Name: characterization, quantification and compound-specific isotopic analysis of pyrogenic carbon using benzene polycarboxylic acids (bpca)
Uploaded: 2016-05-16T15:00:00.000+00:00
Duration: 8 min 12 s
Description: Watch this Scientific Journal Video about Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA at JoVE.com

The authors thankfully acknowledge support by the following funding sources: the University of Zurich Research Priority Program "global change and biodiversity", the Swiss National Science Foundation projects 134452, 131922, 143891, 119950 and 134847, and the Deep Carbon Observatory - Deep Energy award 60040915.

1.一般预防措施和准备工作<ol><li>使用干净的，脱钙（10％盐酸浴），燃烧玻璃器皿（500℃，5小时），彻底清洁工具和超纯，高压液相色谱（HPLC）级的水和溶剂在整个过程。 </li><li>冷冻干燥，并用无碳球磨机58均匀样品，并确定它们的总的有机碳（TOC）的通过元素分析59-60的内容。 注:化学品及实验室设备的纯度要求是BPCAs的特定化合物-14 C-分析特别高。包括空白评估49和刷卡测试61，监测样品污染的潜在来源。 </li></ol> 2. HNO 3消解<ol><li>称取冻干并均质化样品（ 比照 1.2）到石英消化管并覆盖针对与铝箔灰尘。 <ol><li>对于热解碳quantificati并定性的目的，使用含有&gt; 1毫克TOC 42个样品。因此，在土壤和沉积物，使用约 200的情况下- 400毫克，并富含有机质的样品，如纯木炭，用约 10时- 20％的消化毫克管。 </li><li>对于热解碳随后的特定化合物同位素分析（13℃和14℃），确保样品中含有足够的BPCA-C，以满足将第6步之后，如果要使用的特定同位素比质谱的检出限没有关于样本的可用（ 例如，从以前的测量）热解碳量的先验信息，第一量化其热解碳含量（步骤1 - 5），后来准备更多的样品，如果BPCA-C收益率太低同位素分析。 注:包括与已知的热解碳和13 C和14 C的含量（ 如空白和参考样本，从"黑碳参考资料"， 比照结果本身ction）。这将允许以检查热解碳量化的重复性和分析后使特定化合物同位素测量空白校正计算。 </li></ol></li><li>加入2毫升65％HNO 3到消化管中，使用涡旋混合器，以协助将样品完全润湿，然后插入消化管到压力室中。根据手册62关闭压力室并把它们放在一个预加热的炉中在170℃8小时。 注意:消化后，让室内降温的烤箱内，只打开它们通风柜下他们达到室温后，由于有害气体可能逃过。 </li><li>过滤，用水将样品成用一次性玻璃纤维过滤器（&lt;0.7微米），例如，在玻璃注射器容量瓶，并调整体积至25ml。需要稀释停止进一步消化。 注意:含有BPCAs 25毫溶液可以储存在长达2个月前，冰箱进一步处理。消化可以在原则上也可以用其它的仪器与加压微波系统16执行，例如。在这种情况下，测试应参考材料来运行以检查BPCA回收率和方法的再现性（ 参见代表性结果部分）。 </li></ol> 3.去除阳离子<ol><li>对于每个样品，以11 3g的柱的阳离子交换树脂的准备两个玻璃柱（400毫米的高度，15毫米直径）。水2柱体积，1柱体积的2 M氢氧化钠，水2倍柱体积中和pH值，2 M盐酸1柱体积，并且水最终2倍柱体积:通过用连续漂洗调理列内的树脂。 </li><li>检查水，这是通过其调节后的树脂洗涤的导电性。树脂被认为是正确的空调时的电导率低于2μS厘米<suP&gt; -1。 </li><li>把样品的一半（ 即12.5毫升， 比照步骤2.3）每一列上，冲洗，用10ml水依次5倍，并冷冻后干燥该水溶液。样品是在冷冻干燥后稳定，如果它是在阴凉处保持干燥可以存储最多一个星期进一步处理之前。 注意:使用液氮冷冻样品（&#39;卡扣冻结"），因为它避免了冻结出的HNO 3中 ，这可能会导致的强的非冷冻酸溶液水坑的。确保冷冻干燥机是耐酸由真空泵油烟度好并测试潜在的污染，如果BPCAs的特定化合物-14 C-分析的目的。 </li></ol> 4，拆卸非极性化合物<ol><li>根据制造商的说明手册调理C18固相萃取柱， 即 ，连续地用2.5毫升甲醇，2.5毫升的水一漂洗它们ND最终与2.5甲醇/水的混合物（1:1体积/体积）。 </li><li>重新溶解的冷冻干燥残留物的3ml甲醇/水（1:1体积/体积）。洗脱它各占一半（1.5毫升），在一个单独的C18固相萃取柱到2.5毫升试管。与另一个1毫升甲醇/水的冲洗盒（1:1体积/体积）。 </li><li>干燥使用真空浓缩与样品溶液的试管，例如，加热至45℃，并用约 50毫巴的真空。蒸发的其他方法也可以使用，例如，用N 2气吹式系统，在第6步。 </li><li>再溶解在试管中的残余物用1毫升水。支持解散与旋涡混合器并转移到1.5毫升自动进样瓶。 注:样品可以在此阶段42被存储在冰箱中长达3个月。 </li></ol> 5.色谱<ol><li>由980毫升水混合20毫升85％的正磷酸制备溶剂A和过滤标使用真空通过一次性玻璃纤维过滤olution。不要溶剂A暴露在阳光下，为了避免藻类生长24小时之内使用它。使用纯HPLC级乙腈作为溶剂B. </li><li>准备市售BPCAs（连苯三，偏苯三，均苯，pentacarboxylic和苯六甲酸）的标准溶液，以产生一个外部标准浓度系列（ 例如 ，含有5，20，60，100，150和各BPCA 250微克6小瓶一起混合在1毫升水，分别）。 </li><li>进行表1和表2中的设置的色谱法和由相应BPCA峰面积进行比较，以外部标准系列63的测量量化BPCA内容。 </li><li>表达样本[克/公斤]或BPCA-C / TOC [％]的BPCA-C /干重热解碳量的调查结果。此外，热解碳的样品中的质量特性可以用individ的比例进行说明UAL BPCAs，例如 B6CA的比例（B6CA / BPCA [％]）表示热解碳44的芳族缩合度。 </li></ol>纯化BPCAs为随后13 C 6.湿式氧化和14 C-分析<ol><li>以下步骤5.3，收集足够数量的个体BPCAs（ 例如，&gt; 30微克BPCA-C的当前加速器质谱仪49,64）使用连接到HPLC 49，然后一个馏分收集器通过用吹下来的级分除去溶剂一个温柔的N 2流，同时它们加热到70℃。只有极少量的液体的磷酸，包括BPCAs，将留在瓶中。 </li><li>通过将2克的 Na 2 S 2 O 8在50ml水中，用24小时内新鲜制备制备氧化剂。 注意:重​​结晶的过硫酸钠两次通过完全溶解几克，以提高其纯度在热水中，然后收集水后的固体冷却65-66。 </li><li>重新溶解吹倒残余物（步骤6.1）用4ml水和转让样品12毫升气密硼硅酸盐瓶。加入1 ml的氧化剂和关闭含丁基橡胶隔膜的标准上限。 </li><li>吹扫气密小瓶包括与他的水溶液8分钟从小瓶并将该溶液66除去CO 2。 </li><li>通过在100℃加热60分钟氧化在气密小瓶样品。 </li><li>直接分析从同位素比质谱氧化的CO 2的13 C含量65-66和加速质谱仪14 C含量67-68。 注意:氧化样品可以前13 C和/或14 C-分析被存储为至少一个66周 。 </li></ol>

The authors have nothing to disclose.

相比其他可用的热解碳的方法78-79，当BPCA方法有几个重要的优点:ⅰ）检测热解碳在宽范围燃烧连续体， 即 ，它是敏感稍向烧焦生物质以及高温字符和烟尘42 ，70％，ii）其能够同时表征16,44,80-81，量化20,42和同位素分析热解碳49-50,66,73,82-83，ⅲ）它适用于很宽的范围内的环境样品的材料42,70，和iv）其方法已强烈审查，并且可以放置成与其他热解碳的方法44,47,70,84-85评估一个一致的框架。对于所有这些原因，BPCA的做法可以说是目前可用的，其基本假设是很好的约束，并不断对其他方法测试的最通用的方法热解碳。 上述协议巩固了STREN先前BPCA方法成一个单一的过程gths，是高度可再现的，简单采用，并且可以很容易地扩展和修改，以特定的要求。例如，当层析用pH梯度，而不是有机溶剂中进行，在线BPCAs的同位素比率监测是可能42，消除了对湿式氧化步骤的需要。同样地，去除阳离子和/或非极性化合物（步骤3和4）时，已知特定的样品不含有任何这样的化合物可以被跳过（ 例如 ，在实验室生产的字符的某些情况下）。 像每一个热解碳的方法，该方法BPCA具有一定的局限性，太。在这方面，需要注意的是BPCA做法本质上低估了总热解碳量的样品中是很重要的:该方法破坏了热解碳环结构的较大部分，以提取其BPCA积木，因此不能定量地恢复所有热解碳的形式BPCAs的20,86。转换系数在过去已经提出来翻译BPCA产量为总热解碳含量。然而，找到一个正确的转换因子是因为在大多数字符41,48,80,86异构度芳香缩合的实际上是不可能的。在许多情况下，样品的热解碳量是相对于彼此42,81,87-88比较。然后，我们建议不要使用任何转换系数和"作为衡量"48只是简单地报告BPCA数据。在特殊情况下，采取BPCA收益率来估算绝对量热解碳24,89-90，最初公布的换算系数2.27 20似乎是恰当的，因为它转换BPCA产生成热解碳86含量保守估计。 与热解碳的方法的另一个困难是，它们是与干扰，非热解碳材料和/或热解碳在分析本身期间产生，导致的过高估计潜在的敏感实际的热解碳含量在样品70。该BPCA方法是针对这样的干扰物质70很坚固，自身并不16,70,86产生任何热解碳和在性质上是保守的（上文第比照 ）。即使石墨，化学性质非常类似物质热解碳，但成岩出身，不BPCA干扰测量（施耐德，MPW 未公布结果。苏黎世，（2013））。到目前为止，唯一已知的非热解碳干扰的方法BPCA都是一些凝结，真菌91芳香颜料，这应该是绝大多数研究86定量忽略不计。该方法BPCA与其同时定性，定量和13℃和14 C-同位素信息，从而为热解碳的各学科调查的优秀工具。

在一个完整的燃烧过程中，生物质或矿物燃料被转换成CO 2，H 2 O和无机残余物（灰）。然而，在本地或时间限制的氧气，燃烧变得不完全和热解发生，产生被称为焦炭1固体有机残留物。这些烧焦残基也被称为热解的有机物（PyOM）和主要由热解碳（PYC）或，同义，黑碳（BC）2-4。炭化过程是无所不在的，可以是自然和人为燃烧5-6的一部分。野火是一个重要的自然过程，固有的最生态系统，它产生每年4,7-10热解碳的显著量。同样，矿物燃料的工业和交通能源生产的燃烧热解碳呈现11-13的一个重要的人为来源。两个源向热解碳的环境中的普及:热解碳是存在于空气中，在气溶胶13-14的形式，在水为颗粒状或溶解的有机物质15-17，以及在冰芯​​18-19，土壤20-21，和沉积物22-24在尺寸改变从微米到纳米（ 如大烧焦的树干森林火灾或逃避柴油发动机尾气纳米尺度的烟尘颗粒后）。热解碳的环境中的普及不仅是由于大的生产速度，而且其长期持久性和抗降解25-26相对稳定。虽然确切的周转时间尚未确定，可能取决于具体的环境条件27-28，它似乎很清楚，热解碳是不太容易分解成二氧化碳比大多数其他形式的有机碳29-30。这种看法对全球碳循环的一个重要含义:如烧焦的​​材料热解碳卖场在相当长的时间，他们在有机形式，否则将被迅速隔离řçespired为二氧化碳 ，从而减少随着时间的推移31-32大气温室气体浓度。 除了气候缓和方面，字符有进一步的与环境相关的属性。其孔隙率高，比表面积大，表面负电荷可以固定有害化合物33和提高土壤肥力34-35。字符作为一个潜在的有利土壤改良剂识别导致所谓的生物炭技术36的新兴领域。生物炭很可能会在大尺度上在未来几年产生的，因而显著增加丰富的热解碳在土壤中37。此外，森林火灾的发生和化石燃料的燃烧，也预计将保持高在21 世纪的过程中，不断地贡献大量的热解碳对环境11,38-39。热解碳的另一越来越重要的来源可能是纳米技术也使用小号热解碳类化合物40-41。它是如此重要，以检测，表征和为了研究它们的性质和了解其在环境中的角色精确地量化这些致热材料。 在这里，我们目前使用的国家的最先进的特定化合物的方法来分析热解碳各种样品中:最新一代的苯聚羧酸（BPCA）方法42。这种方法是热解碳研究中广泛适用的，因为它直接靶向热解碳的"骨架":其在热处理43-45中形成和该环稠合的结构，因此固有的所有各种形式的热解碳5,46。然而，这些结构不是通过色谱手段直接评，由于它们的大小和异质性。以色谱分析这种热解的化合物，热解碳是首先用硝酸消化高的温度和压力下，打破了大的多环结构分解成它的组成部分，各个BPCAs（ 比照 图1）。该BPCAs随后，经过几次纯化步骤，适合进行色谱分析20,42。由此热解碳分离并在分子水平上分析，可以用来量化热解碳丰度在环境室20,42。当B3-，B4-，B5-和B6CA的相对产量进行比较（ 参见 图 1）的BPCA方法另外表征所研究的热解碳:不同羧BPCAs的各自比例被链接到原始的多环结构的大小，是因此，热解碳指示的质量和热解温度44,47-48的。此外，所提出的方法允许对C同位素组成热解碳（13 C和14 C）的判定，因为个人BPCAs，直接从纯热解碳结构推导，可以是同位素ANA之后隔离裂解（ 参见 图1，步骤5和6）49。热解碳的特定化合物的同位素分析是极大的兴趣50的，因为它可以使用， 例如，在热带地区51-52个字符的前体的生物量来区分，以导出烧焦材料53-54的年龄或跟踪热解碳在用同位素标记26,55-56碳循环的研究。关于热解碳还有BPCA方法的历史，发展，特别是应用程序的更多信息可Wiedemeier可以发现，2014年57，从那里上述段落和讨论的一部分的一部分进行编译。

<table><tbody><tr><td>ball mill</td><td>Retsch</td><td>N/A</td><td>ball mill with carbon-free grinding jars and balls (Retsch MM 200 with agate grinding jars and balls)&amp;nbsp;</td></tr><tr><td>combustion oven</td><td>Nabertherm</td><td>N/A</td><td>combustion oven/muffle furnace with a temperature of 500 &amp;deg;C (Nabertherm L40/11 or similar)</td></tr><tr><td>pressure bombs with PTFE pressure chambers,&lt;br/&gt; quartz digestion tubes with quartz lids</td><td>Seif Aufschlusstechnik, Unterschleissheim, Germany</td><td>N/A</td><td>Helma U. Rudolf Seif Aufschlusstechnik&lt;br/&gt; Fastlingerring 67&lt;br/&gt; 85716 Unterschleissheim&lt;br/&gt; Germany&lt;br/&gt; Tel: (+49) 89 3108181</td></tr><tr><td>vortex mixer</td><td>common lab supply</td><td>N/A</td><td></td></tr><tr><td>oven&amp;nbsp;</td><td>Thermo Scientific</td><td>50051010</td><td>drying oven with constant temperature (Thermo Scientific Heraeus or similar)</td></tr><tr><td>vacuum manifold system&lt;br/&gt; with PTFE connectors</td><td>Machery Nagel</td><td>Chromabond&lt;br/&gt; 730151&lt;br/&gt; 730106</td><td>ftp://ftp.mn-net.com/english/Instruction_leaflets/Chromatography/SPE/CHROMABOND_VK_DE_EN.pdf</td></tr><tr><td>reusable glass syringes with disposable glass fibre filters</td><td>Machery Nagel&amp;nbsp;</td><td>730172&lt;br/&gt; 730192</td><td>http://www.mn-net.com/SPEStart/SPEaccessories/EmptySPEcolumns/tabid/4285/language/en-US/Default.aspx</td></tr><tr><td>25 ml volumetric glass flasks</td><td>common lab supply</td><td>N/A</td><td>In contrast to all other glassware, do not combust to ensure volumetric accuracy. Instead, clean in acid bath, with ultrasound and with ultrapure water.</td></tr><tr><td>chromatographic glass columns with frit and PTFE stopcock and glass wool</td><td>custom made</td><td>N/A</td><td>dimensions of glass columns:&lt;br/&gt; &lt;em&gt;ca.&lt;/em&gt; 40 cm long, &lt;em&gt;ca.&lt;/em&gt; 1.5&amp;nbsp;cm in diameter</td></tr><tr><td>cation exchange resin</td><td>Sigma Aldrich</td><td>217514</td><td>Dowex 50 WX8 400</td></tr><tr><td>conductivity meter</td><td>WTW</td><td>300243</td><td>LF 320 Set</td></tr><tr><td>100 ml conical flasks for freeze drier</td><td>common lab supply&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>liquid nitrogen</td><td>common lab equipment</td><td>N/A</td><td>for snap-freezing the aequous solution after removal of cations</td></tr><tr><td>freeze dryer</td><td>Christ</td><td>N/A</td><td>Alpha 2 - 4 LD plus</td></tr><tr><td>C18 solid phase extraction cartridges</td><td>Supelco</td><td>52603-U</td><td>http://www.sigmaaldrich.com/catalog/product/supelco/52603u?lang=de&amp;amp;region=CH</td></tr><tr><td>2.5 ml glass test tubes</td><td>Agilent Technologies</td><td>5022-6534</td><td>http://www.chem.agilent.com/store/en_US/Prod-5022-6534/5022-6534?navAction=push&amp;amp;navCount=0</td></tr><tr><td>concentrator&amp;nbsp;</td><td>Eppendorf</td><td>5305000.100</td><td></td></tr><tr><td>1.5 ml HPLC autosampler vials</td><td>depending on HPLC</td><td>N/A</td><td></td></tr><tr><td>6 ml fraction collector vials</td><td>depending on HPLC</td><td>N/A</td><td></td></tr><tr><td>high purity N&lt;sub&gt;2&lt;/sub&gt; gas</td><td>common lab equipment</td><td>N/A</td><td></td></tr><tr><td>12 ml borosilicate gas tight vials</td><td>Labco</td><td>538W</td><td>http://www.labco.co.uk/europe/gas.htm#doublewad12ml</td></tr><tr><td>needles</td><td>B Braun</td><td>4665643</td><td>http://www.bbraun.ch/cps/rde/xchg/cw-bbraun-de-ch/hs.xsl/products.html?prid=PRID00000510</td></tr><tr><td>high purity He gas</td><td>common lab equipment</td><td>N/A</td><td></td></tr><tr><td>HNO&lt;sub&gt;3&lt;/sub&gt; (65%) p.a.</td><td>Sigma Aldrich</td><td>84378</td><td>http://www.sigmaaldrich.com/catalog/product/sial/84378?lang=de&amp;amp;region=CH</td></tr><tr><td>2 M HCl</td><td>Sigma Aldrich</td><td>258148</td><td>mix with ultrapure water to achieve 2 M solution</td></tr><tr><td>2 M NaOH</td><td>Sigma Aldrich</td><td>71691</td><td>mix with ultrapure water to achieve 2 M solution</td></tr><tr><td>methanol</td><td>Sigma Aldrich</td><td>34860</td><td>http://www.sigmaaldrich.com/catalog/product/sial/34860?lang=de&amp;amp;region=CH</td></tr><tr><td>water</td><td>Milli-Q</td><td>Z00QSV0WW</td><td>Type 1 grade, optimized for low carbon</td></tr><tr><td>orthophosphoric acid</td><td>Sigma Aldrich</td><td>79606</td><td>http://www.sigmaaldrich.com/catalog/product/fluka/79606?lang=de&amp;amp;region=CH</td></tr><tr><td>acetonitrile</td><td>Sigma Aldrich</td><td>34851</td><td>http://www.sigmaaldrich.com/catalog/product/sial/34851?lang=de&amp;amp;region=CH</td></tr><tr><td>C18 reversed phase column</td><td>Agilent Technologies</td><td>685975-902</td><td>Agilent Poroshell 120 SB-C18 (4.6 x 100 mm)</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;8, &lt;/sub&gt;sodium persulfate</td><td>Sigma Aldrich</td><td>71890</td><td>http://www.sigmaaldrich.com/catalog/product/sial/71890?lang=de&amp;amp;region=CH</td></tr><tr><td>&lt;strong&gt;BPCA standards&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>trimellitic acid</td><td>Sigma Aldrich</td><td>92119</td><td>http://www.sigmaaldrich.com/catalog/product/fluka/92119?lang=de&amp;amp;region=CH</td></tr><tr><td>hemimellitic acid</td><td>Sigma Aldrich</td><td>51520</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/51520?lang=de&amp;amp;region=CH</td></tr><tr><td>pyromellitic acid</td><td>Sigma Aldrich</td><td>83181</td><td>http://www.sigmaaldrich.com/catalog/search?&lt;br/&gt;
term=83181&amp;amp;interface=All&amp;amp;&lt;br/&gt;N=0&amp;amp;mode=match%20partialmax&amp;amp;&lt;br/&gt;lang=de&amp;amp;region=CH&amp;amp;focus=product</td></tr><tr><td>benzenepentacarboxylic acid</td><td>Sigma Aldrich</td><td>S437107</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/s437107?lang=de&amp;amp;region=CH</td></tr><tr><td>mellitic acid</td><td>Sigma Aldrich</td><td>M2705</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/m2705?lang=de&amp;amp;region=CH</td></tr><tr><td>&lt;strong&gt;oxidation standards&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>phtalic acid</td><td>Sigma-Aldrich</td><td>80010</td><td>http://www.sigmaaldrich.com/catalog/product/sial/80010?lang=de&amp;amp;region=CH</td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/s7903?lang=de&amp;amp;region=CH</td></tr><tr><td>black carbon reference materials</td><td>University of Zurich</td><td>N/A</td><td>http://www.geo.uzh.ch/en/units/physische-geographie-boden-biogeographie/services/black-carbon-reference-materials</td></tr></tbody></table>

characterization, quantification and compound-specific isotopic analysis of pyrogenic carbon using benzene polycarboxylic acids (bpca)

火衍生的热解碳（PYC），有时也被称为黑碳（BC）是生物量和化石燃料的燃烧，如炭和烟灰的含碳固体残留物。热解碳是普遍存在于环境中，由于其持续时间长，其丰度甚至可能在全球野火活动预计增加和化石燃料的燃烧持续增加。热解碳也越来越多地从工业裂解有机废弃物，这将产生烧焦的土壤改良剂（炭）生产。此外，纳米技术的出现，也可能导致在热解碳状化合物的释放到环境中。因此，这是一个高优先级，以可靠地检测，表征和以调查他们的环境性能，并了解他们在碳循环中的作用量化这些烧焦的材料。 这里，我们提出的苯多羧酸（BPCA）方法，它允许热解碳的characteri的同时评估STICS，数量和在分子水平上的同位素组合物（13℃和14℃）。该方法适用于很宽的范围内的环境样品材料的，并检测热解碳在宽范围燃烧连续体， 即，它是敏感稍向烧焦生物质以及高温字符和烟灰。这里介绍的BPCA协议是简单的使用，高度重现，以及容易可扩展和可修改具体要求。因此它提供了热解碳的各学科调查，从考古学和环境取证，以生物炭和碳循环研究的多功能工具。

我们建议以测试通过测量套件已广泛在文献44,48,69-77用于各种方法的发展和比较良好描述热解碳材料（"黑碳参考材料"），该方法的建立。在参考资料信息可从苏黎世大学(http://www.geo.uzh.ch/en/units/physische-geographie-boden-biogeographie/services/black-carbon-reference-materials). 所描述的过程允许通过HPLC所有BPCA目标化合物的基线​​分离。参考材料的黑钙土&#39;（具有显著热解碳含量粉土）和草炭（来自稻制）的色谱图显示在图2，通过调节在表1和2的色谱参数（ 例如，色谱法温度，溶剂A的pH值或流速等 ），分离可进一步修饰为特定需要42,63。 参考材料的外部标准（步骤5.3）。色谱定量分析应该产生在图3中所示的热解碳的值。请注意，该过程是轻微的变化（ 例如，步骤3或4在特定情况下的省略），可以导致更高的热解碳值。通常，回收率应与纯BPCA标准进行检查:掺入参考材料可有助于检测在步骤3和4不成比例损失和得到约在步骤5 42,63的色谱性能信息。 表3示出纯化的参考材料BPCAs时所获得的分析它们的碳同位素含量步骤6之后对于13 C和14 C值可靠的结果，当务之急是要收集足够数量BPCA-C（如 &gt; 30微克BPCA-C当前加速器质谱仪， 参见 图4），并采取一切可能的措施，以尽量减少外来Ç49样品的污染。 除了 ​​检查如上述所描述的方法建立的参考材料，这是非常可取准备和在重复测量样品，都为热解碳量化（步骤5）和随后的特定化合物-13 C和BPCAs（步骤6的14 C-分析）。 <img alt="图1" src="/files/ftp_upload/53922/53922fig1.jpg" /> 图1: 本BPCA分析过程在协议步骤2中 ，热解碳的多环芳族稠结构被消化，产生不同BPCAs，这是个烯进一步清洁（ 步骤 3和4）和色谱分析，分离（ 步骤5）。湿式氧化（ 第6步 ）后，纯化BPCAs适合进行同位素比质谱特定化合物同位素分析（13℃和14℃）。 <a href="https://www.jove.com/files/ftp_upload/53922/53922fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53922/53922fig2.jpg" /> 图 2: 用于色谱分离BPCA显示的是黑碳参考资料"黑钙土"（A）和"草CHAR"（B）。 B5CA; 1,2,4,5- 1,2,3,5-，1,2,3,4- B4CA;基线分离所有BPCA目标化合物（B6CA实现1,2,4-， 1,2,3-B3CA）42。在<html>形成对黑碳的参考材料可从苏黎世大学(http://www.geo.uzh.ch/en/units/physische-geographie-boden-biogeographie/services/black-carbon-reference-materials).这个数字是从Wiedemeier 等修改2013 42，并从爱思唯尔的翻印许可。 <a href="https://www.jove.com/files/ftp_upload/53922/53922fig2large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53922/53922fig3.jpg" /> 图3: 不同 的 黑碳参考材料的复制热解碳测量实验室重复错误条比符号尺寸较小，变异系数平均为5％（最低1％，最高10％），这个数字是从Wiedemeier 等修改人 2013 42和转载爱思唯尔的权限。"https://www.jove.com/files/ftp_upload/53922/53922fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53922/53922fig4.jpg" /> 图4: 放射性碳（14 C）值B5CA和B6CA从现代和煤焦化石中分离出的给定的错误是由更正为仪器加速器质谱仪背景和空白湿式氧化的。固体灰线代表用于各个样品和所确定的平均外部污染的实际F 14 C值的混合物形式的理想化的线路。这个数字是从Gierga 等修改2014 49和转载爱思唯尔的权限。 <a href="https://www.jove.com/files/ftp_upload/53922/53922fig4large.jpg" target="_blank">请点击此处查看次的放大版本</a>是图。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>流动相A </td><td> 20毫升邻位在980毫升超纯水中的磷酸（85％） </td></tr><tr><td>流动相B </td><td>乙腈</td></tr><tr><td>柱</td><td> C18反相（详情参见物料清单） </td></tr><tr><td>柱温</td><td> 15℃ </td></tr><tr><td>流量</td><td> 0.4毫升分钟-1 </td></tr><tr><td>识别</td><td>保留时间，在216nm处的UV吸收</td></tr><tr><td>量化</td><td> BPCAs的外部标准</td></tr><tr><td>压力</td><td> 约 120条</td></tr></tbody></table> 表1: 色谱设置。 <table border="1" fo:keep-together.within-page="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td>时间</td><td>流动相B </td></tr><tr><td> [分钟] </td><td> [体积％] </td></tr><tr><td> 0 </td><td> 0.5 </td></tr><tr><td>五</td><td> 0.5 </td></tr><tr><td> 25.9 </td><td>三十</td></tr><tr><td> 26 </td><td> 95 </td></tr><tr><td> 28 </td><td> 95 </td></tr><tr><td> 28.1 </td><td> 0.5 </td></tr><tr><td>三十</td><td> 0.5 </td></tr></tbody></table> 表2: 混合流动相的梯度。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="2">散焦</td><td colspan="2"> BPCA </td></tr><tr><td></td><td colspan="4"> δ13 ​​C [‰VPDB对比] </td></tr><tr><td>栗炭</td><td> -27.4 一个 </td><tD&gt;±0.4 一 </td><td> -27.7 </td><td> ±0.8 </td></tr><tr><td>玉米CHAR </td><td> -12.9 </td><td> ±0.4 </td><td> -13.0 </td><td> ±0.4 </td></tr><tr><td></td><td colspan="4"> F 14 C [％] </td></tr><tr><td>现代炭</td><td> 1.142 b </td><td> ±0.004 b </td><td> 1.13 </td><td> ±0.013 </td></tr><tr><td>化石CHAR </td><td> 0.003 b </td><td> ±0.001 b </td><td> 0.014 </td><td> ±0.001 </td></tr></tbody></table> 表 3:分别在第5步Howev同时收集到的碳同位素值（δ13 C和F 14 C）参考木炭材料和复合专用相应BPCAs同位素分析的BPCA值代表B6CA和B5CA呃，个别BPCAs的同位素分析，可以类似地，当BPCAs单独收集实现。散装char数据是从Yarnes 等人 （2011）73为栗炭（a）和从Gierga 等人 （2014）49用于化石和现代炭（b）中 。对于δ13 C测量误差是从一式三份的标准误差而歼14 C-测量错误（批量字符:ETH-50456，ETH-50458; BPCA:ETH-62324，ETH-62335）由误 ​​差传播64而得。

Watch this Scientific Journal Video about Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA at JoVE.com

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

表征，气相炭用苯聚羧酸的定量和特定化合物同位素分析（BPCA）

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

characterization-quantification-compound-specific-isotopic-analysis

Nanyang Technological University

Research

JoVE Journal

Chemistry

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

15.3K 次观看.  University of Zurich. We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC. 

视频: Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

PTR-ToF-MS结合自动采样系统和食品研究定制数据分析:生物过程监测，筛选和鼻空间分析

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

科研

化学

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

在线含硫化合物在复杂油气矩阵氮分析

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

该Faix方法:生物油羰基官能团通过电位滴定法测定

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

环境科学

Quantitative Analysis by Thermogravimetry-Mass Spectrum Analysis for Reactions with Evolved Gases

During energy conversion, material production, and metallurgy processes, reactions often have the features of unsteadiness, multistep, and multi-intermediates. Thermogravimetry-mass spectrum (TG-MS) is seen as a powerful tool to study reaction features. However, reaction details and reaction mechanics have not been effectively obtained directly from the ion current of TG-MS. Here, we provide a method of an equivalent characteristic spectrum analysis (ECSA) for analyzing the mass spectrum and giving the mass flow rate of reaction gases as precise as possible. The ECSA can effectively separate overlapping ion peaks and then eliminate the mass discrimination and temperature-dependent effect. Two example experiments are presented: (1) the decomposition of CaCO3 with evolved gas of CO2 and the decomposition of hydromagnesite with evolved gas of CO2 and H2O, to evaluate the ECSA on single-component system measurement and (2) the thermal pyrolysis of Zhundong coal with evolved gases of inorganic gases CO, H2, and CO2, and organic gases C2H4, C2H6, C3H8, C6H14, etc., to evaluate the ECSA on multi-component system measurement. Based on the successful calibration of the characteristic spectrum and relative sensitivity of specific gas and the ECSA on mass spectrum, we demonstrate that the ECSA accurately gives the mass flow rates of each evolved gas, including organic or inorganic gases, for not only single but multi-component reactions, which cannot be implemented by the traditional measurements.

Precise determination of the evolved gases&#39; flow rate is key to study the details of reactions. We provide a novel quantitative analysis method of equivalent characteristic spectrum analysis for thermogravimetry-mass spectrum analysis by establishing the calibration system of the characteristic spectrum and relative sensitivity, for obtaining the flow rate.

热重分析-质谱分析对演化气体反应的定量分析

During energy conversion, material production, and metallurgy processes, reactions often have the features of unsteadiness, multistep, and ...

工程学

A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps.

This method for two-step pyrolysis online coupled to gas chromatography with mass spectrometric detection and data evaluation protocol can be used for multi-component analysis of tattoo inks and discrimination of counterfeit products.

一种用于检测纹身油墨成分和假冒产品的两步热解-气相色谱法

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

生物工程

Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is crucial. There are significant challenges to overcome when measuring trace compounds in a complex matrix such as fuels. Background interferences and matrix effects can create limitations to routine analytical instrumentation, such as GC-MS. In order to facilitate specific and quantitative measurements of trace nitrogen compounds in fuels, a nitrogen-specific detector is ideal. In this method, a nitrogen chemiluminescence detector (NCD) is used to detect nitrogen compounds in fuels. NCD utilizes a nitrogen-specific reaction that does not involve the hydrocarbon background. Two-dimensional (GCxGC) gas chromatography is a powerful characterization technique as it provides superior separation capabilities to one-dimensional gas chromatography methods. When GCxGC is paired with a NCD, the problematic nitrogen compounds found in fuels can be extensively characterized without background interference. The method presented in this manuscript details the process for measuring different nitrogen-containing compound classes in fuels with little sample preparation. Overall, this GCxGC-NCD method has been shown to be a valuable tool to enhance the understanding of the chemical composition of nitrogen-containing compounds in fuels and their impact on fuel stability. The % RSD for this method is &#60;5% for intraday and &#60;10% for interday analyses; the LOD is 1.7 ppm and the LOQ is 5.5 ppm.

Here, we present a method utilizing two-dimensional gas chromatography and nitrogen chemiluminescence detection (GCxGC-NCD) to extensively characterize the different classes of nitrogen-containing compounds in diesel and jet fuels.

多维气相色谱在燃料中的氮化合物表征

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...