A method for detecting the isotopic abundance in acetic acid

Abstract

The present application relates to a kind of detection methods of isotopic abundance in acetic acid, comprising the following steps: stable isotope labeled acetic acid is decomposed under vacuum and high temperature conditions, generates stable isotope labeled methane or carbon dioxide, can determine 13 C labeled in carboxyl site acetic acid CH3 13 COOH in 13 C isotopic abundance, 13 C labeled in methyl site acetic acid 13 CH3COOH in 13 C isotopic abundance, 18 O labeled acetic acid CH3C 18 O 18 OH in 18 O isotopic abundance, methyl deuterium isotopic abundance in deuterium labeled methyl site acetic acid CD3COOH;Compared with prior art, the present application has the advantages that the isotopic abundance in acetic acid labeled by stable isotope in different sites is detected, can be used for deuterium, 13 C or 18 O labeled acetic acid isotopic abundance determination, test data accuracy and precision etc.

Description

Technical Field

[0001] This invention relates to the field of stable isotope abundance detection technology, specifically to a method for detecting isotope abundance in acetic acid. Background Technology

[0002] Acetic acid is a simple carboxylic acid and a major chemical product, one of the most important organic acids. It is mainly used in the production of cellulose acetate, polyvinyl acetate, etc. Acetic acid is widely distributed in nature, for example, in fruits and vegetable oils, where it mainly exists in the form of esters. In animal tissues, excrement, and blood, acetic acid exists as a free acid. Many microorganisms can convert different organic substances into acetic acid through fermentation.

[0003] Stable isotope-labeled compounds are those that use stable isotope atoms (deuterium, uran ... 13 C 18 Compounds in which one or more atoms are replaced by stable isotopes (e.g., deuterium). The labeling forms of stable isotope-labeled compounds are diverse, and the applications of compounds labeled with different stable isotope atoms vary. For example, acetic acid, also known as vinegar, is commonly used as cooking vinegar. When the hydrogen atoms in acetic acid are replaced by the stable isotope deuterium, stable isotope-labeled acetic acid is formed. It is used as a nuclear magnetic resonance reagent, in pharmaceuticals and synthesis, for modifying optoelectronic materials, and as a laboratory reagent. When stable isotopes are used... 13 When carbon atoms replace carbon atoms in acetic acid, stable isotopes are formed. 13 C-labeled acetic acid can be used in organic or biosynthetic processes. 13 The starting material of C-labeled compounds. When using 18 Stable isotopes formed when oxygen replaces oxygen atoms in acetic acid 18 O-labeled acetic acid can be used for metabolomics research or clinical diagnosis.

[0004] Invention patent CN 110793826 A discloses a sample preparation method for determining the abundance of carbon-13 isotopes. This method belongs to the oxidation method, in which an oxidant reacts with carbon to generate... 13 CO2 tests measure the overall isotopic abundance of carbon. For carbon only labeled in the methyl group... 13 CH3COOH) or carboxyl site (CH3) 13 Acetic acid (COOH) cannot be accurately measured.

[0005] Invention patent CN 115452924 A (A method for detecting isotopic abundance in urea) and industry standard HG / T4138—2010 Stable Isotopes 13 C-Urea both disclose urea- 13 The method for detecting isotope abundance in C, the row standard method, utilizes the strong oxidizing properties of nitrous acid.13 Methods for detecting the abundance of C-urea isotopes [J]. Isotopes, 2010, 23(1):39-43), urea- 13 C oxidation produces 13 C-labeled carbon dioxide. Invention patent CN 115452924 A utilizes the specificity of urease to label urea-... 13 C is ultimately transformed into 13 C-labeled carbon dioxide. Both detection methods were developed for stable isotope-labeled urea, and are for urea labeled only on the methyl group (C). 13 CH3COOH) or carboxyl site (CH3) 13 Acetic acid (COOH) cannot be accurately measured.

[0006] Invention patent CN111257403 A discloses a method for determining the stable hydrogen isotope ratio of the methyl group of pure acetate, comprising the following steps: removing hydrogen from the carboxyl group of acetate with a chemical reagent to convert acetic acid into a non-volatile acetate; drying in an oven or freeze-drying to remove moisture; packaging the sample in a silver cup, and subjecting the acetate to constant-temperature high-temperature decomposition at 1420℃ to convert all hydrogen elements into hydrogen gas, and determining the stable hydrogen isotope ratio; correcting with an acetic acid standard sample to obtain the stable hydrogen isotope ratio of the methyl group of acetate. The reaction product of this method is hydrogen gas.

[0007] Invention patent CN 112305058 A (A method for determining the abundance of deuterium isotopes in water samples) and paper (Determination of the abundance of deuterium isotopes in heavy water by high-resolution gas isotope mass spectrometry [J]. Chemical Reagents, 2021, 43(11): 1541-1545) disclose a method for detecting the abundance of deuterium isotopes in water samples, and the reaction product of the method is hydrogen gas. Summary of the Invention

[0008] The purpose of this invention is to overcome at least one of the defects of the prior art and provide a method for labeling acetic acid at different sites of stable isotopes, calculating isotopic abundance using different formulas, and applicable to deuterium, etc. 13 C or 18 O-labeled acetic acid isotope abundance determination method for detecting isotope abundance in acetic acid.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A method for detecting the abundance of isotopes in acetic acid, the method comprising the following steps:

[0011] The stable isotope-labeled acetic acid is decomposed to generate a stable isotope-labeled gas.

[0012] The gas is introduced into a gas isotope mass spectrometer to detect the ion current intensity;

[0013] After calculation using the formula, the isotopic abundance in stable isotope-labeled acetic acid was determined.

[0014] Furthermore, the stable isotope-labeled acetic acid includes 13 C-labeled acetic acid CH3 at the carboxyl site 13 COOH, 13 C-labeled acetic acid at the methyl site 13 CH3COOH, 18 O-labeled acetic acid CH3C 18 O 18 OH or deuterium-labeled acetic acid CD3COOH at the methyl site;

[0015] The measured isotopic abundances are as follows: 13 C-labeled acetic acid CH3 at the carboxyl site 13 COOH 13 C isotope abundance, 13 C-labeled acetic acid at the methyl site 13 CH3COOH 13 C isotope abundance, 18 O-labeled acetic acid CH3C 18 O 18 OH 18 O isotope abundance or methyl deuterium isotope abundance in acetic acid CD3COOH labeled with methyl sites.

[0016] Furthermore, the gas is methane or carbon dioxide.

[0017] Furthermore, the decomposition reaction is carried out at a temperature of 550-950℃ for 2-6 hours, with a vacuum degree of <10Pa.

[0018] Furthermore, before the decomposition reaction, the stable isotope-labeled acetic acid is frozen before the vacuum is activated.

[0019] Furthermore, stable isotope-labeled acetic acid was frozen using liquid nitrogen prepared with alcohol.

[0020] Further, measurement 13 C-labeled acetic acid CH3 at the carboxyl site 13 COOH 13 In the process of C isotope abundance: 13 CO2 was introduced into a gas isotope mass spectrometer to detect the ion current intensities of mass numbers 44 and 45.

[0021] Through formula Calculate the carboxyl site 13 C isotope abundance.

[0022] Further, measurement 13 C-labeled acetic acid at the methyl site13 CH3COOH 13 In the process of C isotope abundance: 13 CH4 was introduced into a gas isotope mass spectrometer to detect the intensities of ions with mass numbers of 16 and 17.

[0023] Through formula Calculation of methyl sites 13 C isotope abundance.

[0024] Further, measurement 18 O-labeled acetic acid CH3C 18 O 18 OH 18 O isotope abundance process: C 18 O2 was introduced into the gas isotope mass spectrometer to detect the ion current intensities of mass numbers 46 and 48.

[0025] Through formula calculate 18 O isotope abundance.

[0026] Furthermore, in determining the abundance of methyl deuterium isotopes in deuterium-labeled acetic acid CD3COOH at the methyl site: CD4 was introduced into a gas isotope mass spectrometer, and the ion current intensities with mass numbers of 18, 19, and 20 were detected.

[0027] Through formula Calculate the abundance of methyl deuterium isotopes.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] Because acetic acid is poorly ionized in liquid chromatography-mass spectrometry (LC-MS), its overall isotopic abundance can be measured in GC-MS. However, due to the small molecular weight of fragment ions (at different labeling sites), the isotopic abundance at different labeling sites cannot be accurately calculated. This invention targets acetic acid labeled at different sites of stable isotopes, using different formulas to calculate the isotopic abundance, thereby enabling the detection of deuterium. 13 C or 18 O-labeled acetic acid offers advantages such as high accuracy and precision in test data. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the reaction tube used in this invention;

[0031] Figure 2 This is a schematic diagram of the sample inlet tube used in this invention;

[0032] The numbers in the diagram indicate: 1-vacuum connector, 2-high temperature spray gun sealing position, 3-mass spectrometer interface, 4-fragile part of glass reaction tube, 5-round hole, 6-male connecting seal, 7-female connecting seal; Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0034] A method for detecting isotopic abundance in acetic acid involves labeling acetic acid at different sites of stable isotopes, calculating the isotopic abundance using different formulas, and then correspondingly detecting deuterium, etc. 13 C or 18 O-labeled acetic acid. The detection method includes the following steps:

[0035] In the reaction tube and sample injection tube, the vacuum connector 1 is connected to the vacuum system through a vacuum rubber tube, and the mass spectrometer interface 3 is connected to the gas isotope mass spectrometer; the fragile part 4 of the glass reaction tube is placed in the round hole 5, and the fragile part 4 of the reaction tube is broken off by rotation and the gas is released, and the sealing male connector 6 is connected to the sealing female connector 7 for sealing.

[0036] Stable isotope-labeled acetic acid reacts under vacuum and high temperature conditions: vacuum degree <10 Pa, reaction temperature 550-950℃, and reaction time 2-6 h. To prevent the stable isotope-labeled acetic acid from being removed during the vacuuming process, liquid nitrogen prepared with alcohol is used to freeze the stable isotope-labeled acetic acid before the vacuum is activated. The acetic acid decomposes to produce stable isotope-labeled methane or carbon dioxide, which can be measured. 13 C-labeled acetic acid (CH3) at the carboxyl site 13 COOH) 13 C isotope abundance, 13 C-labeled acetic acid at the methyl site ( 13 CH3COOH) 13 C isotope abundance, 18 O-labeled acetic acid (CH3C) 18 O 18 OH) 18 O isotope abundance, methyl deuterium isotope abundance in deuterium-labeled acetic acid (CD3COOH) at methyl sites;

[0037] Measurement 13 C-labeled acetic acid (CH3) at the carboxyl site 13 COOH) 13 During the C isotope abundance process: the contents of the reaction tube... 13 CO2 was introduced into a gas isotope mass spectrometer to detect the ion current intensities of mass numbers 44 and 45.

[0038] Through formula Calculate the carboxyl site 13 C isotope abundance;

[0039] Measurement 13 C-labeled acetic acid at the methyl site ( 13 CH3COOH) 13 During the C isotope abundance process: the contents of the reaction tube... 13 CH4 was introduced into a gas isotope mass spectrometer to detect the intensities of ions with mass numbers of 16 and 17.

[0040] Through formula Calculation of methyl sites 13 C isotope abundance;

[0041] Measurement 18 O-labeled acetic acid (CH3C) 18 O 18 OH) 18 O isotope abundance process: The C in the reaction tube... 18 O2 was introduced into the gas isotope mass spectrometer to detect the ion current intensities of mass numbers 46 and 48.

[0042] Through formula calculate 18 O isotope abundance;

[0043] In determining the abundance of methyl deuterium isotopes in deuterium-labeled acetic acid (CD3COOH) with methyl sites: CD4 from the reaction tube was introduced into a gas isotope mass spectrometer, and the ion current intensities with mass numbers of 18, 19, and 20 were detected.

[0044] Through formula Calculate the abundance of methyl deuterium isotopes;

[0045] Example 1

[0046] 13 C-labeled acetic acid at the carboxyl site (commercially available chemical reagent), 13 C isotope abundance: 99.1 atom%. 13 C) 13 C isotope abundance determination includes the following steps:

[0047] The reaction apparatus used in this embodiment is as follows: Figure 1 The reaction tube shown, such as Figure 2 The sample inlet tube shown.

[0048] Approximately 10 μL of sample was added to the reaction tube. Liquid nitrogen, prepared with alcohol, was used to freeze the sample in the reaction tube before vacuuming was initiated. When the vacuum level was <10 Pa, the sample reaction tube was sealed at point 2 using a high-temperature spray gun. The sealed reaction tube was then placed in a muffle furnace at 800 °C for high-temperature decomposition reaction. After 2 hours, it was cooled to room temperature. The sealed sample reaction tube was then placed into the injection tube, and the injection tube was connected to the gas isotope mass spectrometer via interface 3. Vacuuming was initiated, and the fragile part of the reaction tube was broken off and the sample released through the rotating circular hole 5. 13 CO2 gas, 13 CO2 gas was introduced into the gas isotope mass spectrometer to detect the ion current intensities with mass numbers of 44 and 45.

[0049] Through formula Calculate the carboxyl site 13 C isotope abundance;

[0050] 99.21% respectively 13 C, 99.26% 13 C, average 99.2% 13 C.

[0051] Compare the above test data with 13 The values ​​of the acetic acid reagent labeled with C at the carboxyl site were compared, and the results were comparable. It can be seen that the isotope abundance determination method of the present invention also has very high measurement accuracy.

[0052] Example 2

[0053] 13 C-labeled acetic acid at the methyl site (commercially available chemical reagent, 13 C isotope abundance: 99.0 atom%. 13 C) 13 C isotope abundance determination includes the following steps:

[0054] Add approximately 5 μL of sample to the reaction tube. First, freeze the sample in the reaction tube using liquid nitrogen prepared with alcohol, then begin evacuation. When the vacuum level is <10 Pa, use a high-temperature spray gun to seal the sample reaction tube at point 2. Place the sealed reaction tube in a muffle furnace at 600 °C for high-temperature decomposition reaction. After 6 hours, cool to room temperature. Place the sealed sample reaction tube into the injection tube, connect the injection tube to the gas isotope mass spectrometer via interface 3, evacuate, and break off the fragile part of the reaction tube through the rotating circular hole 5 to release the sample. 13 CH4 gas will purify the gas inside the reaction tube. 13 CH4 was introduced into a gas isotope mass spectrometer to detect the ion current intensities with mass numbers of 16 and 17.

[0055] Through formula Calculation of methyl sites13 C isotope abundance;

[0056] 99.06% respectively 13 C, 99.02% 13 C, average 99.0% 13 C.

[0057] Compare the above test data with 13 The values ​​of the acetic acid reagent labeled with C at the methyl site were compared, and the results were comparable. It can be seen that the isotope abundance determination method of the present invention also has very high measurement accuracy.

[0058] Example 3

[0059] 18 O-labeled acetic acid (commercially available chemical reagent) 18 O isotope abundance ≥ 95.0 atom%. 18 O) 18 O isotope abundance determination includes the following steps:

[0060] Approximately 8 μL of sample was added to the reaction tube. Liquid nitrogen, prepared with alcohol, was used to freeze the sample in the reaction tube before vacuuming was initiated. When the vacuum level was <10 Pa, the sample reaction tube was sealed at point 2 using a high-temperature spray gun. The sealed reaction tube was then placed in a muffle furnace at 950 °C for high-temperature decomposition reaction. After 4 hours, it was cooled to room temperature. The sealed sample reaction tube was then placed into the injection tube, and the mass spectrometer interface 3 of the injection tube was connected to a gas isotope mass spectrometer. Vacuum was applied, and the fragile part of the reaction tube was broken off through the rotating circular hole 5 to release carbon. 18 O2 gas, C 18 O2 was introduced into the gas isotope mass spectrometer to detect the ion current intensities of mass numbers 46 and 48.

[0061] Through formula calculate 18 O isotope abundance;

[0062] 96.70 atom% respectively 18 O, 96.59 atom% 18 O, average 96.6% 18 O.

[0063] Compare the above test data with commercially available chemical reagents 18 The results of comparing the labeling values ​​of O-labeled acetic acid are comparable, demonstrating that the isotope abundance determination method of the present invention also has very high measurement accuracy.

[0064] Example 4

[0065] The determination of deuterium isotope abundance in deuterium-labeled acetic acid (commercially available chemical reagent CD3COOD, deuterium isotope abundance value 99.7 attom% D) with methyl sites includes the following steps:

[0066] Approximately 20 μL of sample was added to the reaction tube. Liquid nitrogen, prepared with alcohol, was used to freeze the sample in the reaction tube before vacuuming was initiated. When the vacuum level was <10 Pa, the sample reaction tube was sealed at point 2 using a high-temperature spray gun. The sealed reaction tube was then placed in a muffle furnace at 550 °C for high-temperature decomposition reaction. After 3 hours, it was cooled to room temperature. The sealed sample reaction tube was then placed into the injection tube, and the mass spectrometer interface 3 of the injection tube was connected to the gas isotope mass spectrometer. Vacuum was applied, and the fragile part of the reaction tube was broken off through the rotating circular hole 5 to release CD4 gas. The CD4 gas was introduced into the gas isotope mass spectrometer, and the ion current intensities at mass numbers 18, 19, and 20 were detected.

[0067] Through formula Calculate the abundance of methyl deuterium isotopes;

[0068] The values ​​were 99.68% atom% D and 99.69% atom% D, respectively, with an average of 99.7% atom% D.

[0069] The test data were compared with the labeled values ​​of commercially available chemical reagent deuterated acetic acid (CD3COOD), and the results were comparable. It can be seen that the isotope abundance determination method of the present invention also has very high measurement accuracy.

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for detecting the isotopic abundance in acetic acid, characterized in that, The method includes the following steps: The stable isotope-labeled acetic acid is decomposed to generate a stable isotope-labeled gas. The gas is introduced into a gas isotope mass spectrometer to detect the ion current intensity; After calculation using the formula, the isotopic abundance in stable isotope-labeled acetic acid was determined. The stable isotope-labeled acetic acid includes 13 C-labeled acetic acid CH3 at the carboxyl site 13 COOH, 13 C-labeled acetic acid at the methyl site 13 CH3COOH, 18 O-labeled acetic acid CH3C 18 O 18 OH or deuterium-labeled acetic acid CD3COOH at the methyl site; The measured isotopic abundances are as follows: 13 C-labeled acetic acid CH3 at the carboxyl site 13 COOH 13 C isotope abundance, 13 C-labeled acetic acid at the methyl site 13 CH3COOH 13 C isotope abundance, 18 O-labeled acetic acid CH3C 18 O 18 OH 18 O isotope abundance or methyl deuterium isotope abundance in acetic acid CD3COOH labeled with methyl sites; Measurement 13 C-labeled acetic acid CH3 at the carboxyl site 13 COOH 13 In the process of C isotope abundance: 13 CO2 was introduced into a gas isotope mass spectrometer to detect the ion current intensities of mass numbers 44 and 45. Through formula Calculate the carboxyl site 13 C isotope abundance; Measurement 13 C-labeled acetic acid at the methyl site 13 CH3COOH 13 In the process of C isotope abundance: 13 CH4 was introduced into a gas isotope mass spectrometer to detect the intensities of ions with mass numbers of 16 and 17. Through formula Calculation of methyl sites 13 C isotope abundance; Measurement 18 O-labeled acetic acid CH3C 18 O 18 OH 18 O isotope abundance process: C 18 O2 was introduced into the gas isotope mass spectrometer to detect the ion current intensities of mass numbers 46 and 48. Through formula calculate 18 O isotope abundance; In determining the abundance of methyl deuterium isotopes in deuterium-labeled acetic acid CD3COOH with methyl sites: CD4 was introduced into a gas isotope mass spectrometer, and the ion current intensities with mass numbers of 18, 19, and 20 were detected. Through formula Calculate the abundance of methyl deuterium isotopes.

2. The method for detecting isotopic abundance in acetic acid according to claim 1, characterized in that, The gas in question is methane or carbon dioxide.

3. The method for detecting isotopic abundance in acetic acid according to claim 1, characterized in that, The decomposition reaction is carried out at a temperature of 550-950℃ for 2-6 hours, with a vacuum degree of <10 Pa.

4. The method for detecting isotopic abundance in acetic acid according to claim 1, characterized in that, Before the decomposition reaction, the stable isotope-labeled acetic acid is frozen before the vacuum is activated.

5. The method for detecting isotopic abundance in acetic acid according to claim 4, characterized in that, Stable isotope-labeled acetic acid was frozen using liquid nitrogen prepared with alcohol.

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