Name: Chemical Analysis of Water-accommodated Fractions of Crude Oil Spills Using TIMS-FT-ICR MS (Video) | JoVE Journal
Uploaded: 2017-03-03T14:00:00.000+00:00
Duration: 8 min 17 s
Description: Learn TIMS-FT-ICR MS analysis for molecular characterization of crude oil water-accommodated fractions. This method enables high-resolution identification of thousands of components with minimal sample usage.

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5 minutes

This work was supported by the National Institute of Health (Grant No. R00GM106414 to FFL). We would like to acknowledge the Advanced Mass Spectrometry Facility of Florida International University for their support.

1. Preparation of the Low-energy Water-accommodated Fractions (LEWAF)
<ol>
	<li>Clean 2-L aspirator bottles by rinsing the bottles with methylene chloride in order to remove any potential contaminants.</li>
	<li>Fill bottles with 50&#160;mL of methylene chloride, close them, and agitate for 30 s. Drain them into the appropriate waste container. Repeat for a total of three washes.</li>
	<li>Use one aspirator bottle for the irradiation exposure and the other bottle as a control sample (perform duplicate sets of each if possible).</li>
	<li>Prepare artificial seawater by mixing 35 g of sea salt mixture per 1 L of water used for a final salinity of 33 parts-per-thousand. Filter the solution under little or no negative pressure to prevent the loss of volatile compounds using a filtration apparatus equipped with a pore-size filter of 0.45 &#181;m or smaller.</li>
	<li>Fill the aspirator bottles with the artificial seawater, leaving ~20%, or 400 mL, of headspace.</li>
	<li>Add the crude oil to the premeasured seawater using a gas-tight syringe at a nominal concentration of 1 g oil/L. 
	NOTE: More viscous oils should be delivered by weight.</li>
	<li>After the addition of the oil, mix the solutions for 24 h in the dark (or by covering the aspirator bottles with aluminum foil) using a stirring plate operated at low speed without the generation of a vortex (approximately 100 rpm) in order to allow all the water-soluble components to go into solution and to avoid the inclusion of particulate oil.</li>
</ol>
2. LEWAF Photo-irradiation, Sample Collection, and Handling
<ol>
	<li>Prepare two sets of bottles for each photo-irradiation experiment.</li>
	<li>Cover one set of aspirator bottles with aluminum foil; this will be the dark control.</li>
	<li>Place the aspirator bottles in a temperature-controlled water bath set to maintain the aspirator bottles at a temperature of 25 &#176;C inside of the solar irradiator equipped with a 1,500-W xenon arc lamp at a light intensity of 765 W/m2.</li>
	<li>Irradiate the samples continuously for the desired time (this experiment lasted 115 h); there is no maximum time.</li>
	<li>After set times, (15, 24, 48, and 115 h in this experiment) collect 150 mL of water from the bottom of the glass aspirator by uncorking the aspirator bottle and opening the drain valve.</li>
	<li>Utilizing a separatory funnel perform a liquid-liquid extraction with methylene chloride by adding 50 mL aliquots of methylene chloride, shaking the sample for 2 min regularly venting the funnel to avoid the stopper from blowing off.</li>
	<li>Allow the phases to separate, and remove the bottom organic phase by removing the stopper from the funnel and slowly open the drain valve. Drain the sample into a flat bottom flask equipped with a funnel filled with 100 g of Na2SO4. Rinse funnel with additional methylene chloride Perform the extraction 3 times in order to increase the extraction efficiency. 
	NOTE: Multiple extractions are performed in order to increase the extraction efficiency of the procedure.</li>
	<li>Remove the funnel, add a Snyder column and place the flat bottom flask in a hot water bath set to 58 &#176;C. Evaporate until 15 mL of sample remains.</li>
	<li>Transfer the sample into a concentrating tube and dry the samples using a gentle stream of nitrogen careful to avoid loss of sample by splashing.</li>
</ol>
3. Preparation of the Sample for Analysis
NOTE: Sample preparation for analysis is key, and care must be taken to avoid the introduction of foreign contaminants, particularly through the use of any plastics, which will cause leaching into the sample.
<ol>
	<li>Prepare a mixture of 1:1 v:v methanol:toluene utilizing LC-MS optima-grade solvents.</li>
	<li>Using a positive-displacement micropipette, add solvent to the glass vials and then add the samples at a ratio of 1:100, first by adding 990 &#181;L of solvent and then by adding 10 &#181;L of sample. 
	NOTE: Glass micropipettes are used in order avoid leeching due to the methylene chloride or toluene.</li>
	<li>Optional: Use tuning mix to calibrate the ion mobility. 
	NOTE: An API source will often leach if it is regularly used as a calibration standard. If the calibration ions are not present in the spectra, then a mixture can be prepared by adding some of the standard solution to the sample at a 1:1,000 dilution.</li>
</ol>

4. Fourier Transform&#8211;Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) Analysis
<ol>
	<li>Set the instrument to the &#34;Operate&#34; mode by clicking the operate button.</li>
	<li>Load the sample into a sample syringe.</li>
	<li>Under the API source tab, set the infusion rate at the 200 &#181;L/h rate.</li>
	<li>Set the nebulizer pressure to 1 bar and the vaporizer temperature to 300 &#176;C.</li>
	<li>Set dry gas rate to 1.2 L/min and the temperature to 220 &#176;C. 
	NOTE: This procedure is applicable to both APPI and APCI.</li>
	<li>Set the instrument to &#34;Tune&#34; mode by clicking the tune button. A spectrum should appear on the screen. 
	NOTE: If needed, the instrument may be optimized in order to improve ion transmission and detection in the m/z range of the analytes by changing the instrumental parameters, such as voltages, rf frequencies, and amplitudes. Note that the mass spectrometer will need to be recalibrated.</li>
	<li>Stop &#34;Tune&#34; mode by clicking the stop button.</li>
	<li>Set the number of scans to average, typically between 20-200 scans per mass spectrum, and press the acquisition button to acquire a mass spectrum. 
	NOTE: Averaging multiple spectra improves the overall signal-to-noise ratio by decreasing the noise and increasing the signal for low-abundance species14.</li>
</ol>
5. Trapped Ion Mobility Spectrometry (TIMS) Analysis
<ol>
	<li>Enable &#34;Chromatography&#34; mode on the FT-ICR acquisition program.</li>
	<li>Enable &#34;TIMS&#34; mode by turning the switch on the external breaker to TIMS. 
	NOTE: The voltages used in TIMS are controlled externally from the instrument. A second software will control the entrance funnel of the instrument to perform the IMS analysis.</li>
	<li>Turn on the supplementary roughing pump and, using the control valve of the TIMS, adjust the inlet and outlet pressures to desired ranges (in this experiment, P1 was set to 2.8 mbar and P2 to 1.4 mbar). 
	NOTE: The external pump should be turned on a few hours prior to analysis to ensure that the pump is warm and that performance is uniform during the IMS analysis, since changes in gas velocity will cause errors in the spectra.</li>
	<li>First identify the voltage range that is required to perform the IMS analysis; this is done with a fast, cursory search over a wide voltage range (250 V) at a low IMS resolution (1 V/frame).</li>
	<li>If the voltages used are sufficient to analyze the whole spectrum, then repeat the experiment, adjusting the TIMS for a higher-resolution analysis. To do so, decrease the V/frame to 0.1 V and change the number of frames to cover all of the &#916;Vramp. 
	NOTE: The user may need to optimize the number of TIMS experiments that are accumulated into the collision cell prior to the FT-ICR MS analysis.</li>
</ol>
6. Data Analysis
<ol>
	<li>Open the MS data in a data analysis software.</li>
	<li>Set the peak-picking criteria to select those above 6 signal-to-noise and use the &#34;Find Peaks&#34; function. 
	NOTE: The resulting spectrum is internally recalibrated using standards present in the solution. This is used to identify chemical series and is then recalibrated again in order to improve the accuracy for broad-range elemental assignment.</li>
	<li>Perform elemental assignment using a formula assignment tools or software. 
	NOTE: Formula limits of C1-100H1-100N0-2O0-7S0-2, odd and even electron configurations, are allowed, and a mass tolerance of 0.5 ppm M+ and [M+H]+ ions forms</li>
	<li>Import the IMS-MS data to a data analysis software and generate an average mass spectrum for the whole analytical range.</li>
	<li>Select peaks above 6 signal-to-noise. Using the series that was identified in the ultrahigh-resolution MS analysis, internally recalibrate the spectrum</li>
	<li>Using the formula identifications from the FT-ICR MS analysis, generate a list of all the target m/z, and save this as &#34;Flist.txt&#34; in the data folder. This list will be used in the first step of data processing</li>
	<li>Load and run the provided script in order to extract an extracted ion chromatogram (EIC) for every target m/z in the list.</li>
	<li>Modify the python script to input the file directory, the starting voltage, the step size, and the number of steps in that target analysis11.</li>
	<li>Run the IMS deconvolution script. 
	NOTE: This will open each EIC and identify the peaks by iterative Gaussian fitting. Results will be output in Excel file format13. 
	NOTE: After fitting, peaks below a certain criterion are excluded, typically based on the height, width, and area of the peak (peaks below a certain width are typically excluded from the analysis because of random noise spikes).</li>
	<li>Utilizing the tuning peaks, calibrate the ion mobility from voltage to mobility for the ions11. This can be converted to a collision cross-section using the Mason-Schamp equation.</li>
</ol>

<ol><li>King, S. M., Leaf, P. A., Olson, A. C., Ray, P. Z., Tarr, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photolytic+and+photocatalytic+degradation+of+surface+oil+from+the+Deepwater+Horizon+spill%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">Photolytic and photocatalytic degradation of surface oil from the Deepwater Horizon spill.</a> Chemosphere. 95 (0), 415-422 (2014).</li>
<li>Ray, P. Z., Chen, H., Podgorski, D. C., McKenna, A. M., Tarr, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sunlight+creates+oxygenated+species+in+water-soluble+fractions+of+Deepwater+Horizon+oil%5BTitle%5D)+AND+%22J+Hazard+Mater%22%5BJournal%5D)">Sunlight creates oxygenated species in water-soluble fractions of Deepwater Horizon oil.</a> J Hazard Mater. 280 (0), 636-643 (2014).</li>
<li>Duesterloh, S., Short, J. W., Barron, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoenhanced+toxicity+of+weathered+Alaska+North+Slope+crude+oil+to+the+calanoid+copepods+Calanus+marshallae+and+Metridia+okhotensis%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">Photoenhanced toxicity of weathered Alaska North Slope crude oil to the calanoid copepods Calanus marshallae and Metridia okhotensis.</a> Environ Sci Technol. 36 (18), 3953-3959 (2002).</li>
<li>Duxbury, C. L., Dixon, D. G., Greenberg, B. M. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/etc.5620160824/abstract">Effects of simulated solar radiation on the bioaccumulation of polycyclic aromatic hydrocarbons by the duckweed Lemna gibba.</a> Environmental Toxicology and Chemistry. 16 (8), 1739-1748 (1997).</li>
<li>Faksness, L. G., Altin, D., Nordtug, T., Daling, P. S., Hansen, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+comparison+and+acute+toxicity+of+water+accommodated+fraction+%28WAF%29+of+source+and+field+collected+Macondo+oils+from+the+Deepwater+Horizon+spill%5BTitle%5D)+AND+%22Mar+Pollut+Bull%22%5BJournal%5D)">Chemical comparison and acute toxicity of water accommodated fraction (WAF) of source and field collected Macondo oils from the Deepwater Horizon spill.</a> Mar Pollut Bull. 91 (1), 222-229 (2015).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biodegradation+of+dispersed+Macondo+crude+oil+by+indigenous+Gulf+of+Mexico+microbial+communities%5BTitle%5D)+AND+%22Science+of+The+Total+Environment%22%5BJournal%5D)">Biodegradation of dispersed Macondo crude oil by indigenous Gulf of Mexico microbial communities.</a> Science of The Total Environment. 557-558, 453-468 (2016).</li>
<li>McKenna, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+of+the+analytical+window+for+oil+spill+characterization+by+ultrahigh+resolution+mass+spectrometry%3A+beyond+gas+chromatography%5BTitle%5D)+AND+%22Environ+Sci+Technol%22%5BJournal%5D)">Expansion of the analytical window for oil spill characterization by ultrahigh resolution mass spectrometry: beyond gas chromatography.</a> Environ Sci Technol. 47 (13), 7530-7539 (2013).</li>
<li>Fernandez-Lima, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Petroleum+crude+oil+characterization+by+IMS-MS+and+FTICR+MS%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Petroleum crude oil characterization by IMS-MS and FTICR MS.</a> Anal Chem. 81 (24), 9941-9947 (2009).</li>
<li>Benigni, P., Marin, R., Fernandez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+unsupervised+polyaromatic+hydrocarbons+structural+assignment+from+SA-TIMS-FTMS+data%5BTitle%5D)+AND+%22Int+J+Ion+Mobil+Spectrom%22%5BJournal%5D)">Towards unsupervised polyaromatic hydrocarbons structural assignment from SA-TIMS-FTMS data.</a> Int J Ion Mobil Spectrom. 18 (3), 151-157 (2015).</li>
<li>Benigni, P., Thompson, C. J., Ridgeway, M. E., Park, M. A., Fernandez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+high-resolution+ion+mobility+separation+coupled+to+ultrahigh-resolution+mass+spectrometry+of+endocrine+disruptors+in+complex+mixtures%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Targeted high-resolution ion mobility separation coupled to ultrahigh-resolution mass spectrometry of endocrine disruptors in complex mixtures.</a> Anal Chem. 87 (8), 4321-4325 (2015).</li>
<li>Benigni, P., Fernandez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oversampling+Selective+Accumulation+Trapped+Ion+Mobility+Spectrometry+coupled+to+FT-ICR+MS%3A+Fundamentals+and+Applications%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">Oversampling Selective Accumulation Trapped Ion Mobility Spectrometry coupled to FT-ICR MS: Fundamentals and Applications.</a> Analytical Chemistry. , (2016).</li>
<li>Castellanos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+Screening+of+Polycyclic+Aromatic+Hydrocarbons+using+Trapped+Ion+Mobility+Spectrometry+Mass+Spectrometry%5BTitle%5D)+AND+%22Anal+Methods%22%5BJournal%5D)">Fast Screening of Polycyclic Aromatic Hydrocarbons using Trapped Ion Mobility Spectrometry Mass Spectrometry.</a> Anal Methods. 6 (23), 9328-9332 (2014).</li>
<li>Benigni, P., DeBord, J. D., Thompson, C. J., Gardinali, P., Fernandez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+Polyaromatic+Hydrocarbon+%28PAH%29+Molecular+Coverage+during+Fossil+Oil+Analysis+by+Combining+Gas+Chromatography+and+Atmospheric-Pressure+Laser+Ionization+Fourier+Transform+Ion+Cyclotron+Resonance+Mass+Spectrometry+%28FT-ICR+MS%29%5BTitle%5D)+AND+%22Energy+%26+Fuels%22%5BJournal%5D)">Increasing Polyaromatic Hydrocarbon (PAH) Molecular Coverage during Fossil Oil Analysis by Combining Gas Chromatography and Atmospheric-Pressure Laser Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS).</a> Energy & Fuels. 30 (1), 196-203 (2016).</li>
<li>Qi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Absorption-Mode+Fourier+Transform+Mass+Spectrometry%3A+the+Effects+of+Apodization+and+Phasing+on+Modified+Protein+Spectra%5BTitle%5D)+AND+%22Journal+of+the+American+Society+for+Mass+Spectrometry%22%5BJournal%5D)">Absorption-Mode Fourier Transform Mass Spectrometry: the Effects of Apodization and Phasing on Modified Protein Spectra.</a> Journal of the American Society for Mass Spectrometry. 24 (6), 828-834 (2013).</li>
<li>Lababidi, S., Schrader, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Online+normal-phase+high-performance+liquid+chromatography%2FFourier+transform+ion+cyclotron+resonance+mass+spectrometry%3A+Effects+of+different+ionization+methods+on+the+characterization+of+highly+complex+crude+oil+mixtures%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Online normal-phase high-performance liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry: Effects of different ionization methods on the characterization of highly complex crude oil mixtures.</a> Rapid Communications in Mass Spectrometry. 28 (12), 1345-1352 (2014).</li>
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The authors declare no competing financial interests.

Critical Steps within the Protocol
The chemical complexity of LEWAFs requires accurate preparation in order for the laboratory experiments to accurately reflect what occurs naturally. A valid assessment of the data hinges on three criteria: minimizing the introduction of artifacts throughout sample handling (e.g., preparation of the LEWAF, sampling, extractions, and preparation of the sample for analysis), validating the experimental protocol (i.e., using dark controls for the p...

There are numerous sources of environmental exposure to crude oil, both from natural causes and from anthropogenic exposure. Upon release to the environment, particularly in the ocean, the crude oil can undergo partitioning, with the formation of an oil slick on the surface, a loss of volatile components to the atmosphere, and sedimentation. However, low-energy mixing of the poorly soluble oil and the water does occur, and this mixture, which is not classically solubilized, forms what is referred to as the low-energy water-accommodated fraction (LEWAF). The solubilization of the oil components in the water is typically enhanced during exposure of the oil-water interface to solar radiation. This photo-solubilization of the crude oil in the ocean can undergo significant chemical changes due to this exposure to solar radiation and/or due to enzymatic degradation1,2. Understanding these chemical changes and how they occur in the presence of the bulk matrix (i.e., the crude oil) is fundamental to mitigating the effects this exposure has on the environment.
Previous studies have shown that crude oil undergoes oxygenation, particularly the polycyclic aromatic hydrocarbons (PAHs), which represent a highly toxic source of contamination that harms organisms, undergoes bio-accumulation, and is bioactive3,5,6. Understanding the products of the different oxygenation processes is challenging because they occur only in the presence of the bulk matrix. Therefore, a single, standard analysis may not be representative of the changes occurring in nature. The preparation of the LEWAF must replicate the natural processes that take place in an environmental setting. Of particular interest is the oxygenation of PAHs, which occurs due to solar radiation.
The second challenge in the study of the water-accommodated fraction is the molecular identification of the different chemical constituents in the sample. Due to the complexity of the sample, caused by its high mass and degree of oxygen, the oxygenation products are typically unsuitable for the traditional analysis carried out by gas chromatography combined with MS analysis7,8. An alternative approach is to characterize the changes in the chemical formula of the sample by utilizing ultra-high mass resolution MS techniques (e.g., FT-ICR MS). By coupling TIMS to FT-ICR MS, in addition to the isobaric separation in the MS domain, the ion mobility spectrometry (IMS) dimension provides the separation and characteristic information for the different isomers present in the sample9,10,11. Combined with an atmospheric pressure laser ionization (APLI) source, the analysis can be selective to the conjugated molecules found in the sample, allowing the changes that the PAHs undergo to be accurately characterized12,13.
In this work, we describe a protocol for the preparation of LEWAFs exposed to photo-irradiation in order to study the transformation processes of the oil components. We also illustrate the changes that occur upon photo-irradiation, as well as the procedure for sample extraction. We will also present the use of APLI with TIMS coupled with FT-ICR MS to characterize the PAHs in the LEWAF as a function of the exposure to light.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>methylene chloride, Optima</td><td>Fisher Scientific</td><td>DS-151-4</td><td>Used as received</td></tr><tr><td>methanol, Optima LC/MS</td><td>Fisher Scientific</td><td>A456-4</td><td>Used as received</td></tr><tr><td>toluene, Optima</td><td>Fisher Scientific</td><td>T291-4</td><td>Used as received</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;, Granular, Anhydrous</td><td>Fisher Scientific</td><td>S415-212</td><td>Combusted at 450&amp;nbsp;&amp;#176;C/Stored at 105&amp;nbsp;&amp;#176;C</td></tr><tr><td>Crude oil</td><td>Various sources</td><td></td><td></td></tr><tr><td>Instant Ocean&amp;reg;</td><td>Aquarium Systems</td><td>SS1200</td><td>33 ppt salinity with 0.45&amp;nbsp;&amp;mu;m pore filtration</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Suntext XLS+</td><td>Atlas Chicalo Ill, USA</td><td></td><td>1,500 W xeon arc lamp, light intensity of 765&amp;nbsp;W/m&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Atmospheric Pressure Laser Ionization</td><td>Bruker Daltonics Inc, MA</td><td></td><td>Note a 266 nm laser is used</td></tr><tr><td>TIMS-FT-ICR MS Instrument</td><td>Bruker Daltonics Inc, MA</td><td></td><td>The set up we had consisted of a 7&amp;nbsp;T magnet with an infinity cell</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DataAnalysis 4.2</td><td>Bruker Daltonics Inc, MA</td><td></td><td></td></tr><tr><td>Python 2.7</td><td></td><td></td><td>Requires Numpy, Scipy, Pandas, glob, oct2py, and os</td></tr><tr><td>Octave 4.0</td><td></td><td></td><td></td></tr></tbody></table>

chemical analysis of water-accommodated fractions of crude oil spills using tims-ft-icr ms

Chemical Analysis of Water-accommodated Fractions of Crude Oil Spills Using TIMS-FT-ICR MS

Multiple chemical processes control how crude oil is incorporated into seawater and also the chemical reactions that occur overtime. Studying this system requires the careful preparation of the sample in order to accurately replicate the natural formation of the water-accommodated fraction that occurs in nature. Low-energy water-accommodated fractions (LEWAF) are carefully prepared by mixing crude oil and water at a set ratio. Aspirator bottles are then irradiated, and at set time points, the water is sampled and extracted using standard techniques. A second challenge is the representative characterization of the sample, which must take into consideration the chemical changes that occur over time. A targeted analysis of the aromatic fraction of the LEWAF can be performed using an atmospheric-pressure laser ionization source coupled to a custom-built trapped ion mobility spectrometry&#8211;Fourier transform&#8211;ion cyclotron resonance mass spectrometer (TIMS-FT-ICR MS). The TIMS-FT-ICR MS analysis provides high-resolution ion mobility and ultrahigh-resolution MS analysis, which further allow the identification of isomeric components by their collision cross-sections (CCS) and chemical formula. Results show that as the oil-water mixture is exposed to light, there is significant photo-solubilization of the surface oil into the water. Over time, the chemical transformation of the solubilized molecules takes place, with a decrease in the number of identifications of nitrogen- and sulfur-bearing species in favor of those with a greater oxygen content than were typically observed in the base oil.

LEWAF analysis by TIMS-FT-ICR MS results in a two-dimensional spectrum based on m/z and TIMS trapping voltage. Each of the samples, taken at different time points, can therefore be characterized based on the changing chemical composition, as observed by the distribution of chemical formulas and the isomeric contribution identified by the IMS (see Figure 1). Typically, the m/z information can be utilized to assign elemental formulas to the analyzed peaks....

The low-energy water-accommodated fraction (LEWAF) of crude oil is a challenging system to analyze, because over time, this complex mixture undergoes chemical transformations. This protocol illustrates methods for the preparation of the LEWAF sample and for performing photo-irradiation and chemical analysis by trapped ion mobility spectrometry&#8211;FT-ICR MS.

Multiple chemical processes control how crude oil is incorporated into seawater and also the chemical reactions that occur overtime. Studying this system ...

The overall goal of these experiments is to illustrate the analytical advantages of enabling emerging technologies based on TIMS-FT-ICR MS for the molecular characterization of complex mixtures, such as water-accommodated fractions for crude oils. The TIMS-FT-ICR MS coupling combines two of the most comprehensive orthogonal and alluvial techniques for the analysis of complex mixtures. TIMS-FT-ICR MS coupling permits in a single analysis the characterization of thousands of molecular components with mobility resolving powers of over 150, mass resolution of 500, 000, and sub-ppm mass accuracy.TIMS-FT-ICR MS enables the separation, identification, and quantitation at the molecular level of complex mixtures, based on differences in size, charge and mass with minimal sample appropriation. To begin the LEGAF preparation, add 50 milliliters of dicholomethane to a two liter glass aspirator bottle. Close the bottle and agitate the bottle for 30 seconds.Discard the dichloromethane wash, and repeat this process two more times. Wash one more two liter aspirator bottle in this way, then add about 1.6 liters of filtered artificial seawater with a salinity of 33 parts per thousand to each bottle. Add a stir bar, and then use a gas-tight syringe to add sufficient crude oil sample to each bottle to achieve a final concentration of one gram per liter of oil and seawater.Wrap the bottles in aluminum foil to exclude light, and stir the mixtures at approximately 100 RPM for 24 hours to finish preparing the LEWAF. It's important that care is used when first mixing the oil and water to avoid over-mixing the solution. Next, unwrap one of the LEWAF bottles in aluminum foil, leaving one as the dark control.Place the bottles in the solar irradiator with the temperature set to maintain the samples at 25 degrees Celsius. Irradiate the mixtures continuously for the desired experiment length. At set intervals during the experiment, obtain 150 milliliter fractions from each bottle by uncorking the bottle and opening the drain valve.Extract each sample aliquot using 50 milliliters of dichloromethane three times using a separatory funnel. Shake the funnel for two minutes each time, remembering to regularly vent the funnel to avoid pressure buildup. Drain the sample into a flat-bottom flask through a funnel containing sulfate and rinse the sodium sulfate with additional DCM.Concentrate the extract to 15 milliliters using a hot water bath set at 58 degrees Celsius. Transfer the concentrate into a concentration tube, rinsing the flask with DCM. Concentrate each sample to one milliliter under a gentle stream of nitrogen gas.For each concentrated sample, using a glass positive displacement micropipette, add 990 microliters of a one to one by volume mixture of methanol and toluene to a glass vial. Then mix in 10 microliters of the sample. To begin the FTR-ICR MS analysis, set the instrument to Operate mode.Draw a sample into a sample syringe. Set the infusion rate to 200 microliters per hour, the nebulizer pressure to two bar, and the vaporizer temperature to 300 degrees Celsius. Set the dry gas rate to 1.2 liters per minute and the temperature to 220 degrees Celsius.Then set the instrument to Tune mode. Based on the resulting spectrum adjust the instrument parameters and recalibrate the mass spectrometer to improve the ion transmission and detection for the desired range of mass to charge ratios. Once the instrument parameters have been optimized, exit Tune mode.Set the desired number of scans to be average per mass spectrum, and then acquire the spectrum. To begin the trapped ion mobility spectrometry analysis turn on the external breaker to the TIMS instrument to engage TIMS mode. Set the FT-ICR instrument to chromatography mode.Turn on the supplementary roughing pump and set the inlet and outlet pressures to the desired values using the TIMS control valve. Allow the pump to warm up for at least three hours, then perform a fast sweep over a wide voltage range at a low IMS and MS resolution to identify the needed voltage range for the analysis. Once the range has been identified, set the voltage per frame to 0.1 volts.Set the number of frames to cover the entirety of the DeltaV ramp and run the analysis. After performing the analyses, process the data with the appropriate software and scripts to determine the chemical composition and mobility values of identified peaks. After sunlight irradiation of an LEWAF of a crude oil sample, changes in the chemical complexity are observed as a function of exposure time.This complexity was detected and visualized in a 2D spectrum based on mass to charge ratio and ion mobility. The use of APLI resulted in greater analytical sensitivity to molecules with double bonds. Four molecular classes were identified in the sample.Distribution plots of double bond equivalents as a function of the number of carbons were used to monitor changes in the molecular composition over time. Two-dimensional plots of inverse mobility versus carbon number were created, with the double bond equivalents represented as the color of each point. Linear trends were observed for each of the four common molecular classes.After watching this video you should have a good understanding of the basic steps and unique advantages of APLI-TIMS-FT-ICR MS for the analysis of LEWAF samples as well as other complex mixtures. While attempting this procedure it's important to remember that sample integrity, such as reducing sources of contamination, is fundamental for accurate results. The characterization at the molecular level of the water-accommodated fractions of crude oils, the transformation products, and their fate during exposure to sunlight in the ocean is of the utmost environmental importance.Compared to other separation techniques such as liquid chromatography and gas chromatography, TIMS separation occurs in a shorter time scale, and is timing dependent, which permits better coupling with ultra high resolution MS analyzers while providing collision cross sections and chemical formulas in a single analysis. The generation of new comprehensive analytical tools for the analysis of complex mixtures at molecular level, such as TIMS-FT-ICR MS has great implications for our understanding of the chemical transformations and fate of crude oil in the ocean.

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Research

JoVE Journal

Environment

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Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

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Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography, Deans Switching and Sulfur-selective Detection

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

A protocol about the characterization and application of five different passive sampling devices is presented.

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

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Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

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Laboratory Production of Biofuels and Biochemicals from a Rapeseed Oil through Catalytic Cracking Conversion

This article describes a setup and method for the in situ visualization of oil samples under a variety of temperature and pressure conditions that aim to emulate refining and upgrading processes. It is primarily used for studying isotropic and anisotropic media involved in the fouling behavior of petroleum feeds.

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions

Here, we present a protocol to simultaneously study the flammability and burning efficiency of fresh and weathered crude oil under conditions that simulate in situ burning operations on the sea.

Experimental Procedure for Laboratory Studies of In Situ Burning : Flammability and Burning Efficiency of Crude Oil

This protocol is presented to characterize the complex wetting conditions of an opaque porous medium (hydrocarbon reservoir rock) using three-dimensional images obtained by X-ray microtomography at subsurface conditions.

Pore-scale Imaging and Characterization of Hydrocarbon Reservoir Rock Wettability at Subsurface Conditions Using X-ray Microtomography

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.

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

We illustrate the methods involved in screening and identification of the biosurfactant producing microbes. Methods for chromatographic characterization and chemical identification of the biosurfactants, determining the industrial applicability of the biosurfactant in enhancing residual oil recovery are also presented.

Enhanced Oil Recovery using a Combination of Biosurfactants

Learn TIMS-FT-ICR MS analysis for molecular characterization of crude oil water-accommodated fractions. This method enables high-resolution identification of thousands of components with minimal sample usage.

Chemical Analysis of Water-accommodated Fractions of Crude Oil Spills Using TIMS-FT-ICR MS

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