Funding support for this work was provided by the Defense Logistics Agency Energy (DLA Energy) and the Naval Air Systems Command (NAVAIR).
This research was performed while an author held an NRC Research Associateship award at the U.S. Naval Research Laboratory.

CAUTION: Please consult relevant safety data sheets (SDS) of all compounds before use. Appropriate safety practices are recommended. All work should be performed while wearing personal protective equipment such as gloves, safety glasses, lab coat, long pants, and closed-toed shoes. All standard and sample preparations should be done in a ventilated hood.
1. Preparation of standards
<ol>
	<li>Prepare a 5,000 mg/kg (ppm) solution of carbazole (calibration standard, minimum of 98% purity) by placing 0.050 g in a vial and bring the total mass of each solution to 10.000 g with isopropyl alcohol. Cap the vial immediately to prevent loss of isopropyl alcohol. This is the calibration stock solution.</li>
	<li>Prepare a carbazole solution with 100 ppm nitrogen content by diluting 1.194 mL of stock solution to 5 mL with isopropyl alcohol. This is designated as &#34;100 ppm nitrogen carbazole&#34; and is used to create the calibration standards. 
	NOTE: The concentrations of the calibration standards indicate the concentration of nitrogen in the standard, not the carbazole concentration</li>
	<li>Prepare the following calibration standards by serial dilution: 
	20 ppm nitrogen carbazole 
	10 ppm nitrogen carbazole 
	5 ppm nitrogen carbazole 
	1 ppm nitrogen carbazole 
	0.5 ppm nitrogen carbazole 
	0.025 ppm nitrogen carbazole</li>
	<li>Put 1 mL of the calibration standards into separate GC vials (6 vials total).</li>
	<li>Prepare individual 10 ppm solutions of each standard compounds listed in Table 1 in isopropyl alcohol. Place 1 mL of each standard solution in separate GC vials (10 vials total). 
	NOTE: The standard compounds listed in Table 1 will be used to classify the unknown nitrogen compounds as &#39;light nitrogen compounds&#39;, &#39;basic nitrogen compounds&#39;, or &#39;non-basic nitrogen compounds&#39;.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Standard Compound</td>
			<td>Elution Time Classification Group</td>
		</tr>
		<tr>
			<td>Pyridine</td>
			<td>Group 1 &#8211; light nitrogen compounds</td>
		</tr>
		<tr>
			<td>Trimethylamine</td>
			<td>Group 1 &#8211; light nitrogen compounds</td>
		</tr>
		<tr>
			<td>Methylaniline</td>
			<td>Group 1 &#8211; light nitrogen compounds</td>
		</tr>
		<tr>
			<td>Quinoline</td>
			<td>Group 2 &#8211; basic nitrogen compounds</td>
		</tr>
		<tr>
			<td>Diethylaniline</td>
			<td>Group 2 &#8211; basic nitrogen compounds</td>
		</tr>
		<tr>
			<td>Methylquinoline</td>
			<td>Group 2 &#8211; basic nitrogen compounds</td>
		</tr>
		<tr>
			<td>Indole</td>
			<td>Group 2 &#8211; basic nitrogen compounds</td>
		</tr>
		<tr>
			<td>Dimethylindole</td>
			<td>Group 2 &#8211; basic nitrogen compounds</td>
		</tr>
		<tr>
			<td>Ethylcarbazole</td>
			<td>Group 3 &#8211; Non-basic nitrogen compounds</td>
		</tr>
		<tr>
			<td>Carbazole</td>
			<td>Group 3 &#8211; Non-basic nitrogen compounds</td>
		</tr>
	</tbody>
</table>
Table 1: Nitrogen standards and their elution classification groups.
2. Sample preparation
<ol>
	<li>For diesel fuels: In a GC vial, add 250 &#181;L of fuel sample and 750 &#181;L of isopropyl alcohol.</li>
	<li>For jet fuels: In a GC vial, add 750 &#181;L of fuel sample and 250 &#181;L of isopropyl alcohol. 
	NOTE: If the total nitrogen concentration of either diesel or jet fuel falls below the calibration curve (0.025 ppm nitrogen) when diluted as instructed above, do not dilute. If the nitrogen concentration in a specific nitrogen group in either diesel or jet fuel falls above the calibration curve (20 ppm nitrogen), dilute sample further.</li>
</ol>
3. Instrument setup
<ol>
	<li>Instrument configuration
	<ol>
		<li>Auto-sampler: Ensure that the autosampler tray and tower are installed with a splitless inlet and wash vials in place.</li>
		<li>Nitrogen Chemiluminescence Detector: Ensure that the nitrogen chemiluminescence detector is installed with the appropriate gas lines (i.e., helium and hydrogen). A hydrogen generator can be utilized instead of a tank, if available.</li>
		<li>Duel Loop Thermal Modulator: Ensure that the duel loop thermal modulator is installed and aligned properly so that the column loop will be centered between the cold and hot jet flows during modulation.</li>
	</ol>
	</li>
	<li>Column installation
	<ol>
		<li>Ensure that the instrument is in maintenance mode (i.e., all burners and gas flows are turned off).</li>
		<li>Insert the 30 m primary column into the GC oven and connect to the splitless inlet.</li>
		<li>Measure and cut 2.75 m of the secondary column. Put a mark on the secondary column at 0.375 m and 1.375 m using a white-out pen.</li>
		<li>Place the secondary column into the Zoex modulator column holder and use the marks as guides for creating a 1 m loop within the holder for modulation.</li>
		<li>Connect the shorter end of the secondary column to the primary column using a micro-union. Check for a successful connection by turning on the gas flow and inserting the open end of the column into a vial of methanol. A successful connection is confirmed by the presence of bubbles.</li>
		<li>Place the column holder into the modulator and adjust the loops as necessary so that the loops line up properly with the cold and hot jets, as pictured in Figure 1.</li>
		<li>Insert the other end of the column into the NCD burner. Then turn on all burners and gas flows to ensure there are no leaks.</li>
		<li>Turn the oven on at the maximum temperature limit for a minimum of 2 h in order to bake-out the columns. Once completed, verify that there are no new leaks. Then, cool the oven.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60883/60883fig1.jpg" /> 
Figure 1: Schematic representation of the GCxGC-NCD instrumentation. This figure has been reprinted from Deese et al. <a href="https://www.jove.com/files/ftp_upload/60883/60883fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Method parameters
	<ol>
		<li>Using the computer software, set the instrument to the parameters listed in Table 2.</li>
		<li>Set the initial oven temperature to 60 &#176;C with a ramp rate of 5 &#176;C/min to 160 &#176;C, and then change the ramp rate to 4 &#176;C until 300 &#176;C. The total run time is 55 minutes per sample.</li>
		<li>Set the hot jet temperature to be 100 &#176;C higher than the oven temperature at any point in time. Thus: set the initial hot jet temperature to 160 &#176;C with a ramp rate of 5 &#176;C/min to 260 &#176;C, and then change the ramp rate to 4 &#176;C until 400 &#176;C.</li>
		<li>Set the ancillary liquid nitrogen Dewar connected to the GC to stay between 20% and 30% full during the run.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Instrument Parameters</td>
		</tr>
		<tr>
			<td>NCD</td>
			<td>Nitrogen Base Temperature</td>
			<td>280 &#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td>Nitrogen Burner Temperature</td>
			<td>900 &#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td>Hydrogen flow rate</td>
			<td>4 mL/min</td>
		</tr>
		<tr>
			<td></td>
			<td>Oxidizer flow rate (O2)</td>
			<td>8 mL/min</td>
		</tr>
		<tr>
			<td></td>
			<td>Data collection rate</td>
			<td>100 Hz</td>
		</tr>
		<tr>
			<td>Inlet</td>
			<td>Inlet Temperature</td>
			<td>300 &#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td>Inlet liner</td>
			<td>Splitless</td>
		</tr>
		<tr>
			<td></td>
			<td>Purge flow to split vent</td>
			<td>15 mL/min</td>
		</tr>
		<tr>
			<td></td>
			<td>Septum Purge Flow</td>
			<td>3 mL/min</td>
		</tr>
		<tr>
			<td></td>
			<td>Carrier gas</td>
			<td>He</td>
		</tr>
		<tr>
			<td></td>
			<td>Carrier gas flow rate</td>
			<td>1.6 mL/min</td>
		</tr>
		<tr>
			<td></td>
			<td>Syringe size</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td>Injection volume</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>Modulator</td>
			<td>Modulation time</td>
			<td>6000 ms</td>
		</tr>
		<tr>
			<td></td>
			<td>Hot pulse duration</td>
			<td>375 ms</td>
		</tr>
		<tr>
			<td>Columns</td>
			<td>Flow</td>
			<td>1.6 mL/min</td>
		</tr>
		<tr>
			<td></td>
			<td>Flow type</td>
			<td>Constant flow</td>
		</tr>
	</tbody>
</table>
Table 2: Instrument parameters.
4. Instrument calibration
<ol>
	<li>Place the GC sample vials containing the prepared carbazole standards and load the previously configured method into the GC software.</li>
	<li>Create a sequence that aliquots the blank (isopropyl alcohol) at the beginning followed by the prepared carbazole standards by increasing concentration.</li>
	<li>Verify that the liquid nitrogen Dewar is between 20-30% full and all instrument parameters are in &#34;ready&#34; mode. Start the sequence.</li>
	<li>Once the calibration standard set analysis is completed, use the GCImage software to load each chromatogram, background correct, and detect each carbazole peak or blob. 
	NOTE: In GCImage, the detected peaks within the chromatogram are termed as &#34;blobs&#34; by the software.</li>
	<li>In a spreadsheet program, plot the response (blob volume) against the nitrogen concentration (ppm) of each calibration standard to create a calibration curve (see Figure 2). The trend line of the curve should have R2 &#8805; 0.99.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60883/60883fig2.jpg" /> 
Figure 2: Example GCxGC-NCD carbazole calibration curve. <a href="https://www.jove.com/files/ftp_upload/60883/60883fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Sample analysis
<ol>
	<li>Place the GC sample vials in the autosampler tray and load the previously configured method.</li>
	<li>Create a sequence that has a blank (isopropyl alcohol) at the beginning and then every 5 subsequent samples to limit any build-up of fuel within the columns.</li>
	<li>Verify that enough liquid nitrogen is available in the modulator&#39;s Dewar and that all instrument parameters are in &#34;ready&#34; mode. Then, start the sequence.</li>
</ol>
6. Data analysis
<ol>
	<li>Open the chromatogram in the GCImage software for data analysis and perform a background correction</li>
	<li>Detect blobs using the following filter parameters: 
	Minimum area = 25 
	Minimum volume = 0 
	Minimum peak = 25 
	NOTE: These parameters are subject to change based on instrument response or sample matrix.</li>
	<li>Use the GCImage template function to create or load a template to group nitrogen compound classes based on the elution times of the known standards (see Table 1). 
	NOTE: Further explanation of template usage can be found in the representative results and Figure 8.</li>
	<li>Once the compounds have been grouped, export the &#34;blob set table&#34; into a spreadsheet program. Use the sum the volume of all blobs/peaks within each compound class group, and the calibration equation determined in Section 4.4 to calculate the concentration in ppm for the nitrogen compounds in each group.</li>
	<li>If desired, use the following density calculations to correct for differences in the injection volume of sample vs. standards for quantitation: 
	<img src="/files/ftp_upload/60883/60883eq2a.jpg" /> 
	NOTE: *percent difference between the ng N injected in the sample matrix vs. the standard matrix</li>
	<li>Sum all nitrogen content in each compound class to obtain the total nitrogen content of the sample, if desired. If the total nitrogen content is determined to be above 150 ppm nitrogen or a compound class bin is outside of the calibration range, dilute the sample further for analysis. Compare these results with total nitrogen content as determined by ASTM D462913 for quantification verification.</li>
</ol>

<ol>
	<li>Garner, M. W., Morris, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Studies+of+Good+Hope+and+Other+Diesel+Fuel+Samples.">Laboratory Studies of Good Hope and Other Diesel Fuel Samples.</a> ARTECH Corp. Report No. J8050.93-FR. , (1982).</li><li>Morris, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fleet+Fuel+Stability+Analyses+and+Evaluations.">Fleet Fuel Stability Analyses and Evaluations.</a> ARTECH Corp. Report No. DTNSRDC-SME-CR-01083. , (1983).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+F-76+Fuels+from+the+Western+Pacific+Region+Sampled+in+2014.">Analysis of F-76 Fuels from the Western Pacific Region Sampled in 2014.</a> Naval Research Laboratory Letter Report 6180/0012A. , (2015).</li><li>Westbrook, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+F-76+Fuel,+Sludge,+and+Particulate+Contamination.">Analysis of F-76 Fuel, Sludge, and Particulate Contamination.</a> Southwest Research Institute Letter Report. Project No. 08.15954.14.001. , (2015).</li><li>Morris, R. E., Loegel, T. N., Cramer, J. A., Leska Myers, K. M., A, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Diesel+Fuels+and+Insoluble+Gums+in+Retain+Samples+from+the+West+Coast-Hawaii+Region.">Examination of Diesel Fuels and Insoluble Gums in Retain Samples from the West Coast-Hawaii Region.</a> Naval Research Laboratory Memorandum Report. No. NRL/MR/6180-15-9647. , (2015).</li><li>Maciel, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Nitrogen+Compounds+in+Diesel+Fuel+Samples+by+Comprehensive+Two-Dimensional+Gas+Chromatography+Coupled+with+Quadrupole+Mass+Spectrometry.">Quantification of Nitrogen Compounds in Diesel Fuel Samples by Comprehensive Two-Dimensional Gas Chromatography Coupled with Quadrupole Mass Spectrometry.</a> Journal of Separation Science. 38 (23), 4071-4077 (2015).</li><li>Deese, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Organic+Nitrogen+Compounds+and+Their+Impact+on+the+Stability+of+Marginally+Stable+Diesel+Fuels.">Characterization of Organic Nitrogen Compounds and Their Impact on the Stability of Marginally Stable Diesel Fuels.</a> Energy &amp; Fuels. 33 (7), 6659-6669 (2019).</li><li>Lissitsyna, K., Huertas, S., Quintero, L. C., Polo, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Simple+Method+for+Quanitation+of+Nitrogen+Compounds+in+Middle+Distillates+using+Solid+Phase+Extraction+and+Comprehensive+Two-Dimensional+Gas+Chromatography.">Novel Simple Method for Quanitation of Nitrogen Compounds in Middle Distillates using Solid Phase Extraction and Comprehensive Two-Dimensional Gas Chromatography.</a> Fuel. 104, 752-757 (2013).</li><li>Machado, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+two-dimensional+gas+chromatography+for+the+analysis+of+nitrogen-containing+compounds+in+fossil+fuels:+A+review.">Comprehensive two-dimensional gas chromatography for the analysis of nitrogen-containing compounds in fossil fuels: A review.</a> Talanta. 198, 263-276 (2019).</li><li>Adam, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Benchmark+for+Basic+and+Neutral+Nitrogen+Compounds+Speciation+in+Middle+Distillates+using+Comprehensive+Two-Dimensional+Gas+Chromatography.">New Benchmark for Basic and Neutral Nitrogen Compounds Speciation in Middle Distillates using Comprehensive Two-Dimensional Gas Chromatography.</a> Journal of Chromatography A. 1148, 55-65 (2007).</li><li>Wang, F. C. Y., Robbins, W. K., Greaney, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speciation+of+Nitrogen-Containing+Compounds+in+Diesel+Fuel+by+Comprehensive+Two-Dimensional+Gas+Chromatography.">Speciation of Nitrogen-Containing Compounds in Diesel Fuel by Comprehensive Two-Dimensional Gas Chromatography.</a> Journal of Separation Science. 27, 468-472 (2004).</li><li>Yan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfur+and+Nitrogen+Chemiluminescence+Detection+in+Gas+Chromatographic+Analaysis.">Sulfur and Nitrogen Chemiluminescence Detection in Gas Chromatographic Analaysis.</a> Journal of Chromatography A. 976 (1), 3-10 (2002).</li></ol>

The authors have nothing to disclose.

The purpose of this method is to provide detailed information on the nitrogen content of diesel and jet fuels without extensive sample preparation such as liquid extractions. This is achieved by pairing a two dimensional GC system (GCxGC) with a nitrogen-specific detector (nitrogen chemiluminescence detector, NCD). The GCxGC provides significant separation of the compounds relative to traditional one-dimensional GC. The NCD provides trace nitrogen compound detection without any background interferences. Other nitrogen-sp...

Before use, fuels undergo extensive quality assurance and specification testing by refineries to verify that the fuel they are producing will not fail or cause equipment problems once disseminated. These specification tests include flash point verification, freeze point, storage stability, and many more. The storage stability tests are important as they determine if the fuels have a tendency to undergo degradation during storage, resulting in the formation of gums or particulates. There have been incidences in the past when F-76 diesel fuels have failed during storage even though they passed all specification tests1. These failures resulted in high concentrations of particulate matter in the fuels that could be detrimental to equipment such as fuel pumps. The extensive research investigation that followed this discovery suggested that there is a causal relationship between certain types of nitrogen compounds and the particulate formation2,3,4,5. However, many of the techniques used to measure nitrogen content are strictly qualitative, require extensive sample preparation, and provide little information on the identity of the suspect nitrogen compounds. The method described herein is a two-dimensional GC (GCxGC) method paired with a nitrogen chemiluminescence detector (NCD) that was developed for the purpose of characterizing and quanitifying trace nitrogen compounds in diesel and jet fuels.
Gas chromatography is used extensively in petroleum analyses and there are over sixty published ASTM petroleum methods associated with the technique. A wide range of detectors are combined with gas chromatography such as mass spectrometry (MS, ASTM D27896, D57697), Fourier-transform infrared spectroscopy (FTIR, D59868), vacuum ultraviolet spectroscopy (VUV, D80719), flame ionization detector (FID, D742310), and chemiluminesence detectors (D550411, D780712, D4629-1713). All these methods can provide significant compositional information about a fuel product. Since fuels are complex sample matrices, gas chromatography enhances compositional analysis by separating out sample compounds based on boiling point, polarity, and other interactions with the column.
To further this separation ability, two-dimensional gas chromatography (GCxGC) methods can be utilized to provide compositional maps by using sequential columns with orthogonal column chemistries. Separation of compounds occur both by polarity and boiling point, which is a comprehensive means to isolate fuel constituents. Although it is possible to analyze nitrogen-containing compounds with GCxGC-MS, the trace concentration of the nitrogen compounds within the complex sample inhibits identification14. Liquid-liquid phase extractions have been attempted in order to use GC-MS techniques; however, it was found that the extractions are incomplete and exclude important nitrogen compounds15. Additionally, others have used solid phase extraction to enhance the nitrogen signal while reducing the potential for the fuel sample matrix interference16. However, this technique has been found to irreversible retail certain nitrogen species, especially low molecular weight nitrogen-bearing species.
The nitrogen chemiluminescence detector (NCD) is a nitrogen-specific detector and has been successfully used for fuel analyses17,18,19. It utilizes a combustion reaction of nitrogen-containing compounds, the formation of nitric oxide (NO), and a reaction with ozone (see Equations 1 &#38; 2)20. This is accomplished in a quartz reaction tube that contains a platinum catalyst and is heated to 900 &#176;C in the presence of oxygen gas.
The photons emitted from this reaction are measured with a photomultiplier tube. This detector has a linear and equimolar response to all nitrogen-containing compounds because all nitrogen-containing compounds are converted to NO. It is also not prone to matrix effects because other compounds in the sample are converted to non-chemiluminescence species (CO2 and H2O) during the conversion step of the reaction (Equation 1). Thus, it is an ideal method for measuring nitrogen compounds in a complex matrix such as fuels.
<img src="/files/ftp_upload/60883/60883eq1a.jpg" />
The equimolar response of this detector is important for nitrogen compound quantitation in fuels because the complex nature of fuels does not allow for calibration of each nitrogen analyte. The selectivity of this detector facilitates the detection of trace nitrogen compounds even with a complex hydrocarbon background.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#181;L syringe</td>
			<td>Agilent</td>
			<td></td>
			<td>gold series</td>
		</tr>
		<tr>
			<td>180 &#181;m x 0.18 &#181;m Secondary Column</td>
			<td>Restek</td>
			<td>Rxi-1MS</td>
			<td>nonpolar phase column, crossbond dimethyl polysiloxane</td>
		</tr>
		<tr>
			<td>250 &#181;m x 0.25 &#181;m Primary Column</td>
			<td>Restek</td>
			<td>Rxi-17SilMS</td>
			<td>midpolarity phase column</td>
		</tr>
		<tr>
			<td>Autosampler tray and tower</td>
			<td>Agilent</td>
			<td>7963A</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbazole</td>
			<td>Sigma</td>
			<td>C5132</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Diethylaniline</td>
			<td>Aldrich</td>
			<td>185898</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>Dimethylindole</td>
			<td>Aldrich</td>
			<td>D166006</td>
			<td>97%</td>
		</tr>
		<tr>
			<td>Duel Loop Thermal Modulator</td>
			<td>Zoex Corporation</td>
			<td>ZX-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylcarbazole</td>
			<td>Aldrich</td>
			<td>E16600</td>
			<td>97%</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td>Agilent</td>
			<td>7890B</td>
			<td></td>
		</tr>
		<tr>
			<td>GC vials</td>
			<td>Restek</td>
			<td>21142</td>
			<td></td>
		</tr>
		<tr>
			<td>GCImage Software, Version 2.6</td>
			<td>Zoex Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indole</td>
			<td>Aldrich</td>
			<td>13408</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Fisher Scientific</td>
			<td>A461-500</td>
			<td>Purity 99.9%</td>
		</tr>
		<tr>
			<td>Methylaniline</td>
			<td>Aldrich</td>
			<td>236233</td>
			<td>&#8805; 99%</td>
		</tr>
		<tr>
			<td>Methylquinoline</td>
			<td>Aldrich</td>
			<td>382493</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Nitrogen Chemiluminescence Detector</td>
			<td>Agilent</td>
			<td>8255</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine</td>
			<td>Sigma-Aldrich</td>
			<td>270970</td>
			<td>anhydrous, 99.8%</td>
		</tr>
		<tr>
			<td>Quinoline</td>
			<td>Aldrich</td>
			<td>241571</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Trimethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>243205</td>
			<td>anhydrous, &#8805; 99%</td>
		</tr>
	</tbody>
</table>

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.

The nitrogen-containing compound, carbazole, was used in this method as the calibration standard. Carbazole elutes at approximately 33 min from the primary column and at 2 s from the secondary column. These elution times will vary slightly depending on the exact column length and instrumentation. In order to obtain a proper calibration curve and, subsequently, good quantitation of nitrogen compounds within a sample, the calibration peaks should not be overloaded nor have any nitrogen cont...

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Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

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 ...

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7.5K Views.  U.S. Naval Research Laboratory. Questo è un metodo per la caratterizzazione dettagliata delle specie di azoto nel jet e nel gasolio. Consente all'utente di generare informazioni a livello di classe composta sulla distribuzione dell'azoto all'interno di un combustibile, che possono fornire una maggiore comprensione del comportamento e delle prestazioni del carburante. Questo metodo utilizza una strumentazione economica e disponibile in commercio, consente la quantificazione da un singolo standard e richiede una preparazione...

Video: Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

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.

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.

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

Reducing Willow Wood Fuel Emission by Low Temperature Microwave Assisted Hydrothermal Carbonization

Biomass is a sustainable fuel, as its CO2 emissions are reintegrated in biomass growth. However, the inorganic precursors in the biomass cause a negative environmental impact and slag formation. The selected short rotation coppice (SRC) willow wood has a high ash content (<img alt="Equation 1" src="/files/ftp_upload/58970/58970eq1.jpg" /> = 1.96%) and, therefore, a high content of emission and slag precursors. Therefore, the reduction of minerals from SRC willow wood by low temperature microwave assisted hydrothermal carbonization (MAHC) at 150 &#176;C, 170 &#176;C, and 185 &#176;C is investigated. An advantage of MAHC over conventional reactors is an even temperature conductance in the reaction medium, as microwaves penetrate the whole reactor volume. This allows a better temperature control and a faster cooldown. Therefore, a succession of depolymerization, transformation and repolymerization reactions can be analyzed effectively. In this study, the analysis of the mass loss, ash content and composition, heating values and molar O/C and H/C ratios of the treated and untreated SCR willow wood showed that the mineral content of the MAHC coal was reduced and the heating value increased. The process water showed a decreasing pH and contained furfural and 5-methylfurfural. A process temperature of 170 &#176;C showed the best combination of energy input and ash component reduction. The MAHC allows a better understanding of the hydrothermal carbonization process, while a large-scale industrial application is unlikely because of the high investment costs.

A protocol for the emission precursor depletion from low quality biomass by low temperature microwave assisted hydrothermal carbonization treatment is presented. This protocol includes the microwave parameters and the analysis of the biocoal product and process water.

Biomass is a sustainable fuel, as its CO2 emissions are reintegrated in biomass growth. However, the inorganic precursors in the biomass cause a negative ...

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. ...

Chromatographic Fingerprinting by Template Matching for Data Collected by Comprehensive Two-Dimensional Gas Chromatography

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass spectrometry. The rich information encrypted in the data may be highly valuable but difficult to access efficiently. Data density and complexity can lead to long elaboration times and require laborious, analyst-dependent procedures. Effective yet accessible data processing tools, therefore, are key to enabling the spread and acceptance of this advanced multidimensional technique in laboratories for daily use. The data analysis protocol presented in this work uses chromatographic fingerprinting and template matching to achieve the goal of highly automated deconstruction of complex two-dimensional chromatograms into individual chemical features for advanced recognition of informative patterns within individual chromatograms and across sets of chromatograms. The protocol delivers high consistency and reliability with little intervention. At the same time, analyst supervision is possible in a variety of settings and constraint functions that can be customized to provide flexibility and capacity to adapt to different needs and goals. Template matching is shown here to be a powerful approach to explore extra-virgin olive oil volatilome. Cross-alignment of peaks is performed not only for known targets, but also for untargeted compounds, which significantly increases the characterization power for a wide range of applications. Examples are presented to evidence the performance for the classification and comparison of chromatographic patterns from sample sets analyzed under similar conditions.

This protocol presents an approach to fingerprint and explore multi-dimensional data collected by comprehensive two-dimensional gas chromatography coupled to mass spectrometry. Dedicated pattern recognition algorithms (template matching) are applied to explore the chemical information encrypted in the extra-virgin olive oil volatile fraction (i.e., volatilome).

Data processing and evaluation are critical steps of comprehensive two-dimensional gas chromatography (GCxGC), particularly when coupled to mass ...

Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including controlled substances, ignitable liquids, and explosives. GC-MS can be coupled with Solid-Phase Microextraction (SPME), in which a fiber with a sorptive coating is placed into the headspace above a sample or immersed in a liquid sample. Analytes are sorbed onto the fiber which is then placed inside the heated GC inlet for desorption. Total Vaporization Solid-Phase Microextraction (TV-SPME) utilizes the same technique as immersion SPME but immerses the fiber into a completely vaporized sample extract. This complete vaporization results in a partition between only the vapor phase and the SPME fiber without interference from a liquid phase or any insoluble materials. Depending upon the boiling point of the solvent used, TV-SPME allows for large sample volumes (e.g., up to hundreds of microliters). On-fiber derivatization may also be performed using TV-SPME. TV-SPME has been used to analyze drugs and their metabolites in hair, urine, and saliva. This simple technique has also been applied to street drugs, lipids, fuel samples, post-blast explosive residues, and pollutants in water. This paper highlights the use of TV-SPME to identify illegal adulterants in very small samples (microliter quantities) of alcoholic beverages. Both gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) were identified at levels that would be found in spiked drinks. Derivatization by a trimethylsilyl agent allowed for conversion of the aqueous matrix and GHB into their TMS derivatives. Overall, TV-SPME is quick, easy, and requires no sample preparation aside from placing the sample into a headspace vial.

Total Vaporization Solid Phase Microextraction (TV-SPME) completely vaporizes a liquid sample whilst analytes are sorbed onto a SPME fiber. This allows for partitioning of the analyte between only the solvent vapor and the SPME fiber coating.

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including ...