WO2021098171A1 - 利用ea-irms分析海水中颗粒有机碳、氮稳定同位素的方法 - Google Patents

利用ea-irms分析海水中颗粒有机碳、氮稳定同位素的方法 Download PDF

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WO2021098171A1
WO2021098171A1 PCT/CN2020/091865 CN2020091865W WO2021098171A1 WO 2021098171 A1 WO2021098171 A1 WO 2021098171A1 CN 2020091865 W CN2020091865 W CN 2020091865W WO 2021098171 A1 WO2021098171 A1 WO 2021098171A1
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sample
standard
samples
instrument
value
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French (fr)
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刘毅
张继红
吴文光
孙科
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中国水产科学研究院黄海水产研究所
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Publication of WO2021098171A1 publication Critical patent/WO2021098171A1/zh
Priority to US17/451,325 priority Critical patent/US11307123B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/12—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using combustion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/18—Water
    • G01N33/188—Determining the state of nitrification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

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  • the invention belongs to the field of water quality detection, and specifically relates to a method for analyzing stable isotopes of particulate organic carbon and nitrogen in seawater by using EA-IRMS. It utilizes the high-efficiency, fast and accurate characteristics of an element analyzer and an isotope mass spectrometer, and combines with the method of the present invention to achieve the purposes of saving consumables, improving data accuracy, and prolonging the service life of the instrument.
  • the stable isotopes of particulate organic carbon and nitrogen in seawater are the key parameters that reveal the material circulation path, trophic level, food source and other key parameters of the ecosystem.
  • the lack of standardized and accurate measurement methods results in low quality and high cost of detection data, which further affects science Reliability of research results.
  • the experimenter usually needs to operate at a high temperature of 950 °C. A little carelessness is very likely to be dangerous, and the combustion tube is filled with toxic chemicals such as lead chromate. , After the combustion tube bursts, it will be completely exposed to the air, and it will be more harmful to the human body at high temperature and easier to contact with the experimenter.
  • the more common analysis method for such samples is to switch to domestic combustion tubes, accumulate a certain amount of samples and then perform centralized measurement.
  • the use of domestic combustion tube Avoid reusing the combustion tube from cracks or explosions to cause greater impact. This can save about 60% of the cost of using original consumables, but it still wastes about 30% of the cost of conventional samples, and domestic consumables are easier than imported consumables
  • the impure quartz and impurities affect the analysis results.
  • the dissolved organic carbon content in the ocean carbon pool can often reach 4-10 times that of particulate organic carbon.
  • the analysis methods of particulate organic carbon and particulate nitrogen specified by the national standard do not consider the attachment of dissolved organic carbon to the filter membrane to affect the sample. The impact of the result.
  • the blank values obtained by different sampling methods have significant differences. Therefore, we have improved the elemental content analysis method of particulate organic carbon and particulate nitrogen on the basis of the national standard, that is, the method of blank value calibration is used in elemental analysis.
  • the instrument completely burns the sample It will all be sent into the detection magnetic field, and the particle state and the dissolved state cannot be separated.
  • the obtained isotope ratio cannot be deducted by the method of elemental analysis and integral calculation to deduct the influence of dissolved organic carbon.
  • the stable isotope detection obviously cannot use the blank value for correction. Therefore, the key to determining the accuracy of the data is to consider how to effectively remove the dissolved organic carbon adsorbed by the filter membrane during the sampling process.
  • the present invention is aimed at the problems encountered and existing in the detection of particulate organic carbon and particulate nitrogen in seawater by the combined use of elemental analyzer-isotopic mass spectrometer, starting from the whole process of sample collection, storage, processing, and detection, and By changing the instrument settings and parameters, the detection cost is reduced by 30%, data accuracy is improved, and work efficiency is improved.
  • the present invention is completed in the following manner:
  • the method of using EA-IRMS to analyze the stable isotopes of particulate organic carbon and nitrogen in seawater includes the following steps:
  • Material preparation Take GF/F glass fiber filter membrane with ⁇ 25mm and burn it at 450°C for 5 hours before use;
  • Sample collection take the water sample at the required station and layer, filter the water sample on the ship or after transporting it back to the laboratory, filter the seawater volume of 200ml, then filter 100ml of distilled water, wrap the membrane sample in tin foil Store at -20°C for later use;
  • Sample pretreatment take the membrane sample, acidify for 30 minutes, put the sample in a drying oven at 60°C for 24 hours, and store it in a desiccator;
  • Sample coating coating the film sample of step c, and the coated sample is ready to be tested on the machine;
  • Sample order on the computer add more than three international atomic energy certified isotope standard materials, or work standard materials with stable isotope values, set 5-6 replicates for each standard material, then add samples, and add a standard for every 10 samples After all the substances and samples are placed, the instrument will be tested;
  • the present invention combines the research of the blank value calibration method in the elemental analysis process, and confirms the sampling method that can easily and effectively remove the influence of dissolved organic carbon on the sample in the sampling stage through experimental comparison. That is, after the sample is suction filtered, 100ml of distilled water is repeatedly suction filtered. This method can realize that the element detection value of the sample is directly equal to the sample value corrected by the blank value calibration method without suction filtered distilled water, and the amount of distilled water is the least. The work intensity is simplified, the accuracy of the analysis results is improved, and the influencing factors that are not considered in the national standard of the element analysis method are improved.
  • the present invention combines the applicant's past device inventions related to this type of instrument, starting with the entire process from sample sampling to analysis, and establishing a complete set of seawater particle organic carbon and particle nitrogen stable isotope analysis and detection procedures, which improves the work
  • the efficiency and data reliability fill the gap that there is no relevant sample detection method at home and abroad.
  • Figure 1C The relationship between the standard substance detection value and the true value of the stable isotope detection value
  • the temperature of the technical solution of the present invention is set to 700°C.
  • the temperature is lower than 700°C, incomplete combustion will occur, and the standard curve will have a problem of poor linearity. After the subsequent inserted standard products are brought into the standard curve, the correction factor will also be relatively high.
  • the operating temperature of the instrument should not be lower than 700°C.
  • the oxygenation time of the instrument should be set at 700°C for 10 seconds to ensure complete combustion.
  • the temperature is higher than 700°C, it is found by statistics that the probability of combustion tube cracks or bursts above 850°C can reach more than 90% as at 950°C.
  • 750°C and 800°C will also have certain probability of explosion phenomenon, 800°C
  • the bursting probability of 750°C can reach about 50%, 750°C is slightly lower than about 20-30%, and when it is set to 700°C, the laboratory has so far carried out more than 20 consecutive experiments, and the phenomenon of blasting has not yet appeared again.
  • the temperature of the technical solution of the present invention was set to 700°C.
  • the oxygen addition amount of the instrument when the oxygen addition amount of the instrument was tested, when the normal oxygen addition amount of the instrument was 70 seconds, the conversion rate of standard product C and N were 99.1% and 99.2% respectively at a temperature of 700 degrees; after changing the oxygen addition amount to 80 seconds, At 700°C, the conversion rates of standard C and N are 100.2% and 99.6%. Therefore, after the temperature is set to 700°C, it is best to add more oxygen for 10 seconds to ensure that the sample is fully burned.
  • the blank film dripping filtrate method that is, after the suction filtration is completed, use a dropper to suck up the filtrate and drop 3-5 drops (to ensure that the surface of the film is wet) on the new blank film, which is used as the blank value
  • the blank membrane is completely soaked method, that is, after the suction filtration is completed, the blank new membrane is completely soaked in the filtrate for 1-2 seconds and then taken out, which is regarded as the blank value
  • the double-layer membrane method that is, the two membranes are stacked and then filtered, Use the second layer of filter membrane as the blank value
  • the second suction filtration method that is, after the suction filtration is completed, take all the filtrate and filter again with a blank new membrane, which is regarded as the blank value
  • the pumping distilled water method that is, after the water sample suction filtration is completed , Continue to suction filter a certain volume of distilled water on the original membrane, and set the volume of distilled water to 50ml, 100m
  • a total of 20 stations were set up in this survey, and after the sampling method was determined, they were filtered and tested according to the method.
  • the pretreatment of samples mainly refers to the process of removing inorganic carbon by acidification.
  • the acidification time of 30 minutes proposed in the national standards and literature is the most appropriate. Too short or too long acidification time will affect the accuracy of the data. .
  • the acidification method is generally to place the sample and concentrated hydrochloric acid in a closed container. This process will cause certain damage to the health of the operator, and will also cause the volatilization and waste of concentrated hydrochloric acid. Therefore, the inventor in the early During the work, a closed multi-layer acid fumigation device (ZL201610938287.1) with controlled release of acid vapor was designed and invented.
  • This device can effectively improve work efficiency and reduce the harm of acid vapor release.
  • the specific operation steps are detailed in the patent description. . After acidification for 30 minutes, the sample was taken out and placed in a drying oven at 60°C for 24 hours as soon as possible, and then placed in an electronic moisture-proof box for storage.
  • the filter membrane needs to be coated before it is put on the machine.
  • the traditional coating tool is inefficient.
  • the inventor also designed and invented a sampler for element analyzer (ZL201410534476. 3) This device can improve work efficiency, increase the success rate of sample coating, and can achieve other functions. For detailed operation steps, see the patent description. The coated sample is waiting to be put on the machine.
  • the element analyzer is a German elementar model EL cube
  • the elemental analyzer is a German elementar model EL cube
  • the blank value of the instrument is tested.
  • the isotope mass spectrometer variant Micro cube-ISOprime100
  • the linearity and stability of the mass spectrometer are tested to make it reach the sample state.
  • modify the general settings of the instrument Firstly, change the oxygen content of the element analyzer from 70s to 80s.
  • change the temperature of the combustion tube from 950°C to 700°C.
  • the filament current (Trap Current) was changed from 200 ⁇ A to 300-400 ⁇ A, and other parameters were conventionally set, and then the sample measurement was started.
  • the samples are first added with three or more international atomic energy certified isotope standard materials or working standard materials with stable isotope values in order.
  • This experiment uses international atomic energy standard materials, IAEA-N-2, IAEA-600, IAEA-CH-6, As well as working standard materials L-phenylalanine, USGS40, EDTA#2, each standard material is set to 5-6 replicates, and then the sample to be tested is added, and one standard material is added for every 10 samples. After the samples are all placed, the instrument starts to run.
  • the sample value and blank value were analyzed and tested to determine the sample collection method for stable isotope analysis.
  • the experimental results are shown in Table 1.
  • the blank value taken by the blank film complete soaking method and the double-layer film method is the highest, and the blank value will be higher than the sample value.
  • the probability of data unavailability due to the blank value higher than the sample value will be greatly increased. Therefore, the blank value sampling method of the above two methods is not advisable; among the other methods, the blank membrane dripping filtrate method has the best effect
  • the blank value of the 6 groups of parallel samples is the most stable.
  • the results of the second filtration filtrate method are similar to the results of the blank membrane dripping filtrate method, but there are abnormal values in the 6 groups of parallel samples; the results of the 4 different distilled water volumes of the pumping distilled water method show After 50ml suction filtration, the calculated value is slightly higher than the average value of the dripping filtrate method.
  • the results of the 100ml-200ml suction filtration volume experimental group are not significantly different, and it is corrected with the blank value of the blank membrane dripping filtrate method and the secondary suction filtration filtrate method. The data is close.
  • the best suction filtration methods are the blank membrane dripping filtrate method, the 100ml distilled water method and the second suction filtration filtrate method.
  • the standard curve that is, the relationship between the detected value of the isotope ratio of the standard substance and the true value. From the standard curve, it can be seen that the measured value of the instrument has a good linear correlation with the true value.

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Abstract

一种利用EA-IRMS分析海水中颗粒有机碳、氮稳定同位素的方法，玻璃纤维滤膜过滤采集的水样后，再过滤蒸馏水100ml；膜样品酸化后上机进行EA-IRMS分析，元素分析仪加氧量设置为80s，燃烧管温度设置为700℃，同位素质谱仪灯丝电流设置为300-400μA。采用该方法可降低检测成本，提高数据准确性，提升工作效率。

Description

利用EA-IRMS分析海水中颗粒有机碳、氮稳定同位素的方法 技术领域
本发明属于水质检测领域，具体的涉及一种利用EA-IRMS分析海水中颗粒有机碳、氮稳定同位素的方法。它利用了元素分析仪和同位素质谱仪的高效、快捷、准确的特性，并结合本发明的方法达到节省耗材，提高数据准确性，延长仪器使用寿命的目的。
背景技术
先进的科研设施和仪器设备是科技创新突破的关键，推动和支撑了科学研究的发展。目前国家正在大力发展海洋蓝色经济，碳汇渔业、海洋牧场等新兴领域正在逐步兴起。
绿色、健康、高效已经成为水产养殖发展的关键词，传统的粗放型养殖模式已经逐渐被集约化、工厂化所代替。但是过去一段时间，受经济利益的驱使，我国的水产养殖经历过一段盲目扩增，以牺牲环境为代价的发展过程，海水养殖的发展必须要遵循绿色健康高效的原则。人工鱼礁、海洋牧场等现代化养殖设施的构建正是发展绿色生态水产养殖的新方法、新措施。而所有生态养殖模式的构建都少不了食物网的相关研究，随着质谱仪的诞生，同位素示踪技术已成功应用于生态系统的食物网结构解析等方面。海水中颗粒有机碳、氮稳定同位素是揭示生态系统物质循环路径、营养级、食物来源等关键参数，然而，由于缺乏规范准确的测定方法，使得检测的数据质量低、成本高，进而影响了科学研究结果的可靠性。
目前海水中颗粒有机碳、颗粒氮稳定同位素的测试方法尚没有国标规定，但是由于同位素质谱仪通常与元素分析仪进行联用，因此可以借鉴颗粒有机碳和颗粒氮的元素分析方法进行操作，此方法在GB/T12763.9-2007海洋调查规范第9部分中有所规定，即使用φ25mm的玻璃纤维滤膜(预先在450℃下灼烧5小时)经海水过滤和酸化后，采用元素分析仪进行测定。
但是，目前此方法尚有较大缺陷，每次样品分析过程中有超过90％以上的概率会出现元素分析仪内部燃烧管裂纹或炸裂的现象。通过我们长时间积累的实验经验分析，主要原因是由于滤膜的玻璃纤维材质所造成的。滤膜在燃烧管内经过元素分析仪高温灼烧后，烧化的玻璃纤维材料可能会产生部分碎屑，并在仪器对燃烧管进行常规氦气吹扫时，接触到石英燃烧管的管壁上，在高温条件下与管壁发生黏连，当仪器工作结束温度降低时，由于热胀冷缩，导致元素分析内部的燃烧管管壁受力不均匀从而产生裂纹，进一步当仪器再次升温过程中，由于受热不均等情况的发生，在仪器内部高温高压力的条件下，形成炸裂现象。这一现象不仅增加了仪器使用成本也影响了仪器使用寿命，同时，样品的检测精度也会降低，对检测人员也带来了更多的危害和工作负担。由于仪器厂家认为，较低的温度会造成样品的燃烧不完全，因此该 仪器的说明书上明确表示工作温度必须为950℃，而燃烧管炸裂通常发生在仪器升温过程中或样品检测过程中，并且炸裂后要及时清理碎屑，否则会影响仪器使用寿命，因此实验人员通常需要在950℃的高温条件下进行操作，稍不小心极可能出现危险，且燃烧管内填充了铬酸铅等有毒化学物，燃烧管炸裂后将完全暴露在空气中，在高温下对人体伤害更大更易与实验人员接触。目前，全国各涉海高校和科研院所，包括国外的仪器公司厂家都没有好的解决办法，针对此类样品比较通用的分析方法是换用国产燃烧管，积攒一定量样品后集中测量，一次性使用国产燃烧管。避免重复利用燃烧管后出现裂纹或炸裂现象造成更大的影响，此举可较使用原厂耗材节省成本约60％，但是仍然较常规样品浪费成本约30％，且国产耗材较进口耗材更容易出现石英不纯有杂质影响分析结果的问题。
另外，海洋碳库中，溶解有机碳含量往往可以达到颗粒有机碳的4-10倍，国标规定的颗粒有机碳、颗粒氮的分析方法中并未考虑溶解性有机碳附着在滤膜上对样品结果带来的影响。在以往颗粒有机碳、颗粒氮元素含量的分析实践中，我们尝试通过各种方法以确定溶解有机碳的影响以及空白值的取样方法，最终我们通过实验验证了溶解有机碳会对样品产生较大影响，且不同的取样方法得到的空白值差异显著。因此，我们在国标的基础上对颗粒有机碳和颗粒氮的元素含量分析方法进行了改进，即在元素分析中采用空白值校准的方法，然而由于同位素分析的特殊性，仪器将样品完全燃烧后会全部送入检测磁场中，无法分离颗粒态和溶解态，得出的同位素比值也不能采用元素分析利用积分计算的方式扣除溶解有机碳的影响，稳定同位素检测显然无法使用空白值进行校正。因此，决定数据准确性的关键是必须在取样过程中就考虑如何有效地将滤膜吸附的溶解性有机碳去除。
发明内容
本发明是针对目前利用元素分析仪-同位素质谱仪联用对海水中颗粒有机碳、颗粒氮检测过程中遇到和存在的问题，从样品的采集、保存、处理、检测的全流程入手，并通过改变仪器设置和参数，实现检测成本降低30％，提高数据准确性，提升工作效率。
本发明的是以下述方式完成的：
利用EA-IRMS分析海水中颗粒有机碳、氮稳定同位素的方法，包括以下步骤：
a、材料的准备：取φ25mm的GF/F玻璃纤维滤膜，预先在450℃下灼烧5小时备用；
b、样品的采集：取所需站位和层位的水样，在船上或运回实验室后立即过滤水样，过滤海水体积200ml后，再过滤蒸馏水100ml，将膜样品用锡箔纸包好置于-20℃保存备用；
c、样品的预处理：取膜样品，酸化30分钟后，将样品立即置于60℃烘干箱中干燥24小时后，在干燥器中保存；
d、样品的包覆：对步骤c的膜样品进行包覆，包覆好的样品准备上机测样；
e、样品的测定：按仪器操作规定清理和调试元素分析仪和同位素质谱仪，使之达到测样状态；调整仪器常规设置，元素分析仪上的加氧量设置为80s，燃烧管温度设置为700℃，最后，修改同位素质谱仪上离子源参数，将灯丝电流(Trap Current)设置为300-400μA，之后开始测样；
f、样品的上机顺序：加入三种以上国际原子能认证同位素标准物质，或同位素值稳定的工作标准物质，每种标准物质设5-6个重复，之后加入样品，每10个样品加入一个标准物质，样品全部放置好后开始运行仪器检测；
g、样品的数据处理：首先观察每个标准物质的5-6个结果是否稳定，标准偏差低于0.2则数据可用；标准物质检查稳定后，将每种标准物质的5-6个结果取平均值；利用Excel以仪器测量平均值为横坐标，真实值为纵坐标绘制标准曲线，将样品测量值代入标准曲线计算后即为样品真实值，同时将所有样品中插入的标准物质测量值也代入标准曲线中计算，并与真实值比较，校正系数范围在0.9-1.1之间，则证明数据可信。
本发明与现有技术相比的有益效果：
1、本发明结合元素分析过程中的空白值校准法研究，通过实验对比确定了在取样阶段即可便捷有效的去除溶解有机碳对样品影响的取样方法。即样品抽滤过后，再重复抽滤蒸馏水100ml，此方法可实现样品的元素检测值直接等于不抽滤蒸馏水的空白值校准法校正后的样品值，并且蒸馏水的用量最少。简化了工作强度，提高了分析结果的准确性，完善了元素分析方法国标中未考虑到的影响因素。
2、通过本发明对仪器参数的设置和标准曲线方法的建立研究，提出了颠覆性的更改仪器温度设置的思路，并通过实验确认了此类样品的最佳仪器设置温度—700℃，此温度即可以保证样品完全燃烧，也可以实现燃烧管的重复利用。在实验验证过程中，破损率几乎降低为0，提高了仪器的使用寿命，每个样品可降低检测成本30％；利用标准曲线对数据校正，以此控制数据质量，保证了更改仪器温度后数据的可靠性，反驳了厂家工程师对更改温度数据不可信的质疑；同时减轻了同类样品对检测人员的工作强度和要求。
3、本发明结合了申请人过去与此类仪器相关的装置发明，从样品取样到分析的全流程入手，建立了一套完整的海水颗粒有机碳、颗粒氮稳定同位素分析检测步骤，提高了工作效率和数据可靠性，填补了目前国内外没有相关样品检测方法的空白。
附图说明
图1C稳定同位素检测值的标准物质检测值与真实值关系；
图2N稳定同位素检测值的标准物质检测值与真实值。
具体实施方式
下面通过实施例来对本发明的技术方案做进一步解释，但本发明的保护范围不受实施例任何形式上的限制。
实施例1
本实施例进行了大量温度对数据结果和燃烧管炸裂的影响的摸索实验，最终得到
本发明技术方案的温度设置为700℃。在实验过程中发现，当温度低于700℃时，会产生燃烧不完全的情况，标准曲线会出现线性不好的问题，将后续插入的标准品带入标准曲线后，也会出现校正因子较大的情况，因此我们认为仪器的工作温度不能低于700℃，需要注意的是，700℃时仪器加氧时间最好多设置10s以保障燃烧的完全。当温度高于700℃时，经统计发现，850℃以上时燃烧管裂纹或炸裂的发生概率与950℃一样可达90％以上，750℃和800℃也会出现一定概率的炸裂现象，800℃的炸裂概率可以达到50％左右，750℃略低在20-30％左右，而设置为700℃时，实验室目前已连续进行实验20余次，尚未再次出现炸裂现象。
实施例2
本实施例进行了加氧量对数据结果的影响的摸索实验，最终得到本发明技术方案的温度设置为700℃。本实施例对仪器加氧量进行测试时，当仪器常规加氧量70s时，在700度温度下，标准品C、N转化率分别为99.1％和99.2％；更改加氧量为80s后，在700度温度下，标准品C、N转化率为100.2％和99.6％，因此温度设置为700℃之后，最好将加氧量多加10s钟以保证样品充分燃烧。
实施例3
1.材料与方法
1.1材料
1.1.1滤膜
2019年6月2日在实验室准备φ25mm的GF/F玻璃纤维滤膜，于马弗炉中450℃灼烧5小时后，取出置于电子防潮箱中放凉备用。
1.1.2水样
2019年6月4日在山东省荣成市东楮岛水产有限公司养殖海域采集表底层海水样品，运回实验室后立即抽滤处理。
1.2方法
1.2.1样品的采集
首先为确定稳定同位素检测采样方法，先进行元素的检测实验，设置4组空白实验组和4组蒸馏水抽滤实验组分别采集样品，以此验证溶解有机碳对结果的元素分析影响以及合理的稳定同位素采样方法。具体为：空白膜滴沾滤液法，即抽滤完成后，用滴管吸取滤液并滴3-5滴(保证膜表面湿润即可)到另取的空白新膜上，以此当做空白值；空白膜完全浸湿法，即抽滤完成后，将空白新膜完全浸湿于滤液1-2秒后取出，以此当做空白值；双层膜法，即两张膜叠放后抽滤，用第二层滤膜做空白值；二次抽滤滤液法，即抽滤完成后，取所有滤液用空白新膜再次过滤，以此当做空白值；抽蒸馏水法，即水样抽滤完成后，继续在原膜上抽滤一定体积蒸馏水，蒸馏水体积分别设置为50ml、100ml、150ml、200ml。每个空白实验组分别进行6次平行实验，蒸馏水抽滤组进行3次平行实验。
本次调查共设置20个站位，确定采样方法后，分别按照方法进行抽滤待测。
1.2.2样品的预处理
样品的预处理主要指酸化去除无机碳的过程，通过查阅文献和国标，并通过实验分析，我们认为国标和文献中提出的酸化时间30分钟最为合适，酸化时间过短或过长都会影响数据准确。但是，酸化的方法，一般都是将样品和浓盐酸一同置于一个密闭容器中，此过程会对操作人员身体健康造成一定损害，也会造成浓盐酸的挥发浪费，因此，发明人在早期的工作中，设计和发明了一种密闭式可控制释放酸蒸气的多层酸熏装置(ZL201610938287.1)，此装置可以有效提升工作效率，减少酸蒸气释放的危害，具体操作步骤详见专利说明。酸化30分钟后，将样品取出，尽快置于60℃烘干箱中干燥24小时后，置于电子防潮箱中保存。
1.2.3样品的包覆
酸化完成后的滤膜在上机前，需要对滤膜进行包覆，传统包覆工具效率低，发明人同样在早期的工作中设计和发明了一种元素分析仪用包样器(ZL201410534476.3)，此装置可提升工作效率，提高样品包覆成功率，并可以实现其他功能，详细操作步骤见专利说明。包覆好的样品等待上机。
1.2.4样品的测定
仪器开机前，首先清理元素分析仪(元素分析仪是德国elementar型号是EL cube)灰分管、检查还原管活性，达到要求后开机，进行系统检漏，检漏通过后进行仪器空白值的检测，之后打开同位素质谱仪(vario Micro cube-ISOprime100)，系统抽真空后对质谱仪进行线性和稳定性的测试，使之达到测样状态。此时修改仪器常规设置，首先将元素分析仪的加氧量 由原来的70s改为80s，其次，将燃烧管温度由950℃改为700℃，最后，修改同位素质谱仪的离子源参数，将灯丝电流(Trap Current)从200μA改为300-400μA，其它参数为常规设置，之后开始测样。样品按顺序先加入三种以上国际原子能认证同位素标准物质，或同位素值稳定的工作标准物质，本次实验选用国际原子能认真标准物质，IAEA-N-2、IAEA-600、IAEA-CH-6，以及工作标准物质L-phenylalanine、USGS40、EDTA#2，每种标准物质设5-6个重复，之后加入待测样品，每10个样品加入一个标准物质，样品全部放置好后开始运行仪器检测。
2.结果与讨论
2.1样品的采集
按照不同的实验组对样品值和空白值进行元素分析检测，以此确定稳定同位素分析时样品的采集方法，实验结果见表1。根据实验结果分析，空白膜完全浸湿法和双层膜法所取的空白值最高，并且会出现空白值高于样品值的情况，由于实际操作中，每个调查站位样品难以做到重复取样，因此由于空白值高于样品值而导致数据不可用的概率也将大幅提高，所以上述两种方法的空白值取样方法不可取；其余几种方法中，空白膜滴沾滤液法效果最好，其6组平行样品空白值最稳定，二次抽滤滤液法结果与空白膜滴沾滤液法结果类似，但也在6组平行中出现异常值；4种不同蒸馏水体积的抽蒸馏水法结果显示，抽滤50ml后，计算值略高于滴沾滤液法平均值，100ml-200ml抽滤体积实验组结果差异不显著，且与空白膜滴沾滤液法和二次抽滤滤液法空白值校正后的数据接近。通过对上述实验分析，效果最好的抽滤方法为空白膜滴沾滤液法、100ml蒸馏水法和二次抽滤滤液法，考虑到同位素分析无法采用空白值校正法，因此检测海水颗粒有机碳、颗粒氮稳定同位素的取样过程中，必须在样品抽滤后再次抽滤100ml蒸馏水。
表1几种空白值取样方法的结果
Figure PCTCN2020091865-appb-000001
Figure PCTCN2020091865-appb-000002
2.2样品的测定
稳定同位素检测完成后，标准曲线即标准物质同位素比值检测值与真实值关系如图1、图2所示，从标准曲线可以看出，仪器检测值与真实值线性相关性较好。本次实验共取海水样品20个，插入标准品2个(本实验选取国际原子能标准物质IAEA-600，δ 15N＝1；δ 13C＝-27.771)，海水样品检测值带入分别带入C、N两条标准曲线，即为样品真实值；同时为验证检测数据的可信度，将标准品检测值带入标准曲线得到碳氮稳定同位素分别为：C(-27.66、-27.74)，N(0.99、1)，校正因子均接近1，所以本发明方法和操作，准确、方便、可行性高。

Claims (1)

  1. 利用EA-IRMS分析海水中颗粒有机碳、氮稳定同位素的方法，其特征在于所述方法包括以下步骤：
    a、材料的准备：取φ25mm的GF/F玻璃纤维滤膜，预先在450℃下灼烧5小时备用；
    b、样品的采集：取所需站位和层位的水样，过滤水样，过滤海水体积200ml后，再过滤蒸馏水100ml，将膜样品用锡箔纸包好置于-20℃保存备用；
    c、样品的预处理：取膜样品，酸化30分钟后，将样品立即置于60℃烘干箱中干燥24小时后，在干燥器中保存；
    d、样品的包覆：对步骤c处理后的膜样品进行包覆，包覆好的样品准备上机测样；
    e、样品的测定：按仪器操作规定清理和调试元素分析仪和同位素质谱仪，使之达到测样状态；调整仪器常规设置，元素分析仪上的加氧量设置为80s，燃烧管温度设置为700℃，修改同位素质谱仪的离子参数，灯丝电流设置为300-400μA，之后开始测样；
    f、样品的上机顺序：加入三种以上国际原子能认证同位素标准物质，或同位素值稳定的工作标准物质，每种标准物质设5-6个重复，之后加入样品，每10个样品加入一个标准物质，样品全部放置好后开始运行仪器检测；
    g、样品的数据处理：首先观察每个标准物质的5-6个结果是否稳定，标准偏差低于0.2则数据可用；标准物质检查稳定后，将每种标准物质的5-6个结果取平均值；利用Excel以仪器测量平均值为横坐标，真实值为纵坐标绘制标准曲线，将样品测量值代入标准曲线计算后即为样品真实值，同时将所有样品中插入的标准物质测量值也代入标准曲线中计算，并与真实值比较，校正系数范围在0.9-1.1之间，则证明数据可信。
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