The objective of my time at UNC was to learn the DT40 bioassay based on chicken cell lines and use it asses the toxicity of Polycyclic Aromatic Hydrocarbon (PAH)-contaminated soil after bioremediation. Though I was quite excited about the opportunity, I was initially intimidated about leaving the familiarity of the chemistry lab at Oregon State University (OSU) and flying cross country to immerse myself in the unfamiliar (and very sterile!) world of cells and assays. It was a definite humbling learning experience; working with living cells taught me just how much of a virtue patience is –something that has helped me develop personally and as a researcher.
The KC Donnelly Externship created a platform on which we were able to combine analytical chemistry, biological and environmental engineering, and toxicology to address a shared concern. I was really inspired by the integration of the different ideas and mindsets from the various fields as we developed this project.
Before the externship, I was analyzing PAHs in remediated soil samples. At UNC, I learned about the DT40 assay and actually got to see how a lab-scale bioreactor (meant to simulate ex situ bioremediation) operated. I feel I now have a better understanding of how bioremediation works and the toxicity concerns often associated with PAHs. The experience has really added more depth to my research at OSU.
The externship was a very intense three months, but I really believe it was a pivotal moment in my development as an environmental health scientist; and has made me more appreciative of my research project. I also just had a great time interacting with everyone at the UNC Superfund Research Program (SRP).
A very powerful and sensitive instrument used to study trace amounts of chemicals is a gas chromatograph connected to a mass spectrometer, or GCMS. GCMS is especially useful for air samples, but it is also used to detect, quantify, and identify chemicals in water, soil, plant and animal tissue, and many other substances.
The GCMS can detect chemicals in amounts as small as a picogram. That is 0.000000000001 gram. One picogram is the equivalent of one drop of detergent in enough dishwater to fill a trainload of railroad tank cars ten miles long. Many of the pollutants found in air are present at concentrations lower than one picogram in a cubic meter of air. It is important for an the instrument to be able to detect these low concentrations.
The GCMS instrument is made up of two parts.
The gas chromatography (GC) portion separates the chemical mixture into pulses of pure chemicals
The mass spectrometer (MS) identifies and quantifies the chemicals.
The GC separates chemicals based on their volatility, or ease with which they evaporate into a gas. It is similar to a running race where a group of people begin at the starting line, but as the race proceeds, the runners separate based on their speed. The chemicals in the mixture separate based on their volatility. In general, small molecules travel more quickly than larger molecules.
The MS is used to identify chemicals based on their structure. Let’s say after completing a puzzle, you accidentally drop it on the floor. Some parts of the puzzle remain attached together and some individual pieces break off completely. By looking at these various pieces, you are still able to get an idea of what the original puzzle looked like. This is very similar to the way that the mass spectrometer works.
Gas chromatography (GC)
Injection port – One microliter (1 µl, or 0.000001 L) of solvent containing the mixture of molecules is injected into the GC and the sample is carried by inert (non-reactive) gas through the instrument, usually helium. The inject port is heated to 300° C to cause the chemicals to become gases.
Oven – The outer part of the GC is a very specialized oven. The column is heated to move the molecules through the column. Typical oven temperatures range from 40° C to 320° C.
Column – Inside the oven is the column which is a 30 meter thin tube with a special polymer coating on the inside. Chemical mixtures are separated based on their votality and are carried through the column by helium. Chemicals with high volatility travel through the column more quickly than chemicals with low volatility.
Mass Spectrometer (MS)
Ion Source – After passing through the GC, the chemical pulses continue to the MS. The molecules are blasted with electrons, which cause them to break into pieces and turn into positively charged particles called ions. This is important because the particles must be charged to pass through the filter.
Filter – As the ions continue through the MS, they travel through an electromagnetic field that filters the ions based on mass. The scientist using the instrument chooses what range of masses should be allowed through the filter. The filter continuously scans through the range of masses as the stream of ions come from the ion source.
Detector – A detector counts the number of ions with a specific mass. This information is sent to a computer and a mass spectrum is created. The mass spectrum is a graph of the number of ions with different masses that traveled through the filter.
The data from the mass spectrometer is sent to a computer and plotted on a graph called a mass spectrum.
The Unsolved Mysteries of Human Health web site was developed by the Environmental Health Sciences Center, another NIEHS-funded Center at OSU. The GCMS section of the web site was developed in collaboration with Dr. Staci Simonich, Superfund Center Project 5 leader. The interactive image above received about 37,000 pageviews this past year (up about 10,000 from the previous year). It is the most popular page coming out of our Centers.
Unfortunately, the interactive image does not currently work on an iPhone or iPad.
This year Carlos Manzano received his PhD from the Simonich lab (Project 5), and moved on from the OSU Superfund Research Center (SRC).
His research with the SRC focused on the development of new analytical techniques for the analysis of PAHs in complex environmental samples. With Dr. Simonich, he developed an analytical method using comprehensive two dimensional GC (GCxGC/ToF-MS) to analyze around 90 PAHs in one chromatographic run, using a highly orthogonal column combination. For his PhD thesis, they wanted to focus specifically on oxy-PAHs and alkyl-PAHs, which were part of other SRP projects at OSU. They got some standards from other groups, and regularly collaborated with other cores and projects.
During his training, he received a prestigious 2012 Student Paper Award from the American Chemical Society (ACS). His work was published in ES & T.
Manzano’s PhD thesis helped him get his current position. He is now holding a postdoctoral Visiting Fellowship in Canadian Laboratories, working in the Canada Centre for Inland Waters as part of the Aquatics Contaminants Research Division of Environment Canada, located in Burlington, Ontario. His research focuses on novel methods and analysis of polycyclic aromatic compounds in oil sands sediments, precipitation and snow samples. The goals are to expand the list of PACs to match reported industry emissions and to identify new PACs that characterize atmospheric emissions from bitumen upgraders as well as dust from mining and refinery waste.
Thanks to the SRP funding and meetings I was also able to meet my current supervisors and share with them my research interests (I met them at SETAC Long Beach in 2012).
The Superfund Research Program is federally funded and administered by the National Institute of Environmental Health Sciences (NIEHS grant #P42 ES016465), an institute of the National Institutes of Health.