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

Summary

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–FT-ICR MS.

Abstract

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–Fourier transform–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.

Introduction

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 degradation^1,2. 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 bioactive^3,5,6. 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 analysis^7,8. 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 sample^9,10,11. 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 characterized^12,13.

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.

Protocol

1. Preparation of the Low-energy Water-accommodated Fractions (LEWAF)

1. Clean 2-L aspirator bottles by rinsing the bottles with methylene chloride in order to remove any potential contaminants.
2. Fill bottles with 50 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.
3. Use one aspirator bottle for the irradiation exposure and the other bottle as a control sample (perform duplicate sets of each if possible).
4. 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 µm or smaller.
5. Fill the aspirator bottles with the artificial seawater, leaving ~20%, or 400 mL, of headspace.
6. 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.
7. 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.

2. LEWAF Photo-irradiation, Sample Collection, and Handling

1. Prepare two sets of bottles for each photo-irradiation experiment.
2. Cover one set of aspirator bottles with aluminum foil; this will be the dark control.
3. Place the aspirator bottles in a temperature-controlled water bath set to maintain the aspirator bottles at a temperature of 25 °C inside of the solar irradiator equipped with a 1,500-W xenon arc lamp at a light intensity of 765 W/m².
4. Irradiate the samples continuously for the desired time (this experiment lasted 115 h); there is no maximum time.
5. 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.
6. 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.
7. 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.
8. Remove the funnel, add a Snyder column and place the flat bottom flask in a hot water bath set to 58 °C. Evaporate until 15 mL of sample remains.
9. 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.

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.

1. Prepare a mixture of 1:1 v:v methanol:toluene utilizing LC-MS optima-grade solvents.
2. 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 µL of solvent and then by adding 10 µL of sample.
   NOTE: Glass micropipettes are used in order avoid leeching due to the methylene chloride or toluene.
3. 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.

4. Fourier Transform–Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) Analysis

1. Set the instrument to the "Operate" mode by clicking the operate button.
2. Load the sample into a sample syringe.
3. Under the API source tab, set the infusion rate at the 200 µL/h rate.
4. Set the nebulizer pressure to 1 bar and the vaporizer temperature to 300 °C.
5. Set dry gas rate to 1.2 L/min and the temperature to 220 °C.
   NOTE: This procedure is applicable to both APPI and APCI.
6. Set the instrument to "Tune" 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.
7. Stop "Tune" mode by clicking the stop button.
8. 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.

5. Trapped Ion Mobility Spectrometry (TIMS) Analysis

1. Enable "Chromatography" mode on the FT-ICR acquisition program.
2. Enable "TIMS" 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.
3. 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.
4. 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).
5. 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 ΔV_ramp.
   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.

6. Data Analysis

1. Open the MS data in a data analysis software.
2. Set the peak-picking criteria to select those above 6 signal-to-noise and use the "Find Peaks" 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.
3. Perform elemental assignment using a formula assignment tools or software.
   NOTE: Formula limits of C_1-100H_1-100N_0-2O_0-7S_0-2, odd and even electron configurations, are allowed, and a mass tolerance of 0.5 ppm M⁺ and [M+H]⁺ ions forms
4. Import the IMS-MS data to a data analysis software and generate an average mass spectrum for the whole analytical range.
5. Select peaks above 6 signal-to-noise. Using the series that was identified in the ultrahigh-resolution MS analysis, internally recalibrate the spectrum
6. Using the formula identifications from the FT-ICR MS analysis, generate a list of all the target m/z, and save this as "Flist.txt" in the data folder. This list will be used in the first step of data processing
7. Load and run the provided script in order to extract an extracted ion chromatogram (EIC) for every target m/z in the list.
8. Modify the python script to input the file directory, the starting voltage, the step size, and the number of steps in that target analysis¹¹.
9. 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).
10. 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.

Results

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....

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
methylene chloride, Optima	Fisher Scientific	DS-151-4	Used as received
methanol, Optima LC/MS	Fisher Scientific	A456-4	Used as received
toluene, Optima	Fisher Scientific	T291-4	Used as received
Na2SO4, Granular, Anhydrous	Fisher Scientific	S415-212	Combusted at 450 °C/Stored at 105 °C
Crude oil	Various sources
Instant Ocean®	Aquarium Systems	SS1200	33 ppt salinity with 0.45 μm pore filtration
Name	Company	Catalog Number	Comments
Equipment
Suntext XLS+	Atlas Chicalo Ill, USA		1,500 W xeon arc lamp, light intensity of 765 W/m2
Atmospheric Pressure Laser Ionization	Bruker Daltonics Inc, MA		Note a 266 nm laser is used
TIMS-FT-ICR MS Instrument	Bruker Daltonics Inc, MA		The set up we had consisted of a 7 T magnet with an infinity cell
Name	Company	Catalog Number	Comments
Software
DataAnalysis 4.2	Bruker Daltonics Inc, MA
Python 2.7			Requires Numpy, Scipy, Pandas, glob, oct2py, and os
Octave 4.0