A method for measuring sample δ based on EA-IRMS 13 C-value method

Abstract

The present application relates to a kind of based on EA-IRMS measurement sample δ 13 C method, comprising the following steps: dilution is carried out to the sample to be measured to obtain sample aqueous solution, and a predetermined volume of sample aqueous solution is placed in a vessel to carry out drying treatment;The δ 13 C value of the sample after pretreatment is measured by using element analysis-isotope mass spectrometer instrument, and a plurality of δ 13 C measurement value is obtained after calibration by standard curve;The plurality of δ 13 C measurement value is drift corrected based on the instrument drift function determined in advance, and a plurality of corrected sample δ 13 C value is obtained, and the final sample δ 13 C value is obtained after averaging. The present application can ensure that the mass deviation of the sample is smaller by using dissolution technology to prepare the sample, so as to improve the measurement accuracy of the final sample δ 13 C value;The present application can further determine the minimum sample peak value and drift correct the EA-IRMS measurement, to improve the result accuracy.

Description

Technical Field

[0001] This invention relates to sample δ 13 In the field of C-value measurement technology, this invention relates in particular to a method for measuring sample δ based on EA-IRMS. 13 The method of C value. Background Technology

[0002] Plants exhibit different photosynthetic cycles, resulting in varying carbon isotope mass fractionation effects. Based on the different carbon cycle pathways in photosynthesis, these can be categorized into the C-3 cycle, the C-4 cycle, and the Crassulacean acid metabolism cycle (CAM). Different photosynthetic processes lead to different carbon isotope ratios; for example, the δ¹⁸O values ​​of plants undergoing the C-3, C-4, and CAM cycles are significantly different. 13 The C values ​​range from -22‰ to -32‰, -8‰ to -16‰, and -11‰ to -13.5‰, respectively. Bees primarily produce honey from plants with a C-3 cycle, while adulterated honey is made by adding C-4 sugars (such as sucrose). Therefore, elemental analysis-isotope ratio mass spectrometry (EA-IRMS) has been developed and is widely used.

[0003] However, the EA-IRMS technology still has the following problems: (1) For EA-IRMS technology, the sample amount can be less than 1 mg, which means that a very small error during sampling will lead to a large result deviation, or even a completely wrong analysis result.

[0004] Therefore, to address the above shortcomings, there is a need to provide a method for measuring the δ of a sample. 13 The C-value method effectively improves δ 13 The accuracy of C-value measurement. Summary of the Invention

[0005] The technical problem to be solved by this invention is that, in view of the prior art, δ 13 To address the drawback of low accuracy in C-value detection, a method based on EA-IRMS for measuring sample δ is provided. 13 The C-value method, through improved sample preparation methods, can increase the δ value. 13 The accuracy of C-value measurement.

[0006] To address the aforementioned technical problems, this invention provides a method for measuring sample δ based on EA-IRMS. 13 The method for calculating the C value includes the following steps:

[0007] The sample pretreatment steps include diluting the sample to be tested to obtain a sample aqueous solution, and taking a predetermined volume of the sample aqueous solution and placing it in a container for drying.

[0008] Measurement steps: Elemental analysis-isotope mass spectrometry was used to analyze the δ-values ​​of the pretreated sample. 13The C value was measured, and multiple δ values ​​were obtained after calibration using a standard curve. 13 C measurement value;

[0009] The drift correction step, based on a predetermined instrument drift function, applies the following to the multiple δ values. 13 The C measurement value was adjusted for drift correction to obtain multiple corrected sample δ values. 13 The C value, after averaging, yields the final sample δ. 13 C value.

[0010] In the EA-IRMS-based sample δ measurement according to the present invention 13 In the C-value method, preferably, the sample to be tested is honey, and the sample pretreatment step includes:

[0011] The honey sample was liquefied in a water bath at 55-65℃ for 8-12 minutes, then homogenized in a homogenizer for 2-3 minutes, and then ultrasonicated at (55-65)℃ for 4-6 minutes for further homogenization.

[0012] The homogenized honey sample was dissolved in degassed ultrapure water to prepare a solution with a concentration of 45–55 mg / g. -1 The solution was sampled at room temperature using a microsyringe and placed in a silver bag, then dried in an oven at 55-65°C for 1.5-2.5 hours.

[0013] In the EA-IRMS-based sample δ measurement according to the present invention 13 In the C-value method, preferably, the amount of sample aqueous solution taken in the sample pretreatment step is determined according to the following steps:

[0014] (1) The δ-values ​​of the standard substance were measured in advance using the same elemental analysis-isotope mass spectrometry instrument and matching silver bag. 13 The C value is used to obtain the original measured value δ of the standard substance. 13 C M And measure the δ of the blank sample. 13 The C-value and peak area are used to obtain the original measured value δ of the blank sample. 13 C B and blank sample peak area Area B ;

[0015] (2) Determine the blank correction formula:

[0016]

[0017] Where y is the blank-corrected sample δ 13 C value; x is the peak area ratio, representing Area M Indicates the peak area of ​​the standard substance;

[0018] (3) Obtain the functional relationship between the sample measurement difference D and the peak area ratio according to the blank correction formula, and determine the minimum peak area ratio according to the pre-set reliability upper limit; wherein the sample measurement difference D refers to the original measurement value δ of the standard substance. 13 C M δ of the blank-corrected sample 13 The difference between C and y;

[0019] (4) Based on the minimum peak area ratio and the peak area of ​​the blank sample Area B Obtain the minimum sample peak area and calculate the sample quantity based on the minimum sample peak area.

[0020] In the EA-IRMS-based sample δ measurement according to the present invention 13 In the method for determining the C value, preferably, the blank correction formula is:

[0021]

[0022] In the EA-IRMS-based sample δ measurement according to the present invention 13 In the C-value method, preferably, the pre-set reliable upper limit of the sample measurement difference is -0.03‰, and the calculated minimum peak area ratio is 100.

[0023] In the EA-IRMS-based sample δ measurement according to the present invention 13 In the C-value method, preferably, the drift correction step includes:

[0024] (1) Using the same elemental analysis-isotope mass spectrometry instrument and matching silver bag, the δ-values ​​of multiple batches of quality control standard substances were continuously measured in advance. 13 The C value was obtained, and multiple δ values ​​were obtained after calibration using a standard curve. 13 C is measured, and δ is calculated for each batch. 13 C-difference:

[0025] Y i =ref.–V i

[0026] Among them, Y i For the i-th batch δ 13 C-difference, ref. is the δ value of the standard substance used for quality control. 13 C standard value; V i δ for the quality control standard substance of the i-th batch 13 C measurement value;

[0027] (2) δ corresponding to multiple batches 13 C difference Y i As the dependent variable, batch number Xi As the independent variable, the batch number and δ were fitted to obtain the results. 13 C-value of the instrument drift function;

[0028] (3) Enter the batch number x of the sample to be tested k Substituting the values ​​into the instrument drift function, we obtain the drift correction difference y. k Then the δ of the sample to be tested 13 C measured value v k and drift correction difference y k Add them together to obtain the corrected sample δ. 13 C value.

[0029] In the EA-IRMS-based sample δ measurement according to the present invention 13 In the method for determining the C value, preferably, the instrument drift function is:

[0030] y k =a×x k 2 +b×x k +c;

[0031] Where a, b, and c are the fitted parameters, x k Let y be the batch number of the kth batch of samples to be tested. k This represents the drift correction difference for the kth batch of samples to be tested.

[0032] In the EA-IRMS-based sample δ measurement according to the present invention 13 In the method for determining the C value, preferably, the method further includes the following steps:

[0033] The uncertainty assessment steps are as follows: Calculate the combined relative uncertainty based on the relative uncertainties of repeated measurements, the relative uncertainties of the standard substance, and the relative uncertainties of the standard curve; then obtain the sample δ based on the combined relative uncertainty. 13 The absolute uncertainty of the C value.

[0034] Implementing the present invention for measuring sample δ based on EA-IRMS 13 The C-value method has the following characteristics:

[0035] Beneficial effects:

[0036] 1. This invention uses a dissolution technique to prepare samples. By diluting the sample to be tested to a certain concentration and then using a micro-syringe to extract the sample, it is possible to ensure smaller deviations in the mass of the weighed sample, thereby improving the final sample's δ value. 13 The measurement accuracy of the C value;

[0037] 2. The present invention also determines the minimum sample peak value, which ensures the detection effect while reducing the sample concentration to ensure sampling accuracy;

[0038] 3. This invention also performs drift correction on EA-IRMS measurements. By using a fitted instrument drift function, the corrected results are closer to the average value, further improving the accuracy of the results. Attached Figure Description

[0039] Figure 1 To measure the δ of a sample based on EA-IRMS according to a preferred embodiment of the present invention 13 Flowchart of the C-value method;

[0040] Figure 2 This is a hyperbola corresponding to the blank correction formula in the embodiment of the present invention;

[0041] Figure 3 This is a graph showing the functional relationship between the difference in incremental values ​​obtained by the blank correction formula in this embodiment of the invention and the peak area ratio;

[0042] Figure 4 To repeatedly measure δ according to Embodiment 1 and Comparative Example 1 of the present invention 13 Distribution graph of C measurement values;

[0043] Figure 5 The final sample δ of the 14 / R1 / A standard material measured according to Example 2 of the present invention. 13 A comparison chart of the C-value with values ​​reported by several other institutions. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] This invention provides a method for measuring sample δ based on EA-IRMS. 13 The C-value method is an improvement on the traditional elemental analysis-isotope ratio mass spectrometry method, used to determine the δ of a sample. 13 C value, especially δ value of honey samples 13 The value of C. Please refer to [link / reference]. Figure 1 To measure the δ of a sample based on EA-IRMS according to a preferred embodiment of the present invention 13 The flowchart for the C-value method is shown below. Figure 1 As shown, the method of the present invention includes the following steps:

[0046] First, in step S1, a sample pretreatment step is performed to dilute the sample to be tested to obtain a sample aqueous solution, and a predetermined volume of the sample aqueous solution is placed in a container for drying.

[0047] Preferably, the sample to be tested is honey, and step S1 specifically includes:

[0048] (1) Place the honey sample in a water bath at 55℃-65℃ (e.g., 55℃, 60℃ or 65℃) for liquefaction for 8-12 min (e.g., 8 min, 10 min, 12 min), then homogenize it in a homogenizer for 2-3 min (e.g., 2 min, 3 min), and then sonicate the honey sample at 55-65℃ (e.g., 55℃, 60℃ or 65℃) for 4-6 min (e.g., 4 min, 5 min, 6 min) to further homogenize it;

[0049] (2) Dissolve the homogenized honey sample in degassed ultrapure water to prepare a solution with a concentration of 45–55 mg / g. -1 (e.g. 45mg g) -1 50mg g -1 55mg g -1 The solution is prepared by sampling it using a microinjector at room temperature and placing it in a container (e.g., a silver pouch). The sample is then dried in an oven at 55-65°C (e.g., 55°C, 60°C, or 65°C) for 1.5-2.5 hours (e.g., 1.5h, 2h, 2.5h). In this step, the prepared solution can be sampled multiple times using a microinjector, injecting the liquid into different silver pouches from the same batch for subsequent instrument measurements.

[0050] Subsequently, in step S2, a measurement step is performed, using an elemental analysis-isotope mass spectrometry instrument to analyze the δ-values ​​of the pretreated sample. 13 The C value was measured, and multiple δ values ​​were obtained after calibration using a standard curve. 13 C measurement value.

[0051] In this step, the prepared silver capsule can be sent into an elemental analysis-isotope mass spectrometry instrument composed of an isotope ratio mass spectrometer (IRMS) and an elemental analyzer (EA) to analyze the sample's δ-axis. 13 The C value is measured to obtain the original measured value δ. 13 C M After calibration using a pre-made standard curve, δ is obtained. 13 C is the measured value v. In this step, the samples in each silver pouch can be measured sequentially to obtain M δ values. 13 C is the measured value v1, v2...v M Among them, the δ of the kth batch 13 The measured value of C is v k1 ≤ k ≤ M, where M is the number of times the sample is repeatedly measured. Finally, in step S3, a drift correction step is performed, adjusting the multiple δ values ​​based on a predetermined instrument drift function. 13 The C-measurement value was used to correct for sample drift, resulting in multiple corrected sample δ values. 13 The C value, after averaging, yields the final sample δ. 13 C value δ 13 C0.

[0052] This invention employs a dissolution technique to prepare samples. By diluting the sample to be tested to a certain concentration and then using a microsyringe for sampling, it is possible to ensure smaller deviations in the mass of the weighed sample, thereby improving the final sample's δ value. 13 The accuracy of C-value measurement.

[0053] Based on the above embodiments, the present invention further considers that after diluting the sample, the sample concentration will decrease. If the concentration is too low, the sample mass obtained from the silver capsule will be too small, and the measurement results will not be satisfactory. Therefore, how to ensure the detection effect while reducing the sample concentration is a challenge faced by those skilled in the art. In a more preferred embodiment of the present invention, a scheme for determining the minimum sample peak value to calculate the sample concentration and sampling volume is also studied.

[0054] The amount of sample aqueous solution taken in the above sample pretreatment step S2 is determined according to the following steps:

[0055] (1) The δ-values ​​of the standard substance were measured in advance using the same elemental analysis-isotope mass spectrometry instrument and matching silver bag. 13 The C value is used to obtain the original measured value δ of the standard substance. 13 C M And measure the δ of the blank sample. 13 The C-value and peak area are used to obtain the original measured value δ of the blank sample. 13 C B and blank sample peak area Area B For example, CCQM K140 can be selected as the standard substance. After dissolving the sample using the aforementioned sample pretreatment steps, samples are taken from the same batch of silver capsules, and the original measured value δ of the standard substance is measured. 13 C M The raw measurement value is the instrument's original measurement value, without data processing such as standard curve calibration and drift correction. This process allows for multiple repeated measurements of the same sample, obtaining multiple measurement data points. The average value is then calculated to obtain the raw measurement value δ of the standard substance. 13 C M Simultaneously, an empty silver capsule (without any sample) was used as a blank sample, and its δ value was measured. 13 From the C value and peak area, the corresponding original measured value δ of the blank sample is obtained. 13 CB and blank sample peak area Area B .

[0056] (2) Determine the blank correction formula:

[0057]

[0058] Where y is the blank-corrected sample δ 13 C value; x is the peak area ratio, representing Area M Indicates the peak area of ​​the standard substance.

[0059] In existing technologies, the average peak area and δ are typically measured using blank measurements. 13 The C value is used to correct blank data, and the formula is as follows:

[0060]

[0061] Among them, Area M and Area B These represent the peak areas of the standard substance and the blank sample, respectively, δ 13 C M The original measured value of the standard substance, δ 13 C B These are the original measurements for the blank sample.

[0062] During the research process, the inventors discovered that the correction effect of the above formula (2) is very small and unpredictable. Therefore, this invention defines the empirical minimum sample peak based on the intensity of the average blank area. The numerator and denominator on the right side of formula (2) are both divided by Area. B Then respectively Defined as x, formula (2) can be simplified to the aforementioned formula (1). Substituting the parameters measured in advance in step (1) into the formula, we can obtain the specific equation.

[0063] For example, in one embodiment, the original measured value δ of the standard substance was obtained using CCQM K140 as a sample. 13 C M The value is (-35.254±0.048)‰ (n=175); the original measured value δ of the blank sample. 13 C B The value is (-37.928±3.093)‰ (n=35). n is the number of repeated measurements. Substituting into formula (1), the simplified blank correction formula is obtained as follows:

[0064]

[0065] The relationship between x and y in the above blank correction formula can be approximated by a hyperbola, for example... Figure 2 The hyperbola approaches but cannot reach -35.254‰, meaning the measured value of honey CCQM K140 is -35.254‰.

[0066] (3) Obtain the functional relationship between the sample measurement difference D and the peak area ratio according to the blank correction formula, and determine the minimum peak area ratio x according to the pre-set reliable upper limit of the sample measurement difference. min ; .

[0067] Specifically, a series of x values ​​can be set, and a series of y values ​​can be obtained according to the blank correction formula, with the sample measurement difference D (D = δ) being the result. 13 C M The ratio of -y=-35.254-y) to the peak area, i.e., x, can be obtained as follows: Figure 3 The relationship diagram is shown. Then, the minimum peak area ratio is determined based on a pre-set reliability upper limit; that is, the peak area ratio corresponding to the sample measurement difference D equaling this reliability upper limit is the determined minimum peak area ratio. For example, when x = 50, the calibrated δ... 13 The C value yields a difference of -0.048‰, which is equal to the δ value in CCQM K140 honey. 13 The standard deviation of C over 175 repeated measurements, which represents δ 13 C is basically reliable. In a more preferred embodiment of the invention, the preset reliability upper limit is -0.03‰, thereby obtaining the minimum peak area ratio x. min Setting it to 100 ensures that the sample measurement difference D is less than or equal to the preset reliability upper limit of -0.03‰.

[0068] (4) Based on the minimum peak area ratio x min and the peak area of ​​the blank sample Area B Obtain the minimum sample peak area and calculate the sample quantity based on the minimum sample peak area.

[0069] Among them, Area M_min =Area B ×x min (4)

[0070] In the above embodiments, the peak area Area of ​​the blank sample was measured. B The value is 30 mV (n = 35). Therefore, the minimum sample peak area is calculated to be 3000 mV. In all embodiments of this invention, samples are taken using a peak value of 3000 mV. Those skilled in the art can control the sampling amount by controlling the volume and concentration of the sample aqueous solution based on this target sample peak area.

[0071] During the measurement process, this invention discovered that the sample δ 13The C-value detection result can vary over time (or batch number), causing a shift in the result. Existing technologies do not account for this bias, leading to inaccurate results. This drift can be caused by various factors, including changes in instrument conditions for elemental analysis-isotope mass spectrometry, the accumulation of water or other impurities, and deterioration of ion source conditions. Therefore, in a more preferred embodiment of the present invention, drift correction is also performed on the instrument and its accompanying silver bag over time.

[0072] The drift correction steps include:

[0073] (1) Using the same elemental analysis-isotope mass spectrometry instrument and matching silver bag, the δ-values ​​of multiple batches of quality control standard substances were continuously measured in advance. 13 The C value was obtained, and multiple δ values ​​were obtained after calibration using a standard curve. 13 C is measured, and δ is calculated for each batch. 13 C difference Y i ;

[0074] Y i =ref.–V i (5)

[0075] Among them, Y i For the i-th batch δ 13 C-difference, ref. is the δ value of the standard substance used for quality control. 13 C standard value; V i δ for the quality control standard substance of the i-th batch 13 C measurement value.

[0076] This invention can use IAEA 600 (caffeine, International Atomic Energy Agency, Vienna, Austria) as a quality control standard for drift correction. For example, multiple sets of IAEA 600 samples are prepared following the same sample pretreatment steps as in step S1, and samples of the same mass are taken using the same silver sac. The obtained multiple standard sample (IAEA 600) are sequentially sent to an elemental analysis-isotope mass spectrometry instrument, and the instrument assigns batch numbers X1, X2, ... X to each set of standard sample according to the measurement time. N In other words, the batch number is determined based on the measurement time and increases over time. Therefore, the δ of N batches of standard substances can be obtained. 13 C is the measured value V1, V2, ... V N δ of the i-th batch 13 The measured value of C is V. i 1≤i≤N, where N is the number of times the standard substance is measured repeatedly. Table 1 below shows the difference calculation table for each batch according to the present invention.

[0077] Table 1

[0078]

[0079] (2) δ corresponding to multiple batches 13 C difference Y i As the dependent variable, batch number X i The batch number and δ were obtained by fitting the data as independent variables. 13 C is the instrument drift function of the difference. Where Y... i This represents the δ of the i-th batch calculated in step (1). 13 C difference, X i This represents the batch number of the i-th batch.

[0080] This invention can fit the batch number X in Table 1 using linear or polynomial methods. i and δ 13 C difference Y i The relationship. Preferably, a function fitting is used:

[0081] Y i =a×X i 2 +b×X i +c; (6)

[0082] Where a, b, and c are the fitted parameters, representing the slope and intercept, respectively.

[0083] Therefore, the instrument drift function can be obtained as follows:

[0084] y k =a×x k 2 +b×x k +c;

[0085] x k Let y be the batch number of the kth batch of samples to be tested. k This represents the drift correction difference for the kth batch of samples to be tested.

[0086] (3) Enter the batch number x of the sample to be tested k Substituting the above instrument drift function, we obtain the drift correction difference y. k Then the δ of the sample to be tested 13 C measured value v k and drift correction difference y k Add them together to obtain the corrected sample δ. 13 C value.

[0087] For example, when formally measuring the samples to be tested, the elemental analysis-isotope mass spectrometry instrument assigns batch numbers x1, x2, ... x to the samples in each silver capsule according to the same numbering rule. MTherefore, the δ values ​​of M batches of the test samples after calibration with the standard curve can be obtained. 13 C's δ 13 C measured values ​​v1, v2, ... v M The batch numbers x1, x2, ... x are sequentially assigned. M Substituting the values ​​into the instrument drift function, we obtain the drift correction differences y1, y2, ... y M Finally, the corrected sample δ of the i-th batch is obtained. 13 The value of C is: v i +(a×x i 2 +b×x i +c). This allows us to obtain the corrected sample δ for M batches. 13 C value. Table 2 below is the drift correction calculation table according to the present invention.

[0088] Table 2

[0089]

[0090] Calculate the δ values ​​of the M corrected samples mentioned above. 13 The average value of C can be used to obtain the final sample δ. 13 C value.

[0091] This invention demonstrates through experiments the measurement of sample δ based on EA-IRMS. 13 The results of the C-value test were verified.

[0092] Experimental instruments

[0093] The δ of the present invention 13 C analysis was performed using an elemental analysis-isotope mass spectrometry (IRMS) instrument, consisting of an isotope ratio mass spectrometer (IRMS, model MAT 253, manufactured in Germany) and an elemental analyzer (EA, model flash 2000, manufactured by Thermo Fisher Scientific, Germany). The two instruments are connected via Thermo Fisher Scientific's ConFlo IV universal continuous flow interface. The EA is equipped with a Porapack QS GC column (3m × 4mm inner diameter). All samples and standards were measured under pulsed working gas conditions. The standard deviation (SD) of the working gas CO2 was less than 0.02‰ (n = 10).

[0094] Experimental reagents

[0095] This invention uses NIST 8540 (polyethylene foil), NIST 8542 (sucrose), and NIST 8573 (L-glutamic acid), distributed by the National Institute of Standards and Technology (NIST), as standard materials for standard curve calibration. Simultaneously, IAEA 600 (caffeine, provided by the International Atomic Energy Agency, Austria) is used as a quality control standard material for drift correction. The δ¹⁸O values ​​of NIST 8540 (polyethylene foil), NIST 8542 (sucrose), NIST 8573 (L-glutamic acid), and IAEA 600 (caffeine) are... 13 The standard values ​​for C are (-32.15±0.10)‰, (-10.45±0.07)‰, (-26.39±0.09)‰ and (-27.771±0.043)‰, respectively.

[0096] In this invention, two honey samples were used as test samples to verify the results: 14 / R1 / A and CCQM K 140. 14 / R1 / A is a sample from an interlaboratory comparison in China in 2014. CCQM K140 30 is a sample from an international comparison by the Advisory Committee on Mass of the International Bureau of Weights and Measures in 2015.

[0097] δ 13 C measurement method

[0098] Weigh the sample to be tested or the required standard substance and add it to a small silver capsule (3mm × 2mm × 5mm). Wrap the silver capsule and place it in a desiccator to remove moisture. After the material is completely burned into carbon dioxide, use EA-IRMS to analyze the δ-ray concentration of the material. 13 Carbon isotope measurements were performed in the reactor using chromium oxide, silver-plated cobalt-cobalt oxide, and copper. The reactor was heated to 1000°C, and the chromatograph oven temperature was set to 50°C. The carrier gas (e.g., He) flow rate was set to 100 mL / min. -1 The O2 flow rate is 200 mL / min. -1 At the start of each measurement, three pulses of CO2 working gas are introduced (each lasting 20 seconds). Stable carbon isotope values ​​are typically represented by δ, i.e., the carbon isotope concentration of the sample. 13 C / 12 C isotope ratio relative to VPDB standard 13 C / 12 The relative value of C is usually expressed as a difference of thousands (‰).

[0099] The measured values ​​δ of two or more standard substances 13 By fitting a curve between C and the corresponding standard value, a univariate linear equation between the standard value and the measured value can be obtained, which can be used as a standard curve for calibration. Based on the equation, if the measured value δ of the sample to be tested is known... 13 C, then its correction value can be calculated directly.

[0100] Comparative Example 1

[0101] The 14 / R1 / A standard substance was used as a honey sample. It was liquefied in a 60°C water bath for 10 minutes, then homogenized in a vortex mixer for 2 minutes, and then further homogenized in a 60°C ultrasonic water bath for 5 minutes. The homogenized sample was stored in a 60°C water bath, and 100 μg of the sample was weighed and placed in a silver bag. During this period, the sample in the water bath was shaken vigorously by hand periodically. The method of this embodiment is also known as the heating sample preparation method.

[0102] The δ-values ​​of the sample in the silver pouch were measured using the aforementioned elemental analysis-isotope mass spectrometry instrument. 13 The C value was obtained by calibrating using a standard curve, and δ was then calculated. 13 C measurement value. Nine sets of measurements were repeated to obtain nine δ values. 13 C measurement value (uncorrected for drift).

[0103] Example 1

[0104] Take 14 / R1 / A standard material as honey sample, place it in a 60℃ water bath for 10 min to liquefy, then homogenize it in a vortex mixer for 2 min, and then treat it in a 60℃ ultrasonic water bath for 5 min to further homogenize it.

[0105] The homogenized honey sample was dissolved in degassed ultrapure water (Mill-Q) to prepare a honey concentration of 50 mg / g. -1 The solution was prepared by taking 2 μL of the solution using a microsyringe and placing it in a silver bag at room temperature and in the dark. The bag was then dried in an oven at 60°C for 2 hours. This method is also known as the dissolution sample preparation method.

[0106] The δ-values ​​of the sample in the silver pouch were measured using the aforementioned elemental analysis-isotope mass spectrometry instrument. 13 The C value was obtained by calibrating using a standard curve, and δ was then calculated. 13 C measurement value. Nine sets of measurements were repeated to obtain nine δ values. 13 C measurement value (uncorrected for drift).

[0107] Some substances in honey crystallize gradually at lower temperatures, and these substances have significantly different isotopic compositions. For example, in pure honey, glucose has a δ¹⁸O₂ concentration... 13 The C value ranges from -22‰ to -17‰, while reports indicate that the δ values ​​of proteins and honey... 13 The difference in C values ​​ranges from 1.2‰ to 3.5‰. Therefore, slight differences in sample preparation can lead to variations in δ values. 13 The C-value varies considerably, and the standard deviation between test results is large. Please refer to [link / reference]. Figure 4 δ is the result of repeated measurements according to Embodiment 1 and Comparative Example 1 of the present invention.13 The distribution of C measurements is shown in the figure. The figure illustrates the distribution of the nine δ values ​​measured in Comparative Example 1 above. 13 The average value of C measured was -20.78‰. The nine δ values ​​measured in Example 1... 13 The average value of C measurements is -20.79‰; where δ 13 The C measurement value was not adjusted for drift. Although the δ values ​​of the samples obtained by the two different sample preparation methods were... 13 The average value of C measurements does not differ significantly, but from Figure 4 It can be seen that the standard deviation of repeated measurements of the sample prepared in Comparative Example 1 is significantly higher than that in Example 1. This is because:

[0108] (1) Compared with viscous solutions, fluid solutions are easier to distribute uniformly and achieve homogeneity.

[0109] (2) Compared with the weighing method for viscous solutions, it is easier to weigh the mass of fluid solutions.

[0110] In summary, compared with the dissolution sample preparation method in Example 1, the heating sample preparation method in Comparative Example 1 has a larger standard deviation, which can lead to inaccurate measurement results if the number of repeated measurements is not sufficient.

[0111] Example 2

[0112] The sample pretreatment and measurement steps in Example 2 are the same as in Example 1. The difference is that the corrected sample δ is obtained through the following drift correction step. 13 C value:

[0113] (1) Using the same elemental analysis-isotope mass spectrometry instrument and matching silver capsule, IAEA 600 samples were prepared according to the same sample pretreatment steps as in Example 1, and 100 μg of sample was taken using the same batch of silver capsules. The obtained IAEA 600 samples were sequentially sent to the elemental analysis-isotope mass spectrometry instrument, and the instrument assigned batch numbers X1, X2, ... X to each IAEA 600 sample according to the measurement time. N In other words, the batch number is determined based on the measurement time and increases over time. This allows us to obtain the δ values ​​for N batches of standard material. 13 C is the measured value V1, V2, ... V N (N=16 in this experiment.)

[0114] The δ of the IAEA 600 sample was calculated using formula (5). 13 C standard value and δ for each batch 13 The difference between the measured C values ​​is taken as δ 13 C difference Y i .

[0115] (2) δ corresponding to multiple batches13 C difference Y i As the dependent variable, batch number X i As the independent variable, the batch number and δ were fitted to obtain the results. 13 C-value of the instrument drift function;

[0116] y k = -0.003×x k 2 +0.0258×x k -0.4098;

[0117] (3) The batch number x of the 14 / R1 / A sample obtained by the same method in Example 1 k Substituting the values ​​into the instrument drift function, we obtain the drift correction difference y. k Then the δ of the sample to be tested 13 C measured value v k and drift correction difference y k Add them together to obtain the corrected sample δ. 13 C value. Then, δ values ​​from multiple batches of calibrated samples... 13 The final sample δ is obtained by averaging the C values. 13 C value.

[0118] Please see Figure 5 δ, the final sample measured according to Embodiment 2 of the present invention 13 The C value is consistent with the δ values ​​of 14 / R1 / A samples reported by several other institutions. 13 A comparison chart of C values. δ measured in Example 2. 13 C measured value v k The average value (without drift correction) is -20.83‰; after drift correction using the instrument drift function pre-defined for the IAEA 600 sample, the final sample δ is obtained. 13 The C-value is -20.80‰, which is 0.03‰ different from the value before drift correction. The final sample δ measured in this example is... 13 The C value is closer to the arithmetic mean of the 14 / R1 / A samples measured by multiple institutions, which is -20.76‰.

[0119] In carbon isotope analysis, memory effect, blank, 17 Factors such as O-correction will affect the accuracy of the results, and the uncertainty assessment process for the measurement results has not been reported. This invention may further include an uncertainty assessment step: calculating the combined relative uncertainty based on the relative uncertainty of repeated measurements, the relative uncertainty of the standard substance, and the relative uncertainty of the standard curve, and then obtaining the sample δ based on the combined relative uncertainty. 13 The absolute uncertainty of the C value.

[0120] Preferably, the relative uncertainty of repeated measurements is calculated based on the standard deviation of the repeated measurements of the sample, specifically using the following formula:

[0121]

[0122] Among them, u rep-rel The relative uncertainty of repeated measurements is given by SD, where SD is the standard deviation of repeated measurements of the sample, p is the number of repeated measurements of the sample, and δ is the relative uncertainty of repeated measurements. 13 C0 represents the final sample δ obtained by averaging after p repeated measurements. 13 C value.

[0123] Preferably, the expanded uncertainty of the standard substance used for calibration and the final sample δ are determined according to the standard curve. 13 The C-value, used to determine the relative uncertainty of a standard substance, includes the following steps:

[0124] (1) Determine the type of standard substance used for standard curve calibration and its corresponding δ. 13 C standard value, determine δ 13 C standard value and final sample δ 13 The type of standard substance with the closest C value shall be used as the master standard substance;

[0125] (2) Calculate the relative uncertainty of the standard substance using the following formula:

[0126]

[0127] Among them, u ref-rel u represents the relative uncertainty of the standard substance. ref Let g = 3, δ be the expanded uncertainty of the master standard material. 13 C ref The δ of the master standard material 13 C standard value.

[0128] Preferably, the δ of the standard substance used for calibration is determined according to the standard curve. 13 C measured value and δ 13 C standard value, and final sample δ 13 The C-value is used to calculate the relative uncertainty of the standard curve, including the following steps:

[0129] (1) Calculate the uncertainty of the standard curve using the following formula:

[0130]

[0131] where u(δ 13 C0) represents the uncertainty of the standard curve, a represents the slope of the standard curve, s represents the standard deviation of multiple measurements, p represents the number of repeated measurements of the sample, and n represents the total number of measurements of the standard substance used for standard curve calibration; δ13 C0 represents the final sample δ 13 C value, δ values ​​of various standard substances used for standard curve calibration 13 The average of the standard C values, x j δ of the j-th standard substance used for standard curve calibration 13 C standard value.

[0132] (2) Based on the uncertainty of the standard curve and the final sample δ 13 The C-value is used to calculate the relative uncertainty of the standard curve:

[0133]

[0134] Preferably, the combined relative uncertainty is calculated based on the relative uncertainty of repeated measurements, the relative uncertainty of the standard substance, and the relative uncertainty of the standard curve, including the following steps:

[0135]

[0136] Where u rep-rel The relative uncertainty of repeated measurements, u ref-rel The relative uncertainty of the standard substance, u(δ) 13 C0) rel This represents the relative uncertainty of the standard curve.

[0137] Preferably, the final sample δ is obtained based on the synthesis relative uncertainty. 13 The absolute uncertainty of the C value includes the following steps:

[0138] U = u rel ×δ 13 C0;

[0139] Where U is the final sample δ 13 The absolute uncertainty of C, δ 13 C0 represents the final sample δ 13 C value.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for measuring sample δ based on EA-IRMS 13 C The method of value, characterized in that, Includes the following steps: The sample pretreatment steps include diluting the sample to be tested to obtain a sample aqueous solution, and then taking a predetermined volume of the sample aqueous solution and placing it in a container for drying; the sample to be tested is honey. Measurement steps: Elemental analysis-isotope mass spectrometry was used to analyze the δ-values ​​of the pretreated sample. 13 C The values ​​were measured and calibrated using a standard curve to obtain multiple δ values. 13 C Measured value; The drift correction step, based on a predetermined instrument drift function, applies the following to the multiple δ values. 13 C The measured values ​​were corrected for drift to obtain multiple corrected sample δ values. 13 C The values ​​are averaged to obtain the final sample δ. 13 C value; The amount of sample aqueous solution taken in the sample pretreatment step is determined according to the following steps: (1) The δ of the standard substance was measured in advance using the same elemental analysis-isotope mass spectrometry instrument and matching silver bag. 13 C The value is obtained by measuring the original value of the standard substance. And measure the δ of the blank sample. 13 C By analyzing the values ​​and peak areas, we can obtain the original measured values ​​of the blank sample. Peak area of ​​blank sample ; (2) Determine the blank correction formula: ； in, δ of the blank-corrected sample 13 C value; Peak area ratio represents ; Indicates the peak area of ​​the standard substance; (3) Obtain the functional relationship between the sample measurement difference D and the peak area ratio according to the blank correction formula, and determine the minimum peak area ratio according to the pre-set reliable upper limit of the sample measurement difference; wherein the sample measurement difference D refers to the original measurement value of the standard substance. δ of the blank-corrected sample 13 C value The difference; (4) Based on the minimum peak area ratio and the peak area of ​​the blank sample Obtain the minimum sample peak area and calculate the sampling amount based on the minimum sample peak area; The drift correction step includes: (1) Using the same elemental analysis-isotope mass spectrometry instrument and matching silver bag, the δ of multiple batches of quality control standard substances were continuously measured in advance. 13 C Values ​​were obtained, and multiple δ values ​​were obtained after calibration using a standard curve. 13 C Measured values, and simultaneously calculate δ for each batch. 13 C Difference ： in, Y i For the first i Batch δ 13 C Difference, ref. δ for quality control standard substances 13 C Standard value; V i δ for the quality control standard substance of the i-th batch 13 C Measured value; (2) δ corresponding to multiple batches 13 C Difference Y i As the dependent variable, the batch number X i As the independent variable, the batch number and δ were fitted to obtain the results. 13 C The instrument drift function of the difference; (3) The batch number of the sample to be tested x k Substituting the values ​​into the instrument drift function, we obtain the drift correction difference. y k Then the δ of the sample to be tested 13 C Measured values v k and drift correction difference y k Add them together to obtain the corrected sample δ. 13 C value; The instrument drift function is: y k =a×x k 2 +b×x k +c； in, a , b and c The parameters obtained from the fitting, x k For the first k Batch number of the sample to be tested. y k For the first k Drift correction difference of batch of test samples.

2. The method for measuring sample δ based on EA-IRMS according to claim 1 13 C The method of value, characterized in that, The sample pretreatment steps include: The honey sample was liquefied in a water bath at 55℃-65℃ for 8-12 minutes, then homogenized in a homogenizer for 2-3 minutes, and then ultrasonicated at 55℃-65℃ for 4-6 minutes for further homogenization. The homogenized honey sample was dissolved in degassed ultrapure water to prepare a solution with a concentration of 45-55 mg / g. -1 The solution was sampled at room temperature using a microsyringe and placed in a silver pouch, then dried in an oven at 55℃-65℃ for 1.5-2.5 hours.

3. The method for measuring sample δ based on EA-IRMS according to claim 2 13 C The method of value, characterized in that, The blank correction formula is: 。 4. The method for measuring sample δ based on EA-IRMS according to claim 3 13 C The method of value, characterized in that, The pre-set upper limit for reliable sample measurement difference is -0.03‰, and the calculated minimum peak area ratio is 100.

5. The method for measuring sample δ based on EA-IRMS according to claim 1 13 C The method of value, characterized in that, The method further includes the following steps: The uncertainty assessment steps are as follows: Calculate the combined relative uncertainty based on the relative uncertainties of repeated measurements, the relative uncertainties of the standard substance, and the relative uncertainties of the standard curve; then obtain the final sample δ based on the combined relative uncertainty. 13 C The absolute uncertainty of the value.

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