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Equilibrium sampling with mussel Hydrophobic chemicals that persist in the environment can be enriched to high levels in the lipid-rich tissues of animals and humans. Toxicity becomes a problem when these chemicals reach high concentrations in the body; the chemical activity of each compound is also a factor. In a new paper published in AC (DOI 10.1021/ac802261z), Philipp Mayer and colleagues at the University of Aarhus (Denmark) and Lund University (Sweden) describe a new technique that uses a silicone membrane equilibrator to measure chemical activities of nonpolar chemicals directly in tissues and lipids. Researchers have struggled to come up with methods to measure chemical activities in media that contain multiple phases, such as lipid mixtures, or in the even more complex systems of living organisms. “What is very difficult for some other techniques is sampling in complex matrices that contain, for instance, many lipids. Lipid-associated analytes might...bias the measurement,” says Mayer. To avoid this problem, the researchers engineered a device capable of equilibrating the chemicals of interest between a sample and a well-defined medium without coextracting unnecessary matrix components. To do this, they used a silicone tube and a two-step equilibrium process. “If you use a tube, you can do the sampling through the outside surface of the tubing. Compounds partition from the outside into the silicone wall. Step two is that the compounds are partitioned from the silicone wall into a solvent on the inside of the tube,” explains Mayer. “Since we do the measurement on the backside of the silicone, any greasy phase on the outside of the tube will not make it into the extract.” For the first round of samples, methanol or vegetable and fish oils were spiked with 12 different polycyclic aromatic hydrocarbons (PAHs)Oa class of compounds that includes some environmental pollutants. A 6-m-long PDMS 1726
ANALYTICAL CHEMISTRY /
MARCH 1, 2009
microtube was placed in a vial containing the heterogeneous sample.
A schematic of a silicone membrane equilibrator. A lipid-rich sample is placed in the vial (left), and a small methanol plug is sent through 6 m of silicone tubing within the vial. Three types of equilibrium are achieved with this method (inset): (1) external equilibrium between sample and silicone, (2) internal equilibrium between silicone and methanol plug, and (3) equilibrium across the silicone wall.
After allowing the sample and silicone tube to equilibrate for varying lengths of time, the researchers sent “a small methanol plug (100 L) on a journey through the tube,” says Mayer. “After 1 minute, it comes out on the other end in equilibrium with the silicone and thus the sample.” But how did the researchers know they had achieved equilibrium in both of these steps? For the first step, equilibrium between the silicone tubing’s outer wall and a sample, the researchers varied the contact time between the tube and a PAH-containing methanol sample. Then, they sent a methanol plug through the tube and plotted the ratio of the PAH concentration in the methanol plug versus its concentration in the methanol sample as a function of sampling time. After 10 minutes of contact time, the plug and sample concentrations of PAH stabilized at a ratio close to unity, indicating that equilibrium had been reached.
For the second equilibration, it was critical to get the plug volume and the tube length just right. Mayer and colleagues found that a 6-m-long tube allowed the solvent plug to reach equilibrium with the silicone before exiting the tube. The researchers also established that a 100 L plug volume was not too large to deplete the silicone of PAHs but was still large enough for analysis by high-performance liquid chromatography. “If you use too much methanol, you change the concentration in the silicone,” says Mayer. “We want to have an undisturbed equilibrium, which is the prerequisite for measurements of chemical activity.” Equilibrium was established between the plug and silicone tubing with residence timesOthe amount of time the plug spends in the tubeOas brief as 39 seconds, indicating that there was no need to control for this variable. After being expelled from the tube, the solvent plug was fed directly into an HPLC to determine the concentrations of PAHs. Detection limits were established to be 0.2⫺0.3 g/L. Chemical activities were then calculated by multiplying these concentrations by the analyte-specific activity coefficients in methanol. To check their method’s capacity for measuring chemical activities of PAHs in highly heterogeneous samples, the researchers analyzed mussels with known PAH exposure levels. They removed the mussels from their lab’s freezer, thawed them out, and then prepared them by “putting them into a food processor and making ground mussels,” says Mayer. They then ran their tube through this gristly mixture and successfully determined its PAH chemical activities. According to Mayer, this method is suitable for everything from on-line industrial applicationsOlike checking the safety of olive oilsOto detecting the exposure levels of dangerous chemicals in whale blubber. —Erika Gebel
10.1021/AC802731A 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 01/15/2009
