The SBO project &#8220;Bioleum&#8221; (IWT-SBO 130039) supported by the Institute for Promotion of Innovation through Science and Technology in Flanders (IWT) and the &#8216;Long Term Structural Methusalem Funding by the Flemish Government&#8217; are acknowledged.

Caution: Please consult relevant material safety data sheets (MSDS) of all compounds before use. Appropriate safety practices are recommended. Solutions and samples should be prepared in the fume hood, while using personal protective equipment. Best practice implies use of safety glasses, protection laboratory gloves, lab coat, full length pants, and closed-toe shoes. The reactor should be properly sealed as several reactants and reaction products can be acutely toxic and carcinogenic.
1. Offline GC &#215; GC&#8211;NCD Analysis
<ol>
	<li>Sample Preparation for offline GC &#215; GC analysis
	<ol>
		<li>Select 2-chloropyridine as an internal standard. Make sure that the concentration of the internal standard in the prepared sample is within the range of the concentrations of nitrogen containing compounds present in the analyzed mixture. The concentration of the internal standard must be at least ten times higher than the detection limit of the NCD. Furthermore the concentration of the internal standard should not be more than two times higher than the concentration of the nitrogen compound with the highest concentration in the sample.</li>
		<li>Prepare and weigh two glass vials using an analytical balance.</li>
		<li>Add 20.498 mg of 2-chloropyridine to the first vial using an analytical balance.</li>
		<li>Tare the balance with the first vial and add 1024.287 mg of a shale oil sample. Gently mix the vial content.</li>
		<li>Transfer 23.369 mg of the prepared mixture into a second vial and add 435.195 mg of the original shale oil sample. The obtained concentration of the internal standard in the sample is approximately 1,000 ppmw.</li>
	</ol>
	</li>
	<li>GC &#215; GC&#8211;NCD analysis and operating conditions 
	<ol>
		<li>Perform the analysis using a GC &#215; GC equipped with a dual-stage cryogenic liquid CO2 modulator45.
		<ol>
			<li>Connect the first dimension non-polar column (PONA, 50 m L &#215; 0.25 mm I.D. &#215; 0.5 &#181;m df) directly to the second dimension mid-polar column (BPX-50, 2 m L &#215; 0.25 mm I.D. &#215; 0.25 &#181;m df). Apply the GC &#215; GC operating conditions optimized for detection of nitrogen containing compounds in shale oil, with the parameters presented in Table 1.</li>
			<li>Inject the sample using the auto sampler, with the settings stated in the Supplementary Table.</li>
		</ol>
		</li>
		<li>For the NCD, set the flow rates at 5 and 11 ml min-1 for the H2 and O2 respectively, while keeping the burner temperature at 1,198 K. Use a data acquisition rate of 100 Hz. The detector settings are presented in Table 1.</li>
	</ol>
	</li>
	<li>Reprocessing of results
	<ol>
		<li>Perform data acquisition using the data system for the NCD. Export the raw data to a .cdf file and import into an analytical software (e.g., GCImage).</li>
		<li>Use the analytical software to construct the contour plot, to determine the retention times, and to perform peak fitting and blob integration according to the software protocols. Identify each blob as a compound of a specific nitrogen group with a specific carbon number. 
		Note: Response signal of NCD is equimolar6,46,47,48, hence the procedure for quantification of nitrogen containing compounds in the sample is straightforward.</li>
		<li>Calculate the concentration of detected compounds using the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/54236/54236eq1.jpg" /> 
		where Vi and Vst are the peak volumes of the nitrogen containing compound i and the internal standard respectively. Wst is the concentration of the internal standard compound, Ni is the number of nitrogen atoms in compound i, Mi and Mst are the molar masses of the nitrogen containing compounds and of the nitrogen internal standard respectively6.</li>
	</ol>
	</li>
</ol>
2. On-line Analysis
<ol>
	<li>Pilot plant steam cracking unit 
	Note: The pilot plant steam cracker consists of three sections: the feed section, the furnace/reactor section and the analysis section. The unit is explained in detail elsewhere18,49,50,51. A schematic description of the setup can be found in the Supplementary Information.
	<ol>
		<li>During the experiments, as heptane is fed using a computer controlled rotary pump by setting the flowrate using the Programmable Logical Controller (PLC), introduce pyridine to the reactor convection section by manually setting the speed of the piston pump. The diluent used depends on the purpose of the test and can be varied. Depending on the requirements of the experiment, use the gas controllers for helium and nitrogen dilution, and regulate the water feeding pump frequency for steam dilution.</li>
		<li>Prior to the reaction section of the furnace, evaporate and mix the feedstock in two separately heated zones kept at a temperature of 773 K.</li>
		<li>Impose a temperature profile to the Incoloy 800HT 12.4 m long and 9 mm internal diameter reactor by setting the temperatures in the five heated zones using the PLC. Different experiments with varying process conditions can be performed. Use the process conditions applied to perform method testing as outlined in Table 2.</li>
	</ol>
	</li>
	<li>Analysis methodology
	<ol>
		<li>Use the refinery gas analyzer (RGA) equipped with three detectors working on parallel channels in order to detect as many compounds as possible. Two TCD&#39;s and a FID allow detection of CO, CO2, H2, and hydrocarbons up to four carbon atoms, using three different columns for separating the reaction products. The RGA setting are presented in Table 3.</li>
		<li>For the GC &#215; GC set-ups, use a non-polar PONA (50 m L &#215; 0.25 mm I.D. &#215; 0.5 &#181;m df) column in the first dimension and a mid-polar BPX-50 (2 m L &#215; 0.15 mm I.D. &#215; 0.15 &#181;m df) column in the second dimension. Ensure that the temperature in the oven can be lowered to 233 K by addition of liquid nitrogen inside the GC oven. Use the GC &#215; GC settings given in Table 4 for the on-line analyses.</li>
		<li>Ensure that TOF-MS electron impact ionization is 70 eV, and detector voltage 1,700 V by setting these values in the software. Additionally set the acquisition frequency to 30 spectra/sec in a mass range of 15-400 amu in the same software.</li>
		<li>For the NCD, manually set flow rates at 5 and 11 ml min-1 for the H2 and O2 respectively, while keeping the burner temperature at 1,198 K. The data acquisition rate needs to be 100 Hz.</li>
		<li>For FID H2, air and N2 (make-up gas) flow rates are respectively 35, 350 and 35 ml min-1. Ensure that the FID temperature is set at 573 K with a data acquisition rate of 100 Hz.</li>
		<li>Sample the pilot plant effluent on-line, i.e., during pilot plant operation, and at high temperature (673 - 773 K). Use a valve-based sampling system, positioned in the sampling oven kept at 573 K, see Figure 1.
		<ol>
			<li>Turn the pneumatic 6-port 2-way valve to the position shown in Figure 1a using the PLC, flush the sample loop for at least 2 min.</li>
			<li>Turn the pneumatic 6-port 2-way valve to the position shown in Figure 1b using the PLC, a gaseous sample from the sample loop is carried with helium and injected onto selected GC &#215; GC. Keep the valve in the injection position for 20 sec. Turn the pneumatic 6-port 2-way valve back in the position shown in Figure 1a.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Internal standard addition
	<ol>
		<li>Use 2-chloropyridine as an internal standard for the NCD analyses. The introduced amount of this compound depends on the purpose of the experiment (values illustrated in Table 2). To add 2-chloropyridine precisely and uniformly, dissolve the compound in n-hexane (values illustrated in Table 2) and gentle stir the mixture after preparation.</li>
		<li>Add the internal standard 2-chloropyridine continuously by pumping the prepared mixture through a capillary transfer line, which is centrally positioned in the product stream before the sampling system. For this purpose use a peristaltic pump and keep the flow rate stable (values indicated in Table 2).</li>
	</ol>
	</li>
	<li>Data acquisition and quantification 
	<ol>
		<li>Acquire the data obtained with TOF-MS, and use the results for product identification and establishing the retention times of the reaction products. Acquisition and reprocessing of data obtained from FID is similar to the procedure applied to reprocessing of results gathered using the NCD, as explained in section 1.3.2.</li>
		<li>Recalculate the product yields using an internal standard18. Introduce a fixed amount of nitrogen (N2) continuously to the product stream and use it as a reference compound as shown in Figure 2.</li>
		<li>Combine the data obtained on the different instruments. Apply the calibration factors for each compound depending on the detector.
		<ol>
			<li>Set the methane calibration factor to 1.</li>
			<li>Obtain response factors relative to methane for permanent gases detectable on RGA by calibration. Ensure that for all major compounds detected on the GC &#215; GC the relative response factors are determined by calibration. For minor products use the FID calibration factors reported by Dietz52. If the response factors for a compound are not available, calculate values using the group contribution method developed by Dierickx et al.53.</li>
			<li>Methane functions as a secondary internal standard, and its concentration is needed to determine the concentrations based on the peaks of the GC &#215; GC&#8211;FID chromatogram. Therefore determine the methane mass flow rate using equation 2: 
			<img alt="Equation 2" src="/files/ftp_upload/54236/54236eq2.jpg" /> 
			Where, fCH4 and fN2 are the calibration factors of methane and nitrogen on TCD respectively. ACH4 and AN2 are peak areas obtained during analysis, while <img alt="Equation 3" src="/files/ftp_upload/54236/54236eq3.jpg" /> is the nitrogen mass flow rate added to the effluent stream.</li>
		</ol>
		</li>
		<li>Carry out quantification of the nitrogen containing products by applying the procedure explained in section 1.3.3.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The described experimental procedures enabled a successful comprehensive off-line and on-line identification and quantification of nitrogen containing compounds in the studied samples.
The separation of nitrogen containing compounds in shale oil was accomplished using GC &#215; GC&#8211;NCD, as shown in Figure 3. Since the NCD cannot be used for identification, the retention times of the observed species need to be established in advance by carrying out analyses on the GC &#21...

The reserves of light sweet crude oils are gradually diminishing, and hence, alternative fossil resources are being considered to be used in the energy and petrochemical industry. In addition, renewables such as bio-oils produced by fast pyrolysis of biomass are becoming a more attractive resources of bio-based fuels and chemicals. Nevertheless, heavy crude oil is a logical first choice because of the large proven reserves in Canada and Venezuela1-3. The latter are being recognized as the largest crude oil reserves in the world and their composition is similar to the composition of natural bitumen. Similar to bio-oils, heavy crude oils differ from light crude oils by their high viscosity at reservoir temperatures, high density (low API gravity), and significant contents of nitrogen, oxygen, and sulfur containing compounds4,5. Another promising alternative is shale oil, derived from oil shale. Oil shale is a fine-grained sedimentary rock containing kerogen, a mixture of organic chemical compounds with a molar mass as high as 1,000 Da6. Kerogen can contain organic oxygen, nitrogen, and sulfur in the hydrocarbon matrix; depending on the origin, age, and the extraction conditions. Global characterization methods have shown that the concentration of heteroatoms (S, O and N) in shale oil and heavy crude oils is typically substantially higher than the specifications set for the products used in for example the petrochemical industry6. It is well documented that nitrogen containing compounds present in heavy conventional crude oil and shale oil have a negative effect on the catalyst activity in hydrocracking, catalytic cracking and reforming processes7. Similarly, it has been reported that the presence of nitrogen containing compounds are a safety concern because they promote gum formation in the cold-box of a steam cracker8.
These processing and safety challenges are a strong driver to improve the current methods for off-line and on-line characterization of nitrogen containing compounds in complex hydrocarbon matrices. Two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD) is a superior characterization technique compared to one-dimensional gas chromatography (GC) for analyzing conventional diesels or liquefied coal samples7. Recently a method has been developed and applied to the offline characterization of nitrogen content in shale oil6, the identification of extracted nitrogen compounds present in middle distillates9, and the determination of the detailed composition of plastic waste pyrolysis oil10.
It is thus clear that GC &#215; GC analysis is a powerful offline processing technique for analyzing complex mixtures11-17. However, on-line application is more challenging due to the need for a reliable and non-discriminating sampling methodology. One of the first developed methodologies for comprehensive on-line characterization was demonstrated by analyzing steam cracking reactor effluents using a TOF-MS and a FID18. The optimization of the GC settings and an appropriate column combination enabled analysis of samples consisting of hydrocarbons ranging from methane up to polyaromatic hydrocarbons (PAHs)18. The present work takes this method to a new level by extending it to the identification and quantification of nitrogen compounds present in the complex hydrocarbon mixtures. Such a method is among others needed to improve fundamental understanding of the role these compounds play in several processes and applications. To the authors&#39; best knowledge, information concerning kinetics of conversion processes of nitrogen containing compounds is scarce19, partly due to the lack of an adequate method to identify and quantify nitrogen containing compounds in the reactor effluent. Establishing the methodology for offline and on-line analyses is thus a prerequisite before one can even attempt feedstock reconstruction20-27 and kinetic modeling. One of the fields which would benefit from the accurate identification and quantification of nitrogen containing compounds is steam cracking or pyrolysis. Bio and heavy fossil feeds for steam cracking or pyrolysis reactors contain thousands of hydrocarbons and compounds that contain heteroatoms. Moreover, because of the complexity of the feed and the radical nature of the occurring chemistry, ten thousands of reactions can occur among the thousands free radical species28, which makes the reactor effluent even more complex than the starting material.
In hydrocarbon mixtures nitrogen is mainly present in aromatic structures, e.g., as pyridine or pyrrole; hence most experimental efforts have been dedicated to the decomposition of these structures. Hydrogen cyanide and ethyne were reported as major products for the thermal decomposition of pyridine studied in a temperature range of 1,148-1,323 K. Other products such as aromatics and nonvolatile tars were also detected in minor quantities29. The thermal decomposition of pyrrole was investigated in a broader temperature range of 1,050-1,450 K using shock wave experiments. The main products were 3-butenenitrile, cis and trans 2-butenenitrile, hydrogen cyanide, acetonitrile, 2-propenenitrile, propanenitrile, and propiolonitrile30. Additionally thermal decomposition shock tube experiments were performed for pyridine at elevated temperatures resulting in comparable product spectra31,32. Product yields in these studies have been determined by applying GC&#39;s equipped with a FID, a nitrogen-phosphorus detector (NPD)31, a mass spectrometer (MS)32 and a Fourier transform infrared (FTIR) spectrometer32. A similar methodology implementing the FID and the NPD was applied to analyze the shale oil pyrolysis products in a continuous flow reactor8. Using a cold trap at 273.15 K and GC-MS, Winkler et al. 33 showed that during pyridine pyrolysis heteroatom-containing aromatic compounds are formed. Zhang et al.34 and Debono et al.35 applied the method of Winkler et al. for studying the pyrolysis of organic waste. The nitrogen rich reaction products were analyzed on-line, using a GC coupled to a thermal conductivity detector (TCD)34. The collected tars were analyzed offline using GC-MS34,35. Simultaneous pyrolysis of toluene and pyridine showed a difference in soot formation tendency compared to pyridine pyrolysis, indicating the complex nature of the free-radical reactions31,36.
One of the most comprehensive analytical methodologies was developed by Nathan and co-workers37. They used FTIR, nuclear magnetic resonance (NMR) and GC-MS for analyzing decomposition products of pyridine and diazine and electron paramagnetic resonance (EPR) spectroscopy for tracing free radical species. FTIR analysis can be a very effective approach for the identification of a large range of products, even PAHs38-40, nevertheless quantification is extremely challenging. Calibration requires a full set of infrared spectra at different concentrations for each target species at a specific temperature and pressure41. Recent work of Hong et al. demonstrated the possibilities of using molecular-beam mass spectrometry (MBMS) and tunable synchrotron vacuum ultraviolet photoionization for determination of products and intermediates during pyrrole and pyridine decomposition42,43. This experimental method enables selective identification of isomeric intermediates and near-threshold detection of radicals without inflicting fragmentation of the analyzed species44. However, the uncertainty on the measured concentrations using MBMS analysis is also substantial.
In this work, first the offline comprehensive characterization results of the complex shale oil are reported. Next, the limitations of using an on-line GC &#215; GC-TOF-MS/FID for the analysis of nitrogen compounds in a complex hydrocarbon matrix are discussed. Finally, the newly developed methodology for the on-line quantification of nitrogen containing compounds by GC &#215; GC&#8211;NCD is demonstrated. The qualitative analysis of products was carried out using TOF-MS, while FID and NCD were used for quantification. The application of the NCD is a substantial improvement compared to using the FID because of its higher selectivity, lower detection limit and equimolar response.

<table><tbody><tr><td>2-Chloropyridine, 99%</td><td>Sigma Aldrich</td><td>C69802</td><td>Highly toxic</td></tr><tr><td>Shale oil</td><td>Origin Colorado, US</td><td>Piceance Basin in&lt;br/&gt; Colorado, USA</td><td>Toxic</td></tr><tr><td>Pyridine, 99.8%</td><td>Sigma Aldrich</td><td>270970</td><td>Highly toxic</td></tr><tr><td>Carbon Dioxide, industrial grade refrigerated liquid</td><td>PRAXAIR</td><td>CDINDLB0D</td><td>Wear safety gloves and glasses</td></tr><tr><td>Helium, 99.99%</td><td>PRAXAIR</td><td>6.0</td><td></td></tr><tr><td>Hydrogen, 99.95%</td><td>Air Liquide</td><td>695A-49</td><td>Flammable</td></tr><tr><td>Oxygen</td><td>Air Liquide</td><td>905A-49+</td><td>Flammable</td></tr><tr><td>Air</td><td>Air Liquide</td><td>365A-49X</td><td></td></tr><tr><td>Nitrogen</td><td>Air Liquide</td><td>765A-49</td><td></td></tr><tr><td>Hexane, 95+%</td><td>Chemlab</td><td>CL00.0803.9025</td><td>Toxic</td></tr><tr><td>Heptane, 99+%</td><td>Chemlab</td><td>CL00.0805.9025</td><td>Toxic</td></tr><tr><td>Nitrogen, industrial grade refrigerated liquid</td><td>PRAXAIR</td><td>P0271L50S2A001</td><td>Wear safety gloves and glasses</td></tr><tr><td>Autosampler</td><td>Thermo Scientific, Interscience</td><td>AI/AS 3000</td><td></td></tr><tr><td>High temperature 6 port/2 position valve</td><td>Valco Instruments Company Incorporated</td><td>SSACGUWT</td><td></td></tr><tr><td>Gas chromatograph</td><td>Thermo Scientific, Interscience</td><td>Trace GC ultra</td><td></td></tr><tr><td>Rafinery Gas Analyzer</td><td>Thermo Scientific, Interscience</td><td>KAV00309</td><td></td></tr><tr><td>rtx-1-PONA column</td><td>Restek Pure Chromatography</td><td>10195-146</td><td></td></tr><tr><td>BPX-50 column</td><td>SGE Analytical science</td><td>54741</td><td></td></tr><tr><td>TOF-MS</td><td>Thermo Scientific, Interscience</td><td>Tempus Plus 1.4 SR1 Finnigan</td><td></td></tr><tr><td>NCD</td><td>Agilent Technologgies</td><td>NCD 255</td><td></td></tr><tr><td>Chrom-card</td><td>Thermo Scientific, Interscience</td><td>HyperChrom 2.4.1</td><td></td></tr><tr><td>Xcalibur software</td><td>Thermo Scientific, Interscience</td><td>1.4 SR1</td><td></td></tr><tr><td>Chrom-card software</td><td>Thermo Scientific, Interscience</td><td>HyperChrom 2.7</td><td></td></tr><tr><td>GC image software</td><td>Zoex Corporation</td><td>GC image 2.3</td><td></td></tr></tbody></table>

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.

The chromatogram obtained using the offline GC &#215; GC&#8211;NCD for characterization of nitrogen containing compounds in a shale oil sample is given in Figure 3. The following classes were identified: pyridines, anilines, quinolines, indoles, acridines, and carbazoles. Moreover, detailed quantification of the individual compounds was possible. The gathered data was used to determine the individual compound concentrations, and the obtained values are presented in <stron...

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

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 ...

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Chemistry

10.6K Views.  Ghent University. 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.

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

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Cancer Research

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

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Environment

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 ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

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 ...

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of cyanobacteria and related cyanotoxins in water samples and in an organic matrix, such as bivalve extracts. FDS combines remote/proximal sensing techniques with analytical/bioinformatics analyses. Sampling spots are chosen through multi-disciplinary, multi-scale, and multi-parametric monitoring in a three-dimensional physical space, including remote sensing. Microscopic observation and taxonomic analysis of the samples are performed in the laboratory setting, which allows for the identification of cyanobacterial species. Samples are then extracted with organic solvents and processed with LC-MS/MS. Data obtained by MS/MS are analyzed using a bioinformatic approach using the online platform Global Natural Products Social (GNPS) to create a network of molecules. These networks are analyzed to detect and identify toxins, comparing data of the fragmentation spectra obtained by mass spectrometry with the GNPS library. This allows for the detection of known toxins and unknown analogues that appear related in the same molecular network.

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of ...

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 ...

Bioengineering

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Biology

Carbon and Nitrogen Analysis of Environmental Samples

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Elemental Analysis is a method used to determine elemental composition of a material. In environmental samples such as soils, scientists are particularly interested in the amounts of two ecologically important elements, nitrogen and carbon. Elemental analysis by the flash combustion technique works by oxidizing the sample with a catalyst through combustion in a high-temperature chamber. The products of combustion are then reduced to N2 and CO2 and detected with a thermal conductivity detector.
Unlike other methods for total nitrogen determination (Kjeldahl method) and total carbon determination (Walkley-Black, Heanes or Leco methods), the flash combustion technique does not use toxic chemicals and is therefore much safer to use.
This video will demonstrate combustion-based elemental analysis using the Flash EA 1112 instrument from Thermo Fisher Scientific.

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Elemental Analysis is a method used to determine elemental composition of a ...

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

Summary

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Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

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

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Carbon and Nitrogen Analysis of Environmental Samples