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.
