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. General Precautions and Preparations&#160;
<ol>
	<li>Use only clean, decalcified (10% HCl bath) and combusted glassware (500 &#176;C for 5 hr), thoroughly cleaned tools and ultrapure, high pressure liquid chromatography (HPLC) grade water and solvents for the entire procedure.</li>
	<li>Freeze dry and homogenize samples with a carbon-free ball mill58 and determine their total organic carbon (TOC) content by elemental analysis59-60 . 
	Note: Purity requirements for chemicals and laboratory equipment are especially high for compound-specific 14C analysis of BPCAs. Include blank assessments49 and swipe tests61 to monitor potential sources of sample contamination.</li>
</ol>
2. HNO3 Digestion
<ol>
	<li>Weigh freeze-dried and homogenized samples (cf. 1.2.) into quartz digestion tubes and cover against dust with aluminum foil.
	<ol>
		<li>For PyC quantification and characterization purposes, use samples containing &#62; 1 mg TOC42. Thus, in the case of soils and sediments, use ca. 200 - 400 mg and in the case of organic-rich samples, such as pure charcoals, use ca. 10 - 20 mg per digestion tube.</li>
		<li>For subsequent compound-specific isotopic analysis of PyC (13C and 14C), make sure the sample contains enough BPCA-C to meet the detection limits of the particular isotope-ratio mass spectrometer that will be used after step 6. If there is no a priori information about a sample&#39;s PyC quantity available (e.g., from previous measurements), first quantify its PyC content (steps 1 - 5) and prepare more sample later if the BPCA-C yields are too low for isotopic analysis. 
		Note: Include blank and reference samples with known PyC and 13C and 14C content (e.g., from the &#34;black carbon reference materials&#34;, cf. results section). This will allow to check the reproducibility of the PyC quantification and enable blank correction calculations of the compound-specific isotopic measurements after analysis.</li>
	</ol>
	</li>
	<li>Add 2 ml of 65% HNO3 into the digestion tubes, use a vortex mixer to assist thorough wetting of the sample and then insert the digestion tubes into the pressure chamber. Close the pressure chambers according to the manual62 and put them into a pre-heated oven at 170 &#176;C for 8 hr. 
	CAUTION: After digestion, let the chambers cool down inside the oven and only open them under the fume hood after they reached room temperature because harmful gases may escape.</li>
	<li>Filter the samples with water into volumetric flasks using disposable glass fiber filters (&#60; 0.7 microns), for instance in glass syringes, and adjust volume to 25 ml. The dilution is needed to stop further digestion. 
	Note: The 25 ml solutions containing the BPCAs can be stored in the refrigerator for up to 2 months before further processing. Digestion can in principle also be performed using other instrumentation, for example with a pressurized microwave system16. In that case, tests should be run with reference materials to check BPCA recoveries and method reproducibility (cf. representative results section).</li>
</ol>
3. Removal of Cations&#160;
<ol>
	<li>For each sample, prepare two glass columns (400 mm height, 15 mm diameter) with 11 g of cation exchange resin per column. Condition the resin inside the columns by consecutively rinsing it with: 2 column volumes of water, 1 column volume of 2 M NaOH, 2 column volumes of water for neutralizing pH, 1 column volume of 2 M HCl, and eventually 2 column volumes of water.</li>
	<li>Check the conductivity of the water, which is rinsed through the resin after its conditioning. The resin is considered as properly conditioned when the conductivity is below 2 &#181;S cm-1.</li>
	<li>Put one half of the sample (i.e., 12.5 ml, cf. step 2.3) on each column, rinse sequentially 5 times with 10 ml water and freeze dry the aqueous solution afterwards. The sample is stable after the freeze-drying and can be stored up to a week before further processing if it is kept dry in a dark and cool place. 
	Note: Use liquid nitrogen to freeze the samples (&#39;snap freezing&#39;) as it avoids the freezing out of HNO3, which can result in a puddle of strong non-freezing acid solution. Make sure the freeze drier is acid proof to a good degree and test for potential contamination by vacuum pump fumes if compound-specific 14C analysis of BPCAs is intended.</li>
</ol>
4. Removal of Apolar Compounds
<ol>
	<li>Condition the C18 solid phase extraction cartridges according to the manufacturer&#39;s instruction manual, i.e., consecutively rinse them with 2.5 ml of methanol, 2.5 ml of water and eventually with 2.5 ml of methanol/water (1:1 v/v).</li>
	<li>Redissolve the freeze-dried residue in 3 ml methanol/water (1:1 v/v). Elute each half of it (1.5 ml) over a separate C18 solid phase extraction cartridge into 2.5 ml test tubes. Rinse the cartridges with another 1 ml of methanol/water (1:1 v/v).</li>
	<li>Dry the test tubes with the sample solution, for instance using a vacuum concentrator, heated to 45 &#176;C and with a vacuum of ca. 50 mbar. Other means of evaporation can also be used, for instance a blow-down system with N2 gas as in step 6.</li>
	<li>Redissolve the residue in the test tube with 1 ml water. Support dissolution with vortex mixer and transfer to 1.5 ml auto sampler vials. 
	Note: Samples can be stored in the refrigerator for up to 3 months at this stage42.</li>
</ol>
5. Chromatography
<ol>
	<li>Prepare solvent A by mixing 20 ml of 85% orthophosphoric acid with 980 ml of water and filter the solution through a disposable glass fiber filter using vacuum. Do not expose solvent A to sunlight and use it within 24 hr in order to avoid algal growth. Use pure HPLC grade acetonitrile as solvent B.</li>
	<li>Prepare standard solutions of commercially available BPCAs (hemimellitic, trimellitic, pyromellitic, pentacarboxylic and mellitic acid) to produce an external standard concentration series (e.g., 6 vials containing 5, 20, 60, 100, 150 and 250 &#181;g of each BPCA mixed together in 1 ml water, respectively).</li>
	<li>Conduct the chromatography using the settings in Table 1 and Table 2 and quantify the BPCA contents by comparing the respective BPCA peak areas to the measurements of the external standard series63.</li>
	<li>Express findings of PyC quantity in BPCA-C/dry weight of the sample [g/kg] or BPCA-C/TOC [%]. Moreover, the qualitative characteristics of the PyC in the samples can be described using proportions of individual BPCAs, e.g., the proportion of B6CA (B6CA/BPCA [%]) indicates the degree of aromatic condensation of the PyC44.</li>
</ol>
6. Wet Oxidation of Purified BPCAs for Subsequent 13C and 14C Analysis
<ol>
	<li>Following step 5.3., collect the individual BPCAs in sufficient quantity (e.g., &#62; 30 &#181;g BPCA-C for current accelerator mass spectrometers49,64) using a fraction collector connected to the HPLC49 and then remove the solvents by blowing down the fractions with a gentle N2 stream while heating them to 70 &#176;C. Only tiny amounts of liquid phosphoric acid, including the BPCAs, will remain in the vial.</li>
	<li>Prepare the oxidizing reagent by dissolving 2 g of Na2S2O8 in 50 ml of water, freshly prepared within 24 hr of use. 
	Note: Recrystallize the sodium persulfate twice to improve its purity by fully dissolving several grams in hot water and then collecting the solid after the water has cooled65-66.</li>
	<li>Redissolve the blown down residue (step 6.1) with 4 ml water and transfer sample to 12 ml gas-tight borosilicate vial. Add 1 ml of oxidizing reagent and close with standard cap containing a butyl rubber septum.</li>
	<li>Purge the gas-tight vial including the aqueous solution with He for 8 min to remove CO2 from the vial and the solution66.</li>
	<li>Oxidize samples in the gas-tight vials by heating them at 100 &#176;C for 60 min.</li>
	<li>Directly analyze the CO2 from the oxidation on isotope-ratio mass spectrometers for 13C content65-66 and on accelerated mass spectrometers for 14C content67-68. 
	Note: Oxidized samples can be stored for at least one week66 before 13C and/or 14C analysis.</li>
</ol>

The authors have nothing to disclose.

The BPCA method has several important advantages when compared to other available PyC methods78-79: i) it detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot42,70, ii) it can simultaneously characterize16,44,80-81, quantify20,42 and isotopically analyze PyC49-50,66,73,82-83, iii) it is applicable to a very wide range of environmental sample materials42,70, and iv) its methodology has been intensely reviewed and could be put in a consistent framework with the assessments of other PyC methods44,47,70,84-85. For all these reasons, the BPCA approach is arguably the most versatile PyC method available to date, whose underlying assumptions are well constrained and have been continuously tested against other methods.
The above protocol consolidates the strengths of previous BPCA methods into a single procedure, is highly reproducible, simple to employ and can easily be extended and modified to specific requirements. For example, when chromatography is conducted with a pH gradient instead of an organic solvent, on-line isotope-ratio monitoring of BPCAs is possible42, obviating the need for the wet oxidation step. Similarly, the removal of cations and/or apolar compounds (steps 3 and 4) may be skipped when it is known that particular samples do not contain any such compounds (e.g., in some cases of laboratory-produced chars).
Like every PyC method, the BPCA procedure has some limitations, too. In this regard, it is important to note that the BPCA approach inherently underestimates total PyC quantity in the samples: the method destroys large parts of the PyC polycyclic structures in order to extract their BPCA building blocks, thus not quantitatively recovering all PyC in the form of BPCAs20,86. Conversion factors had been proposed in the past to translate BPCA yields into total PyC contents. However, finding one correct conversion factor is practically impossible because of the heterogeneous degree of aromatic condensation in most chars41,48,80,86. In many cases, PyC quantities of samples are compared relative to each other42,81,87-88. We then suggest not to use any conversion factors and to simply report BPCA data &#34;as measured&#34;48. In particular cases, when BPCA yields are taken to estimate absolute PyC quantities24,89-90, the originally published conversion factor20 of 2.27 seems appropriate as it converts the BPCA yields into conservative estimates of PyC contents86.
Another difficulty with PyC methods is that they are potentially sensitive to interfering, non-PyC materials and/or that PyC is produced during the analysis itself, leading to an overestimation of the actual PyC content in samples70. The BPCA approach is very robust against such interfering materials70, does not produce any PyC by itself16,70,86 and is conservative in nature (cf. above paragraph). Even graphite, a chemically very similar material to PyC but of petrogenic origin, does not interfere with BPCA measurements(Schneider, M. P. W. unpublished results. Zurich, (2013)). So far, the only known non-PyC interferences for the BPCA method are some condensed, aromatic pigments of fungi91, which should be quantitatively negligible for the vast majority of studies86. The BPCA method with its simultaneous qualitative, quantitative and 13C and 14C isotopic information is thus an excellent tool for the investigation of PyC in various disciplines.

In a complete combustion process, biomass or fossil fuel is converted into CO2, H2O and inorganic residues (ash). However, under local or temporal oxygen limitations, combustion becomes incomplete and pyrolysis takes place, producing a solid organic residue known as char1. These charred residues are also referred to as pyrogenic organic matter (PyOM) and mainly consist of pyrogenic carbon (PyC) or, synonymously, black carbon (BC)2-4. Charring processes are omnipresent and can be part of both natural and anthropogenic combustion5-6. Wildfire is an important natural process, intrinsic to most ecosystems, which produces a significant quantity of PyC each year4,7-10. Similarly, the burning of fossil fuel for energy production in industry and transport presents an important anthropogenic source of PyC11-13. Both sources contribute to the ubiquity of PyC in the environment: PyC is present in the air, in the form of aerosols13-14, in water as particulate or dissolved organic matter15-17, as well as in ice cores18-19, soils20-21, and sediments22-24 in sizes varying from m to nm (e.g., large charred tree trunk after a forest fire or nano-scale soot particles that escape a diesel engine exhaust). The ubiquity of PyC in the environment is not only due to large production rates but also to its long persistence and relative stability against degradation25-26. Although exact turnover times have not yet been established and may depend upon specific environmental conditions27-28, it seems clear that PyC is less readily decomposed into CO2 than most other forms of organic carbon29-30. This observation has an important implication for the global C cycle: as charred materials store PyC for a relatively long time, they sequester C in organic forms that would otherwise be rapidly respired as CO2, thus reducing atmospheric greenhouse gas concentrations over time31-32.
Besides the climate mitigating aspect, chars have further environmentally relevant properties. Their high porosity, large surface area and negative surface charge can immobilize hazardous compounds33 and improve soil fertility34-35. The recognition of chars as a potentially beneficial soil amendment led to the emerging field of so-called biochar technology36. Biochar will likely be produced on large scales in the coming years and thus significantly increase PyC abundance in soils37. Moreover, the occurrence of wildfires and the burning of fossil fuels are also projected to remain high over the course of the 21th century, continuously contributing large quantities of PyC to the environment11,38-39. Another increasingly important source of PyC is likely to be nanotechnology that also uses PyC-like compounds40-41. It is thus crucial to detect, characterize and quantify these pyrogenic materials accurately in order to investigate their properties and understand their role in the environment.
Here, we present the use of a state-of-the-art compound-specific approach to analyze PyC in various samples: the most recent generation of the benzene polycarboxylic acid (BPCA) method42. This method is broadly applicable within PyC research as it directly targets the &#34;backbone&#34; of PyC: its polycyclic condensed structures that form during the thermal treatment43-45 and that are therefore inherent to all the various forms of PyC5,46. However, these structures are not directly assessable by chromatographic means, due to their size and heterogeneity. In order to chromatographically analyze such pyrogenic compounds, PyC is first digested with nitric acid under high temperature and pressure, which breaks the large polycyclic structures down into its building blocks, the individual BPCAs (cf. Figure 1). The BPCAs are then, after a few purification steps, amenable to chromatographic analysis20,42. PyC is thus isolated and analyzed on a molecular level and can be used to quantify PyC abundance in environmental compartments20,42. The BPCA method additionally characterizes the investigated PyC when relative yields of B3-, B4-, B5- and B6CA are compared (cf. Figure 1): The respective proportion of differently carboxylated BPCAs is linked to the size of the original polycyclic structures and is therefore indicative of PyC&#39;s quality and pyrolysis temperature44,47-48. Moreover, the presented method allows for the determination of the C isotopic composition (13C and 14C) of PyC because the individual BPCAs, deriving directly from pure PyC structures, can be isotopically analyzed after isolation (cf. Figure 1, steps 5 and 6)49. Compound-specific isotopic analysis of PyC is of great interest50 as it can be used, e.g., to distinguish between the precursor biomass of chars in tropical regions51-52, to derive the age of charred materials53-54 or to trace PyC in C cycling studies with an isotopic label26,55-56. Further information about PyC as well as the BPCA method&#39;s history, development and applications in particular can be found in Wiedemeier, 201457, from where part of the above paragraphs and part of the discussion were compiled.

<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)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

We recommend to test the method set-up by measuring a suite of well-described PyC materials (&#34;black carbon reference materials&#34;) that have extensively been used for various method developments and comparisons in the literature44,48,69-77. Information on the reference materials is available from the University of Zurich (http://www.geo.uzh.ch/en/units/physische-geographie-boden-biogeographie/services/black-carbon-reference-materials).
The described procedure allows baseline separation of all BPCA target compounds by HPLC. The chromatograms of the reference materials &#39;chernozem&#39; (silty soil with a significant PyC content) and grass char (made from Oryza Sativa) are shown in Figure 2. By adjusting the chromatography parameters in Tables 1 and 2 (e.g., chromatography temperature, pH of solvent A or flow rate, etc.), the separation can be further modified for specific needs42,63.
Quantitative analysis of the reference materials&#39; chromatograms with external standards (step 5.3.) should yield the PyC values depicted in Figure 3. Please note that slight changes in the procedure (e.g., the omission of step 3 or 4 in specific cases), can lead to higher PyC values. Generally, recoveries should be checked with pure BPCA standards:spiked reference materials can help to detect disproportionate losses in steps 3 and 4 and yield information about the chromatography performance in step 5 42,63.
Table 3 shows the 13C and 14C values that are obtained when purified BPCAs of reference materials are analyzed for their carbon isotopic content after step 6. For reliable results, it is imperative to collect sufficient amounts of BPCA-C (e.g., &#62; 30 &#181;g BPCA-C for current accelerator mass spectrometers, cf. Figure 4) and to take all possible measures to minimize contamination of the sample by extraneous C 49.
Besides checking the method set-up with reference materials as described above, it is highly advisable to prepare and measure samples in replicates, both for PyC quantification (step 5) and subsequent compound-specific 13C and 14C analyses of BPCAs (step 6).
<img alt="Figure 1" src="/files/ftp_upload/53922/53922fig1.jpg" /> 
Figure 1: The BPCA Analysis Procedure. In the protocol step 2, the PyC polycyclic aromatic condensed structures are digested, producing the different BPCAs, which are then further cleaned (steps 3 and 4) and chromatographically analyzed and separated (step 5). After wet oxidation (step 6), the purified BPCAs are amenable to compound-specific isotopic analysis (13C and 14C) on isotope-ratio mass spectrometers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53922/53922fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53922/53922fig2.jpg" /> 
Figure 2: Chromatograms for BPCA Separation. Shown are the black carbon reference materials &#34;chernozem&#34; (a) and &#34;grass char&#34; (b). Baseline separation is achieved for all the BPCA target compounds (B6CA; B5CA; 1,2,4,5-. 1,2,3,5-, 1,2,3,4-B4CA; 1,2,4-, 1,2,3-B3CA)42. Information on the black carbon reference materials is available from the University of Zurich (http://www.geo.uzh.ch/en/units/physische-geographie-boden-biogeographie/services/black-carbon-reference-materials). This figure was modified from Wiedemeier et al. 201342 and is reprinted with permission from Elsevier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53922/53922fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53922/53922fig3.jpg" /> 
Figure 3: Replicated PyC Measurements of Different Black Carbon Reference Materials. Error bars for laboratory replicates are smaller than symbol size and the coefficient of variation averaged 5% (min: 1%, max: 10%).This figure was modified from Wiedemeier et al. 201342 and is reprinted with permission from Elsevier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53922/53922fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53922/53922fig4.jpg" /> 
Figure 4: Radiocarbon (14C) Values for B5CA and B6CA Isolated from a Modern and a Fossil Char. The given error is composed of corrections for instrumental accelerator mass spectrometer background and of the blank for wet oxidation. The solid gray line represents an idealized line for the mixture of the real F14C value of the respective sample and the determined mean external contamination. This figure was modified from Gierga et al. 201449 and is reprinted with permission from Elsevier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53922/53922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>mobile phase A</td>
			<td>20 ml ortho phosphoric acid (85%) in 980 ml ultrapure water</td>
		</tr>
		<tr>
			<td>mobile phase B</td>
			<td>acetonitrile&#160;</td>
		</tr>
		<tr>
			<td>column</td>
			<td>C18 reversed phase (cf. material list for details)</td>
		</tr>
		<tr>
			<td>column temperature</td>
			<td>15 &#176;C</td>
		</tr>
		<tr>
			<td>flow rate</td>
			<td>0.4 ml min-1</td>
		</tr>
		<tr>
			<td>identification</td>
			<td>retention time, UV absorption at 216 nm</td>
		</tr>
		<tr>
			<td>quantification</td>
			<td>external standards of BPCAs</td>
		</tr>
		<tr>
			<td>pressure&#160;</td>
			<td>ca. 120 bar</td>
		</tr>
	</tbody>
</table>
Table 1: Chromatography Settings.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>time&#160;</td>
			<td>mobile phase B</td>
		</tr>
		<tr>
			<td>[min]</td>
			<td>[vol %]</td>
		</tr>
		<tr>
			<td>0</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>5</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>25.9</td>
			<td>30</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>30</td>
			<td>0.5</td>
		</tr>
	</tbody>
</table>
Table 2: Mixing Gradient of Mobile Phases.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">bulk char</td>
			<td colspan="2">BPCA</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">&#948;13C [&#8240; vs. VPDB]</td>
		</tr>
		<tr>
			<td>chestnut char</td>
			<td>-27.4a</td>
			<td>&#177;0.4a</td>
			<td>-27.7</td>
			<td>&#177;0.8</td>
		</tr>
		<tr>
			<td>maize char</td>
			<td>-12.9</td>
			<td>&#177;0.4</td>
			<td>-13.0</td>
			<td>&#177;0.4</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="4">F14C [%]</td>
		</tr>
		<tr>
			<td>modern char</td>
			<td>1.142b</td>
			<td>&#177;0.004b</td>
			<td>1.13</td>
			<td>&#177;0.013</td>
		</tr>
		<tr>
			<td>fossil char</td>
			<td>0.003b</td>
			<td>&#177;0.001b</td>
			<td>0.014</td>
			<td>&#177;0.001</td>
		</tr>
	</tbody>
</table>
Table 3: Carbon Isotopic Values (&#948;13C and F14C) of Reference Char Materials and Compound-Specific Isotopic Analysis of the Corresponding BPCAs. The BPCA values represent B6CA and B5CA that were collected simultaneously in step 5. However, isotopic analysis of individual BPCAs can be achieved analogously when BPCAs are collected separately. Bulk char data is from Yarnes et al. (2011) 73 for the chestnut char (a) and from Gierga et al. (2014) 49 for the fossil and modern char (b). Errors for the &#948;13C measurements are standard errors from triplicates while errors for the F14C measurements (bulk char: ETH-50456, ETH-50458; BPCA: ETH-62324, ETH-62335) are derived from error propagation64.

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.

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

15.3K Views.  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. 

Video: 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).

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

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.

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.

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.

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

Environment

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.

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

Engineering

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.

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.

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

Bioengineering

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.

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.

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.

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

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

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