METHOD FOR ANALYZING STABLE ISOTOPES OF PARTICULATE ORGANIC CARBON (POC) AND NITROGEN IN SEAWATER USING ELEMENTAL ANALYSIS-ISOTOPE RATIO MASS SPECTROMETRY (EA-IRMS)

Abstract

A method for analyzing stable isotopes of particulate organic carbon (POC) and nitrogen in seawater using elemental analysis-isotope ratio mass spectrometry (EA-IRMS), including: using a glass fiber filter membrane to filter a collected water sample and then filter 100 ml of distilled water; subjecting a resulting membrane sample to acidification; and subjecting an acidified membrane sample to EA-IRMS analysis on a machine, where an oxygen addition time is set to 80 s and a combustion tube temperature is set to 700° C. for an elemental analyzer, and a trap current is set to 300 μA to 400 μA for an isotope ratio mass spectrometer. The method of the present disclosure can reduce a detection cost and improve the data accuracy and work efficiency.

Claims

1. A method for analyzing stable isotopes of particulate organic carbon (POC) and nitrogen in seawater using elemental analysis-isotope ratio mass spectrometry (EA-IRMS), comprising the following steps: a. material preparation: burning a φ25 mm GF/F glass fiber filter membrane at 450° C. for 5 h in advance before use; b. sample collection: collecting a water sample at a desired station and in a desired layer; using the φ25 mm GF/F glass fiber filter membrane to filter 200 mL of the water sample and then filter 100 ml of distilled water; and wrapping a resulting membrane sample with tin foil, and storing the resulting membrane sample at −20° C. for later use; c. sample pretreatment: subjecting the resulting membrane sample to acidification for 30 min to obtain an acidified membrane sample, drying the acidified membrane sample in a drying oven at 60° C. for 24 h immediately after the acidification to obtain a dried membrane sample, and storing the dried membrane sample in a desiccator; d. sample coating: coating the dried membrane sample obtained in step c to obtain a coated membrane sample, and letting the coated membrane sample to be ready for test on a machine; e. sample determination: cleaning and debugging instruments of an elemental analyzer and an isotope ratio mass spectrometer according to operational provisions of the instruments to make the instruments in a sample test status; adjusting general settings of the instruments as follows: setting an oxygen addition time to 80 s and a combustion tube temperature to 700° C. for the elemental analyzer, and modifying ion parameters and setting a trap current to 300 μA to 400 μA for the isotope ratio mass spectrometer; and starting a sample test; f. sample loading order: adding three or more isotopic standards certified by the International Atomic Energy Agency (IAEA) or working standards with stable isotope values, wherein 5 to 6 replicates are set for each standard of the isotopic standards and the working standards; adding the coated membrane sample, wherein a standard is added every 10 samples; and after all samples are added, starting the instruments for test; and g. sample data processing: observing whether 5 to 6 results of the each standard are stable, wherein if a standard deviation (SD) is lower than 0.2, data are available; if test results of the isotopic standards and the working standards are stable, averaging the 5 to 6 results of the each standard; using Excel to plot a standard curve with an average value measured by the instruments as an x-coordinate and a true value as a y-coordinate; and substituting a measured value of a sample into the standard curve for calculation to obtain a true value of the sample, substituting a measured value of a standard inserted among all samples into the standard curve for calculation, and comparing a result with the true value, wherein if a correction coefficient is in a range of 0.9 to 1.1, data are credible.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows a relationship between a measured value and a true value of a C stable isotope standard; and

[0022] FIG. 2 shows a relationship between a measured value and a true value of an N stable isotope standard.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The technical solutions of the present disclosure will be further described below with reference to examples, but the protection scope of the present disclosure is not limited by the examples in any manner.

Example 1

[0024] In this example, a large number of experiments on the influence of temperature on data results and combustion tube burst were conducted, and finally it was determined that a temperature of the technical solution of the present disclosure was set to 700° C. During an experimental process, it was found that, when a temperature was lower than 700° C., incomplete combustion would occur, a resulting standard curve had poor linearity, and after a standard subsequently inserted was substituted into the standard curve, a resulting correction factor was large. Therefore, it was believed that a working temperature of the instrument should not be lower than 700° C. It should be noted that, at 700° C., an oxygen addition time of the instrument should be extended by 10 s to ensure complete combustion. When a temperature was higher than 700° C., it was found by statistics that the occurrence probability of combustion tube crack or burst at 850° C. reached more than 90% as that at 950° C.; and at 750° C. and 800° C., there was also a specified probability to occur burst, where a burst probability reached about 50% at 800° C. and reached about 20% to 30% at 750° C. More than 20 consecutive experiments were conducted at 700° C. in a laboratory, and no burst occurred.

Example 2

[0025] In this example, experiments on the influence of oxygen addition time on data results were conducted, and finally it was determined that a temperature of the technical solution of the present disclosure was set to 700° C. In this example, during the test of oxygen addition time of the instrument, when a normal oxygen addition time was set to 70 s, C and N conversion rates of a standard were respectively 99.1% and 99.2% at 700° C.; and after an oxygen addition time was changed to 80 s, C and N conversion rates of a standard were respectively 100.2% and 99.6% at 700° C. Therefore, when the temperature is set to 700° C., an oxygen addition time should be extended by 10 s to ensure complete combustion of a sample.

Example 3

[0026] 1. Materials and Methods

[0027] 1.1 Materials

[0028] 1.1.1 Filter Membrane

[0029] On Jun. 2, 2019, a φ25 mm GF/F glass fiber filter membrane was prepared in a laboratory, then burnt in a muffle furnace at 450° C. for 5 h, and taken out and cooled in an electronic moisture-proof box for later use.

[0030] 1.1.2 Water Sample

[0031] On Jun. 4, 2019, surface and bottom seawater samples were collected in an aquaculture area of Dongchu Island Aquatic Products Co., Ltd. in Rongcheng City, Shandong Province, and then transported back to the laboratory and immediately subjected to suction filtration.

[0032] 1.2 Method

[0033] 1.2.1 Sample Collection

[0034] First, in order to determine a sampling method for stable isotope detection, an element detection experiment was conducted. 4 blank experimental groups and 4 distilled-water suction-filtration experimental groups were set, and corresponding samples were collected to determine the influence of DOC on an elemental analysis result and a reasonable stable isotope sampling method. Specifically: A method of dipping a blank membrane with filtrate drops: after suction filtration was completed, 3 to 5 drops of a resulting filtrate (ensuring that a membrane surface would be wetted) were pipetted with a pipette and added to another blank new membrane, which served as a blank value. A method of completely immersing a blank membrane: after suction filtration was completed, a blank new membrane was completely immersed in a resulting filtrate for 1 s to 2 s and then taken out, which served as a blank value. A double-layer membrane method: Suction filtration was conducted with two stacked membranes, and the second filter membrane layer served as a blank value. A method of conducting suction filtration two times: after suction filtration was completed, a resulting filtrate was completely filtered through a blank new membrane, which served as a blank value. A distilled-water suction-filtration method: after a water sample was subjected to suction filtration with a membrane, and then a specified volume of distilled water was subjected to suction filtration with the original membrane, where the volume of distilled water was set to 50 ml, 100 ml, 150 ml, and 200 ml. 6 replicates were set for each blank experimental group, and 3 replicates were set for each distilled-water suction-filtration group.

[0035] A total of 20 stations were set for this survey, and after the sampling method was determined, a sample was prepared according to a corresponding method for test.

[0036] 1.2.2 Sample Pretreatment

[0037] The pretreatment of a sample mainly refers to a process of removing inorganic carbon by acidification. With reference to literatures and national standards, it was found through experimental analysis that an acidification time of 30 min proposed in the national standards and literatures was the optimal, and a too short or too long acidification time would affect the data accuracy. However, acidification is generally achieved by placing a sample and concentrated hydrochloric acid in a closed container, which will cause specified damage to the health of an operator and will also cause a waste of concentrated hydrochloric acid due to volatilization. Therefore, in the early work, a closed multi-layer acid fumigation device with controlled release of acid vapor (ZL201610938287.1) was designed and invented by the inventors. This device can effectively improve the work efficiency and reduce the harm of released acid vapor. Specific operation steps are detailed in the description of this patent. A sample was subjected to acidification for 30 min, then dried in a drying oven at 60° C. for 24 h as soon as possible, and stored in an electronic moisture-proof box.

[0038] 1.2.3 Sample Coating

[0039] An acidified filter membrane needs to be coated before being loaded on a machine for test. A traditional coating tool is inefficient. In the early work, a sample coating device for an elemental analyzer (ZL201410534476.3) was also designed and invented by the inventors. This device can improve the work efficiency, increase a success rate of sample coating, and achieve other functions. Operation steps are detailed in the description of this patent. A coated sample is ready for test on the machine.

[0040] 1.2.4 Sample Determination

[0041] Before instruments were started, an ash tube was first cleaned and the activity of a reduction tube was checked for the elemental analyzer (EL cube, German elementar); after requirements were met, the system was started and subjected to leakage detection; after the leakage detection was passed, a blank value of the instrument was tested; the isotope ratio mass spectrometer (vario Micro cube-ISOprime 100) was started; and after the system was subjected to vacuum-pumping, linearity and stability tests were conducted for the mass spectrometer to make it in a sample test state. Then the general settings of the instruments were modified as follows: for the elemental analyzer, an oxygen addition time was changed from 70 s to 80 s and a combustion tube temperature was changed from 950° C. to 700° C.; and for the isotope ratio mass spectrometer, ion source parameters were modified, a trap current was changed from 200 μA to 300-400 μA, and other parameters were unchanged. The sample test was started. Three or more isotopic standards certified by the International Atomic Energy Agency (IAEA) or working standards with stable isotope values were first added, where the isotopic standards certified by the International Atomic Energy Agency (IAEA) were IAEA-N-2, IAEA-600, and IAEA-CH-6, the working standards were L-phenylalanine, USGS40, and EDTA #2, and 5 to 6 replicates were set for each standard; then a sample to be tested was added, where a standard was added every 10 samples; and after all samples were added, the instruments were started for test.

[0042] 2. Results and Discussion

[0043] 2.1 Sample Collection

[0044] According to different experimental groups, a sample value and a blank value were obtained through elemental analysis to determine a sample collection method for stable isotope analysis. Experimental results were shown in Table 1. According to analysis of the experimental results, blank values obtained by the method of completely immersing a blank membrane and the double-layer membrane method are the highest, where a blank value may be higher than a sample value. In actual operation, repeated sampling is difficult to achieve at each survey station, and thus the probability that data are unavailable due to a blank value being higher than a sample value is greatly increased. Therefore, the above two blank value sampling methods are inadvisable. Among the remaining methods, the method of dipping a blank membrane with filtrate drops has the optimal effect, where blank values of 6 replicates are the most stable; results of the method of conducting suction filtration two times are similar to results of the method of dipping a blank membrane with filtrate drops, but there are abnormal values in the 6 replicates; and results of the distilled-water suction-filtration method at 4 different distilled water volumes show that, after suction filtration is conducted for 50 ml of distilled water, a calculated value is slightly higher than an average value of the method of dipping a blank membrane with filtrate drops, and results of the experimental groups using 100 ml, 150 ml, and 200 ml of distilled water show no significant difference, and are similar to data corrected with the blank values of the method of dipping a blank membrane with filtrate drops and the method of conducting suction filtration two times. According to the above-mentioned experimental analysis, the method of dipping a blank membrane with filtrate drops, the 100 ml distilled-water method, and the method of conducting suction filtration two times lead to the optimal effect. Given that the isotope analysis cannot use the blank value correction method, in a sample collection process for detecting stable isotopes of POC and PN in seawater, after a sample is subjected to suction filtration with a membrane, 100 ml of distilled water must be subjected to suction filtration with the same membrane.

TABLE-US-00001 TABLE 1 Results of the blank value sampling methods Calculated value Measured value (mg) Blank value (mg) (mg/L) Sample No. C N C N POC PN Method of dipping a blank 12.81 7.50 5.54 2.70 0.3635 0.24 membrane with filtrate drops-1 Method of dipping a blank 12.39 7.42 5.86 2.68 0.3265 0.237 membrane with filtrate drops-2 Method of dipping a blank 12.35 7.75 5.36 2.02 0.3495 0.2865 membrane with filtrate drops-3 Method of dipping a blank 12.50 7.72 5.45 2.21 0.3525 0.2755 membrane with filtrate drops-4 Method of dipping a blank 12.58 7.11 5.15 2.22 0.3715 0.2445 membrane with filtrate drops-5 Method of dipping a blank 12.39 7.53 5.61 2.66 0.339 0.2435 membrane with filtrate drops-6 Average value 12.50 ± 0.17  7.51 ± 0.23 5.50 ± 0.24 2.42 ± 0.30 0.35 ± 0.02 0.25 ± 0.02 Method of completely 12.14 7.89 6.93 16.83 0.2605 / immersing a blank membrane-1 Method of completely 12.28 6.22 6.26 3.96 0.301 0.113 immersing a blank membrane-2 Method of completely 12.31 7.51 15.85 7.48 / 0.0015 immersing a blank membrane-3 Method of completely 12.99 6.81 6.19 3.80 0.34 0.1505 immersing a blank membrane-4 Method of completely 12.22 7.30 9.42 5.25 0.14 0.1025 immersing a blank membrane-5 Method of completely 13.05 7.19 6.48 6.75 0.3285 0.022 immersing a blank membrane-5 Average value 12.49 ± 0.41  7.15 ± 0.58 8.52 ± 3.79 7.35 ± 4.87 0.27 ± 0.20 0.08 ± 0.22 Double-layer membrane method-1 13.27 6.39 14.71 3.18 / 0.1605 Double-layer membrane method-2 13.33 6.67 7.86 3.04 0.2735 0.1815 Double-layer membrane method-3 14.06 5.08 7.29 3.50 0.3385 0.079 Double-layer membrane method-4 13.44 6.03 9.49 4.01 0.1975 0.101 Double-layer membrane method-5 13.24 6.65 3.46 7.46 0.489 / Double-layer membrane method-6 12.44 3.83 12.88 3.88 / / Average value 13.30 ± 0.52  5.78 ± 1.12 9.28 ± 4.06 4.18 ± 1.65 0.32 ± 0.22 0.13 ± 0.09 Method of conducting 12.18 7.11 5.67 2.92 0.3255 0.2095 suction filtration two times-1 Method of conducting 12.72 7.31 5.84 3.05 0.344 0.213 suction filtration two times-2 Method of conducting 12.13 7.82 8.05 3.05 0.204 0.2385 suction filtration two times-3 Method of conducting 12.75 7.22 10.34 2.47 0.1205 0.2375 suction filtration two times-4 Method of conducting 12.49 7.66 5.17 3.12 0.366 0.227 suction filtration two times-5 Method of conducting 12.92 7.22 5.01 2.81 0.3955 0.2205 suction filtration two times-6 Average value 12.53 ± 0.32  7.39 ± 0.28 6.68 ± 2.10 2.90 ± 0.23 0.29 ± 0.11 0.22 ± 0.01 50 ml distilled water-1 7.99 5.73 / / 0.3995 0.2865 50 ml distilled water-2 7.53 5.51 / / 0.3765 0.2755 50 ml distilled water-3 8.00 5.80 / / 0.4 0.29 Average value 7.84 ± 0.27 5.68 ± 0.15 / / 0.39 ± 0.01 0.28 ± 0.01 100 ml distilled water-1 7.09 4.73 / / 0.3545 0.2365 100 ml distilled water-2 6.93 5.01 / / 0.3465 0.2505 100 ml distilled water-3 7.00 5.30 / / 0.35 0.265 Average value 7.01 ± 0.08 5.01 ± 0.29 / / 0.35 ± 0.01 0.25 ± 0.01 150 ml distilled water-1 7.07 5.03 / / 0.3535 0.2515 150 ml distilled water-2 7.10 5.05 / / 0.355 0.2525 150 ml distilled water-3 7.01 5.12 / / 0.3505 0.256 Average value 7.06 ± 0.05 5.07 ± 0.05 / / 0.35 ± 0.01 0.25 ± 0.01 200 ml distilled water-1 7.02 4.77 / / 0.351 0.2385 200 ml distilled water-2 6.91 4.91 / / 0.3455 0.2455 200 ml distilled water-3 6.92 5.02 / / 0.346 0.251 Average value 6.95 ± 0.06 4.90 ± 0.13 / / 0.34 ± 0.01 0.24 ± 0.01

[0045] 2.2 Sample Determination

[0046] After the stable isotope detection was completed, standard curves illustrating a relationship between a measured value and a true value of an isotope ratio of a standard were plotted and shown in FIG. 1 and FIG. 2. It can be seen from the standard curves that a measured value of an instrument had a prominent linear correlation with a true value. In this experiment, a total of 20 seawater samples were collected, and 2 standards were inserted (the standard IAEA-600 certified by the International Atomic Energy Agency (IAEA) was adopted: δ.sup.15N=1 and δ.sup.13C=−27.771). A measured value of a seawater sample was substituted into the C and N standard curves separately to obtain true values of the sample. Moreover, in order to verify the credibility of test data, a measured value of a standard was substituted into the standard curves to obtain stable isotopes of carbon and nitrogen: C (−27.66, −27.74) and N (0.99, 1), and correction factors were both close to 1. Therefore, the method and operation of the present disclosure are accurate, convenient, and highly feasible.