Novel Analytical Techniques

My research group uses stable isotopes (²H, ¹³C, ¹⁵N, ³⁴S, etc) in organic molecules as tracers of biological process in the environment, and through Earth history. Mass spectrometers are our eyes and ears in this pursuit, allowing us to measure the abundance of different isotopes in a variety of samples with incredible precision. Some of these measurements are fairly routine – for example, measuring the bulk carbon isotope abundance (more precisely, the ¹³C/¹²C ratio) of a leaf. However, at the cutting edge of our field is a push to make measurements that are more sensitive, more precise, provide increased levels of molecular resolution, and even extend to new isotopes. These require new technical advances in sample handling, data analysis, instrument hardware, and even (sometimes) the fundamental measurement approach. In our group we spend a lot of time trying to develop "new measurements", a term that includes both building new hardware as well as using existing hardware in new ways. These new measurements open up whole new windows of scientific enquiry for us, helping us solve old problems as well as finding new patterns that have never before been observed. A few examples of analytical advances developed by our lab group are described below.

Hydrogen isotopes in organic molecules have traditionally been measured mainly in lipids. These wax-like molecules have a lot of advantages, but don't provide very much biological specificity, i.e. they are not unique enough in their structure to tell you who made them. We have thus been interested in trying to measure H isotopes in other biomolecules, and two recent projects in the group have addressed these shortcomings. Ph.D. student Shae Silvermann has recently developed a protocol for measuring the hydrogen isotope ratios of amino acid C-bound H using gas chromatograph-pyrolysis-IRMS. A key aspect of her protocol is the derivatization of carboxyl and amine groups, and correcting for added H atoms. She is now applying these measurements to study the origins of marine particulate organic matter (POM). In a separate project, Ph.D. student Elliott Mueller developed a method for measuring the hydrogen isotope ratios of acetate, propionate, and butyrate in aqueous solution using electrospray ionization-Orbitrap mass spectrometry. Elliott is using this measurement to study acetate produced abiotically in deep crustal waters from the Canadian Shield, as well as from fermenting microbes in other environments.

Sulfur is a notoriously difficult element to measure, for a variety of reasons. We have been working on improving techniques for sulfur for over a decade now, and have made several breakthroughs. Postdoc Alon Amrani showed that we could make highly sensitive and precise measurements of three of the four sulfur stable isotopes (³²S, ³³S, ³⁴S) using multi-collector ICPMS (sulfur was traditionally measured using IRMS, a different kind of mass spectrometer). He then went on to attach our gas chromatograph to Jess Adkin's MC-ICP-MS, making the first-ever compound-specific S isotope measurements (see photo in the sidebar). We have since applied these measurements to volatile DMS emitted from the oceans, organosulfur compounds in crude oils, and trace sulfate in rivers and glaciers. In a separate project, Postdoc Tony Wang and Ph.D. student Alex Phillips modified a Thermo Flash elemental analyzer to trap the SO₂ resulting from sample combustion on the GC column. Reducing the carrier gas flow, and releasing the SO² by heating the GC column, then allowed them to elute and measure the SO2 as a sharp peak while reducing the amount of sample required by roughly two orders of magnitude. This opened up many new possibilities for S isotope measurements on the EA-IRMS, including individual amino acids (cysteine and methionine). Alex Phillips used the new technique to measure the S isotopes of dissolved organic matter in seawater, and to prove that sulfurized DOM from sediment porewaters cannot be the source of long-lived, recalcitrant DOM.

Stable isotopes of different elements (e.g., 2H and 13C) have traditionally been measured and considered separately. But the two different isotopes do coexist in the same molecules, and sometimes interact with each other in interesting and useful ways. John Eiler and I, together with Ph.D. student Daniel Stolper and a collaboration with instrument maker Thermo Scientific, developed a whole new approach for looking at the "clumped isotope" species of methane. The new mass spec that Thermo built is called the "Ultra HR-IRMS", and it allowed us to use the mass spectrum of intact methane to measure not only the abundance of ¹³CH₄ and CH₃D, but also ¹³CH₃D, all at the same time. This turned out to be a useful thermometer for the temperature of formation of methane. Other 'clumped isotope' measurements of organic molecules have followed.

Our field is continually seeking out more detailed descriptions of how and where stable isotope fingerprints exist. The first geochemists measured 'bulk' materials, like leaves and sediments; then came 'compound-specific' measurements in which individual compounds were first isolated by chromatography, then measured. The frontier today is 'position-specific' measurements, which tell us about the isotopic composition of each different atomic position within a molecule. Continuing my collaboration with John Eiler, we have been leveraging the techniques developed for methane to investigate variations of isotope abundance within molecules. We do this by fragmenting the molecule into different pieces within the mass spectrometer, then comparing the isotope ratios of the pieces to deduce where within the molecule the interesting signals exist. We are currently using the Orbitrap-MS as our analytical platform, and applying these measurements to a variety of biomolecules including amino acids (from plants, microbes, and meteorites), short-chain acids from fermenting bacteria, and glucose from tree rings. The opportunities for new research ideas here seem almost unlimited!