Figure 2 Based on the phosphorus-hydrogen coupling constant J in Na2HPO3 phosphate solutions of different concentrations. PH Compare the calculated pH value with the pH meter measurement result;

[0029] Figure 3 The phosphorus-fluorine J coupling constant J in 50 mmol / L Na2FPO3 and phosphate solutions. PF The curve showing the relationship between pH and pH. Detailed Implementation

[0030] To facilitate understanding and implementation of the present invention by those skilled in the art, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0031] The main materials used in the following examples are described below: Na2HPO3, Na2FPO3, phosphates (NaH2PO4 and Na2HPO4), etc., were purchased from Adamas.

[0032] All nuclear magnetic resonance spectroscopy-related experiments in the embodiments were performed on a 400M wide-cavity nuclear magnetic resonance spectrometer (Bruker, Switzerland), and the data were processed using Matlab software.

[0033] Example 1

[0034] In this embodiment, sodium phosphite (Na2HPO3) is used as a pH-sensitive compound containing pH.

[0035] Step 1: Prepare a series of Na₂HPO₃ solutions with different pH values. The Na₂HPO₃ concentration is 50 mmol / L, and the phosphate concentration is 100 mmol / L. The phosphate is the collection of all phosphates formed after the acid-base dissociation of Na₂HPO₃. The pH values ​​measured using a pH meter are 0.489, 1.035, 1.483, 1.987, 2.540, 3.043, 3.490, 3.979, 4.530, 5.065, 5.500, 6.052, 6.492, 6.979, 7.473, 7.951, 8.506, 8.976, 9.503, 9.994, 10.564, 11.001, 11.534, and 12.041. The pH values ​​of the different solutions are repeatedly measured using a 400M wide-cavity nuclear magnetic resonance spectrometer. 31 Three P NMR spectra were obtained (298 K, zg sequence, sampling parameters: D1 = 5 s, spectral width SW = 81521.742 Hz, 90° pulse width 30 μs, power -13.62 dB, number of sampling points TD = 65536, number of accumulations NS = 64, receiver gain = 2050). The J coupling constant J in solutions with different pH values ​​was measured. PH As shown in Table 1. A curve showing the relationship between the J coupling constant and pH was also plotted (see...). Figure 1 ), Figure 1 The curve and the corresponding R 2 This indicates that sodium phosphite is based on the phosphorus-hydrogen J coupling constant (J... PH ) Optional compounds for measuring pH.

[0036] Table 1. Coupling constants J for solutions at different pH values. PH value

[0037] pH <![CDATA[J PH / Hz]]> pH <![CDATA[J PH / Hz]]> pH <![CDATA[J PH / Hz]]> 0.489 675.593 4.530 627.340 8.506 567.986 1.035 660.614 5.065 624.467 8.976 567.694 1.483 646.553 5.500 618.944 9.503 567.565 1.987 635.996 6.052 605.406 9.994 567.521 2.540 631.034 6.492 591.178 10.564 567.467 3.043 629.424 6.979 578.370 11.001 567.494 3.490 628.696 7.473 571.620 11.534 567.500 3.979 628.274 7.951 568.736 12.041 567.462

[0038] Step 2, the following acid dissociation equilibrium exists in the Na2HPO3 solution:

[0039]

[0040] pKa1 and pKa2 are the acid dissociation constants for each dissociation step, and the phosphorous groups in solution dissociate as HPO3-. 2― H2PO3 ― The presence of three dissociated substances, including H3PO3, is due to the presence of HPO3. 2― H2PO3 ― H3PO3 and H2PO3 undergo rapid exchange on the NMR timescale, hence the measured J coupling constant J between PH and H2PO3. PH With HPO3 2― H2PO3 ― The J coupling constants J1, J2, and J3 of H3PO3 are expressed as a weighted average:

[0041] J PH =f1*J1+f2*J2+f3*J3 (1)

[0042] Wherein, f1, f2, and f3 are HPO3 in the sample, respectively. 2― H2PO3 ― The molar concentration fraction of H3PO3 is expressed as a function of the molar concentration fraction of the dissociated substance, f. i = f(pKa1, ..., pKa) j pH):

[0043] j = N-1, N = 3, where N is the number of dissociated substances after the acid dissociation of the target compound.

[0044]

[0045] From formulas (1)-(4), the formulas for calculating pH value can be further derived as follows:

[0046]

[0047] Based on formulas (1)-(4) and the nonlinear least squares method, the J coupling constants of different pH solutions obtained in step 1 are input into Matlab software, and the values ​​of pKa1, pKa2, J1, J2 and J3 are obtained by fitting using formulas (1) to (4) (see Table 2).

[0048] Table 2 shows the parameter values ​​in formulas (1) to (4).

[0049] parameter <![CDATA[pKa1]]> <![CDATA[pKa2]]> <![CDATA[J1]]> <![CDATA[J2]]> <![CDATA[J3]]> <![CDATA[R 2 ]]> value 1.149 6.280 567.600 628.400 685.800 0.9999

[0050] Substitute the pKa1, pKa2, J1, J2 and J3 obtained from the fitting in Table 2 into Equation (5).

[0051]

[0052] Step 3: Determine the J coupling constant of the test solution containing the compound and determine the pH value of the test solution.

[0053] Prepare test solutions with Na₂HPO₃ concentrations of 10 mmol / L (pH = 6.163), 15 mmol / L (pH = 6.171), 20 mmol / L (pH = 6.161), 25 mmol / L (pH = 6.171), 30 mmol / L (pH = 6.159), 50 mmol / L (pH = 6.168), 75 mmol / L (pH = 6.167), and 100 mmol / L (pH = 6.164). Measure the J of the solutions at different concentrations using the same method as in step 1. PH The Hz frequencies are 603.279Hz, 602.015Hz, 602.485Hz, 602.291Hz, 602.485Hz, 602.080Hz, 601.756Hz, and 601.610Hz, respectively. Substituting these frequencies into formula (5) in step 2 and referring to Table 2, the calculated pH values ​​are 6.146, 6.183, 6.170, 6.175, 6.170, 6.181, 6.191, and 6.195, respectively. The pH values ​​calculated based on the J coupling constant are compared with the pH values ​​measured by the pH meter. Figure 2 As shown.

[0054] Example 2

[0055] In this embodiment, sodium monofluorophosphate (Na2FPO3) is used as a pH-sensitive compound containing PF.

[0056] Step 1: Prepare a series of sodium monofluorophosphate solutions with different pH values. The Na₂FPO₃ concentration is 50 mmol / L, and the phosphate concentration is 100 mmol / L. The phosphate is the collection of all phosphates formed after the acid-base dissociation of Na₂FPO₃. The pH values ​​measured using a pH meter are 2.051, 2.507, 3.026, 3.539, 4.017, 4.552, 4.958, 5.526, 6.045, 6.510, 7.025, 7.525, and 8.006, respectively. The pH values ​​of the different solutions are repeatedly measured using a 400M wide-cavity nuclear magnetic resonance spectrometer. 31 Three P NMR spectra were obtained (298 K, zg sequence, sampling parameters: D1 = 5 s, spectral width SW = 81521.742 Hz, 90° pulse width 30 μs, power -13.62 dB, number of sampling points TD = 65536, number of accumulations NS = 128, receiver gain = 2050). The J coupling constant J in solutions with different pH values ​​was measured. PF As shown in Table 3. A curve showing the relationship between the J coupling constant and pH was also plotted (see Table 3). Figure 3 ), Figure 3 The curve and the corresponding R2 (See Table 4) This indicates that sodium monofluorophosphate is based on the phosphorus-fluorine J coupling constant (J... PF ) Optional compounds for measuring pH.

[0057] Table 3 J-coupling constants for solutions at different pH values PF value

[0058] pH <![CDATA[J PF / Hz]]> pH <![CDATA[J PF / Hz]]> pH <![CDATA[J PF / Hz]]> 2.051 908.658 4.552 887.296 7.025 868.219 2.507 908.274 4.958 880.249 7.525 868.057 3.026 906.978 5.526 871.410 8.006 868.030 3.539 903.998 6.045 869.131 4.017 896.648 6.510 868.764

[0059] Step 2: In the pH range of 2.051–8.006, sodium monofluorophosphate in the solution exhibits the following acid dissociation equilibrium:

[0060]

[0061] pKa1 is the acid dissociation constant; monofluorophosphate groups in solution mainly exist as FPO3. 2― and HFPO3 ― Both forms exist, due to FPO3 2― and HFPO3 ― Rapid exchange occurs on the NMR timescale, hence the measured J coupling constant J between PFs. PF With FPO3 2― and HFPO3 ― The weighted average of the J coupling constants J1 and J2 is used to express this:

[0062] J PF =f4*J1+f5*J2 (6)

[0063] Wherein, f4 and f5 are the FPO3 in the sample, respectively. 2― and HFPO3 ― The molar concentration fraction.

[0064]

[0065] From formulas (6)-(8), the formulas for calculating pH value can be further derived as follows:

[0066]

[0067] Based on formulas (6)-(8) and the nonlinear least squares method, the J coupling constants and corresponding pH values ​​of different pH solutions obtained in step 1 are input into Matlab software, and the values ​​of pKa1, J1 and J2 are obtained by fitting using formulas (6) to (8) (see Table 4).

[0068] Table 4 shows the parameter values ​​in formulas (6) to (8).

[0069] parameter <![CDATA[pKa1]]> <![CDATA[J1]]> <![CDATA[J2]]> <![CDATA[R 2 ]]> value 4.500 868.050 908.500 0.9989

[0070] Substitute the pKa1, J1, and J2 obtained from the fitting in Table 4 into equation (9):

[0071]

[0072] Step 3: Determine the J coupling constant of the test solution containing the compound and determine the pH value of the test solution.

[0073] Prepare a 50 mmol / L sodium monofluorophosphate solution (pH = 4.563) and measure the J coupling constant using the same method as in step 1. Measure the J of solutions at different concentrations. PF The Hz value is 887.153 Hz. Substituting it into formula (9) in step 2 and combining it with Table 4, the pH value is calculated to be 4.548. The difference between the pH value calculated based on the J coupling constant and the pH value measured by the pH meter is -0.015.

[0074] It should be noted that the specific embodiments described in this invention are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains can make various modifications or additions to the described specific embodiments or use similar methods to replace them, but without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for pH measurement based on multi-core J-coupling constants, characterized by, The J coupling constants of the obtained solutions containing the target compound at different pH values are substituted into the equation set, the J coupling constants of all dissociated substances after acid dissociation of the target compound and the acid dissociation constants of each dissociation step are obtained, the J coupling constants of the obtained solutions containing the target compound and the J coupling constants of all dissociated substances after acid dissociation of the target compound and the acid dissociation constants of each dissociation step are substituted into the equation set, and the pH value of the solution containing the target compound is obtained; the equation set includes: f i = f(pKai,... pKan, pH) j , pH) where N is the number of dissociated species of the target compound after acid dissociation, f i and J i are the mole fraction of the i-th intermediate dissociated species and the J coupling constant, respectively, J PH is the measured J coupling constant, pKa j is the acid dissociation constant of the j-th dissociation step, f i = f(pKa1,... pKa j , pH) is a function of the mole fraction of the 1st to i-th intermediate dissociated species, j = N - 1.

2. The method for measuring pH based on multi-core J-coupling constant according to claim 1, characterized in that, The target compound contains a chemical group, and the J coupling constant of the chemical group changes with the change of the pH value.

3. The method for measuring pH based on multi-core J-coupling constant according to claim 1, characterized in that, The J coupling constant of the solution of the target compound is a phosphorus-hydrogen inter-nuclear coupling constant or a phosphorus-fluorine inter-nuclear coupling constant.

4. The method of claim 3, wherein, The J coupling constant of the solution of the target compound is determined by any of the following two methods: (a) nuclear magnetic resonance spectroscopy (NMR); (b) zero-field-near zero-field J coupling spectrum method.

5. The method of claim 4, wherein, The implementation parameters of the nuclear magnetic resonance spectroscopy include: the pulse sequence is zg standard one-dimensional sequence or P NMR sequence with pre-saturation 31 P NMR sequence.

6. The method of claim 4, wherein, When the zero-field-near zero-field J coupling spectrum method is used for determination, at least one of the following conditions is met: ① The magnetic field strength is <1 μT; ② The detection sensitivity is better than 1 nM; ③ A superconducting quantum interference device (SQUID) or an atomic magnetometer (such as a rubidium atomic magnetometer) is used as a sensor; ④ The signal acquisition frequency range is 0.1 Hz to 10 kHz; ⑤ The concentration of the solution containing the target compound to be measured is 10 mmol / L to 100 mmol / L.

7. A pH measurement system based on multi-core J-coupling constants, characterized by, including: a data acquisition module for obtaining the J coupling constant of the solution containing the target compound; a data processing module for storing and executing equation solving programs for obtaining the J coupling constants of all intermediate dissociated substances after acid dissociation of the target compound and the acid dissociation constants of each dissociation step, each equation solving program being based on an equation set established for a target compound: f i = f(pKai,... pKan, pH) j , pH) where N is the number of dissociated species of the target compound after acid dissociation, f i and J i are the mole fraction of the i-th intermediate dissociated species and the J coupling constant, respectively, J PH is the measured J coupling constant, pKa j is the acid dissociation constant of the j-th dissociation step, f i = f(pKa1,... pKa j , pH) is a function of the mole fraction of the 1st to i-th intermediate dissociated species, j = N - 1; a calculation output module for substituting the J coupling constant of the solution containing the target compound to be measured obtained by the data acquisition module and the output data of the data processing module into the equation set corresponding to the target compound, and outputting the pH value calculation result.

8. A computer readable storage medium storing a computer program, characterized in that, The program is executed by the processor to realize the steps of the method of any one of claims 1-6.

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