by Richard Jack, PhD., Global Market Development Manager, Phenomenex
Ever since the first fluoropolymer compound was patented in 1934, manufacturers have turned to these per- and poly-fluoroalkyl substances (PFAS) to create firefighting foams, nonstick pots and pans, water-repellent clothing, electrical insulation, and more. Over the last 90 years, the number of PFAS molecules has risen to several thousand with a range of chemical properties. Some PFAS are more volatile, others are more hydrophilic. Still, others are branched. To manufacturers, these traits—and the properties they imparted upon the final product—were the characteristics that really mattered.
In the days before the widespread use of gas and liquid chromatography as well as mass spectrometry, chemists identified PFAS molecules by these physical traits. Testing required large amounts of material, and inspecting local wastewater and drinking water supplies for PFAS contamination was almost impossible. For decades, PFAS contamination went unnoticed and unchecked.
On March 16, 2023, the U.S. Environmental Protection Agency (EPA) released the proposed National Primary Drinking Water Regulation (NPDWR) for six PFAS, which puts limits on the concentration of these compounds that can be found in drinking water. Experts in this area expect the agency to finalize guidance by the end of 2023, with additional rules and guidance coming down the pipe soon. Regardless of the specifics that are set by the EPA, complying with these regulations will require testing—and lots of it.
As someone who has been in the chemical analysis field for two decades, I have seen the field evolve from targeted analyses that could only detect specific compounds to current-day sophisticated, untargeted methods that can identify vanishingly small levels of unknown PFAS. Modern testing labs can now detect PFAS in the range of parts per quadrillion. It’s the equivalent of finding a single drop of water in Lake Michigan. And we can perform many more of these tests because sample throughput efficiency has also increased. Five years ago, many contract labs were testing maybe 50 samples each month. Now we can test 100 times that amount.
As PFAS regulations continue to evolve, so will our testing methods. The biggest challenge for PFAS labs now is not sensitivity but rather incremental changes in associated technologies such as automation and sample prep that will have the biggest impact. But to develop better PFAS testing in the future, we first need to understand the field’s history and evolution.
Early production of perfluorinated compounds
For most of the 20th century, PFAS chemists did not set out to design specific molecular structures. The synthesized fluorinated surfactants are made using electrochemistry or telomerization. Electrochemical fluorination was first developed in the 1940s and produces a mixture of varying chain lengths, branching and fluorination. Telomerization was developed in the 1970s and yields mainly even-numbered, straight chain compounds. These bulk products were and are supplied to other companies to make a wide variety of additional products for industrial and commercial uses, including non-stick cookware, stain-resistant fabric, and foams that can help extinguish fires from liquid fuels like gasoline. The chemists measured traits such as viscosity and melting point. Analysis was very limited. Early chemists knew they were making a polymer, such as Teflon, and could use infrared spectroscopy to measure the carbon–fluoride bond.
PFAS compounds contain both polar and non-polar components. This makes them both hydrophobic and hydrophilic, known as amphiphilic, allowing PFAS to associate with both water and oils. The C-F bond is very resistant to breaking under both high chemical and thermal conditions. Before the advent of mass spectrometry, the exact compounds could not accurately be determined with great confidence.
Mass spectrometry (MS) is used to measure the mass-to-charge ratio of ions. Samples are vaporized and bombarded by electrons, which causes some of the sample's molecules to become positively charged. These ions are then separated according to their mass-to-charge ratio and displayed as spectra. The molecules from the sample can be identified by comparing known fragmentation patterns, known as their mass spectra. Early mass spec instruments were extremely specialized machines that could only be operated by highly trained technicians. They were found primarily in research settings rather than commercial analytical labs.
By the late 1990s and early 2000s, however, these machines became more user-friendly, and the speed and sensitivity of sensors improved. And although they never became cheap, the price of mass spectrometers also decreased. These advances dovetailed with advances in both liquid and gas chromatography. Using chromatography to separate the various compounds in a sample made it possible to detect lower concentrations of chemicals within a sample and increased precision.
Searching for a target
The next big milestone in PFAS testing came with the advent of non-targeted testing. In the early days of mass spec analysis, scientists could ask whether a sample contained a certain molecule, but not its concentration without a standard. These were extremely useful questions to answer, but it also required researchers to know what chemical they were looking for. With thousands of potential PFAS chemicals in use, that isn’t always possible. A major challenge is that pure PFAS standards were not available and in many cases are still not available, which is why non-targeted analysis is used for unknown PFAS compounds. Though validated methods exist, and several others are in development, they are only selecting for a very small subset of the potential PFAS compounds that could be contaminating the environment.
Another challenge to consider is that no PFAS chemical is 100 percent pure. Impurities from their synthesis and partial degradation in the environment can occur with every sample. Nor do researchers always know in advance what impurities to identify. To aid in the identification of PFAS and other chemicals, the EPA asked the industry to create a set of PFAS calibration standards. The widespread development and use of these standards in the past 5-10 years have greatly facilitated targeted PFAS testing. However, there is still a long road to go in terms of developing additional PFAS standards used to calibrate MS instrumentation. These standard chemicals are crucial if we need to measure the concentration of all PFAS chemicals in blood or serum, which is a key step in determining a person’s exposure and any resulting health impacts.
As more and more chemicals are characterized using chromatography and mass spec, scientists can build libraries of structures with their associated mass spec ‘fingerprint.’ Not only does each compound have a unique spectrum, but chemists can also distinguish the different isotopes, stereoisomers, and other structural variants of a single chemical. These isotope libraries, then, added another layer of precision because researchers could check their results against known fingerprints. This approach gave us more confidence in the results that we are seeing.
Sample cleanup and other challenges
The most recent major improvement that has impacted PFAS analysis is related to how samples are prepared for testing. To analyze a relatively simple, clean sample such as drinking water takes little preparation. Other types of samples, including sewage sludge, fatty foods like butter, and environmental residues known as biosolids, require extensive purification, and prep before they can be analyzed by mass spec. Testing these items for PFAS requires not only expertise in analytics but also in getting a sample ready to analyze. Fundamentally, these cleanup methods use resins or similar material to trap the PFAS in a sample, while you wash away the matrix using methanol or acetonitrile. All of this serves to concentrate the chemicals of interest.
When the EPA released its PFAS sampling and analysis standards in 2020, they included guidance on this important step. The current recommendation is to use solid phase extraction to separate the chemicals in a mixture by their physical properties. This guidance has allowed some testing companies to begin offering sample prep kits that can do the extraction and cleanup in a single step, which can provide a huge cost and labor savings. These methods also make testing more consistent across labs, which allows utilities and other agencies to compare results across time and space and provide more confidence in the results, which is crucial if national regulations are to be made.
Finally, the sensitivity of modern methods combined with the ubiquity of PFAS means that labs now must be extra careful about contamination in their instrumentation, vials, sample bottles, and reagents. Analytical labs need to make certain that the only PFAS their methods are detecting are the ones found in their samples, not as background in the lab environment.
Testing the future
The incremental improvements that continue to be made in PFAS testing and analysis have opened the eyes of the world to the widespread problem of these forever chemicals. More sophisticated and sensitive techniques have helped make us aware that water sources we thought were clean might not be, since the newer instruments and updated methods allow us to measure lower concentrations more accurately and consistently. In lieu of calibration standards, non-targeted methods will be the leading instruments to identify what PFAS compounds and precursors exist in the environment. This information will subsequently create a demand for the development of new calibration standards that mass spec MS/MS instruments can use to quantify these compounds.
The volume of impending testing can feel discouraging, but it’s also the first step in addressing the PFAS problem. We don’t know what the health impacts are for a lifetime exposure of low levels of PFAS. Technology has fundamentally changed the paradigm about what we conceive of as contaminated or not.
It’s not yet clear how to best address widespread PFAS chemicals in the environment, but regular testing of the world around us will help us ensure that they are being managed effectively.
About the author
Richard F. Jack, Ph.D. is the global market development manager for the environmental and food markets at Phenomenex. He has over 18 years experience with chromatography and mass spectrometry for the environmental, semiconductor, chemical, and pharmaceutical industries. Richard collaborates with global regulatory agencies to develop validated methods through new applications, instrumentation, column chemistries, and software.