Non-target screening enables environmental laboratories to look beyond targeted monitoring lists and detect a broader range of contaminants in complex samples. As its use grows, so does interest in highly polar, persistent, and mobile substances in water systems – compounds that can accumulate across the water cycle yet remain difficult to capture using standard analytical workflows.
However, a key limitation is that no single chromatographic method can encompass the full diversity of polar and semi-polar contaminants. To explore this challenge, Jonathan Zweigle and colleagues compared 12 methods across four major platforms – RP-LC, HILIC, SFC, and ion chromatography – assessing how each extends access to the polar domain. Their findings – summed up by the paper’s title, “Not One Method to Rule Them All” – underscore the value of combining complementary approaches to improve detection of the most mobile compounds.
We spoke with Zweigle about the study’s main results, the practical bottlenecks in multi-method screening, and how this work may help unravel the unknown fraction of fluorinated pollutants often described as “PFAS dark matter.
What do we currently know about highly polar contaminants in water systems?
Persistent and mobile substances (PMs) have become increasingly detectable over the past decade due to advances in chromatographic separation and high-resolution mass spectrometry. These analytical improvements have enabled broader detection in both research and routine laboratories. PMs are now known to be widespread across the water cycle. Their high polarity and water solubility make them difficult, and in many cases impossible to remove, even with advanced treatment processes. PMs originate from different anthropogenic sources and span multiple chemical classes, including industrial chemicals, per- and polyfluoroalkyl substances (PFAS), and pesticides. In many systems, industrial chemicals dominate the PM signature, often including protected compartments such as groundwater. Toxicological data remain limited for most PMs; those with demonstrated toxicity are referred to as PMTs. The persistence and mobility of PMs lead to accumulation within the water cycle, continuous human exposure through drinking water, and irreversible environmental distribution. Furthermore, measured concentrations frequently exceed those of other micropollutants.
Why is it so important for non-target screening approaches to cover this very polar chemical space?
Reversed-phase (RP)-LC has been the dominant platform for non-target screening (NTS) because of its broad applicability and robustness. However, reversed-phase methods inherently underrepresent highly polar substances. Targeted methods exist for selected polar analytes, but until recently NTS provided limited coverage of PMs. The extension of NTS to PMs has been driven by improved chromatographic options (e.g., supercritical fluid chromatography, ion chromatography hyphenated to ESI) and new stationary phases that extend polarity ranges on conventional LC systems without requiring additional instruments. Coverage of polar substances in NTS is important for two reasons. First, authentic reference standards are often unavailable, especially for transformation products (TPs), leaving NTS as the only practical route for their detection. TPs remain one of the largest gaps in current chemical space coverage; they are typically more polar than their parent compounds and therefore require polar chromatographic methods for retention, separation, and discovery. Second, many polar substances and TPs (e.g., PFAS, pesticide TPs) have been shown to be persistent and, in some cases, more toxic than their precursors. Without polar-capable NTS, these compounds remain analytically invisible, delaying identification and hindering downstream exposure and risk assessment.
When you began designing this study, how did you approach the choice of chromatographic platforms?
Selection of chromatographic platforms was primarily guided by the established water-analysis workflows and expertise available at the four participating laboratories. Each laboratory contributed its standard method for aqueous matrices, resulting in the evaluation of RP-LC, HILIC, SFC and IC. Together, these platforms represent the methods most commonly applied for polar and semi-polar compounds in both research and routine environmental analysis.
The main uncertainty did not concern the choice of platforms, but rather the limited chemical space represented by the 123 selected analytes. NTS workflows typically interrogate far broader chemical space than can be represented with authentic standards. Nonetheless, we based the comparison on available standards because they allow unambiguous compound-level alignment between platforms. Alignment of unknown features across four distinct chromatographic methods would have introduced substantial methodological noise and reduced interpretability. Comparative studies of this type are rare; the use of standards provides a rigorous and transparent baseline for evaluating inter-platform coverage and performance.
How would you summarize your main findings?
One major intention of our study was to highlight the need and advantages of using multiple complementary methods to expand chemical space coverage during NTS workflows. We showed that using at least two complementary methods can strongly improve compound detection. As long as one RP-LC method was combined with a method suitable for polar analytes, it did not make a strong difference which exact platform was chosen (e.g., SFC vs. HILIC).
At the same time, our work also underscored that feature detection remains a major challenge in NTS workflows, in terms of both false positives and false negatives. The strong differences between the raw data produced by different chromatographic methods make this issue particularly important when less-studied platforms are used, because most feature detection algorithms were originally developed with RP-LC data in mind. Therefore, we strongly recommend thorough parameter adjustment or optimization when working with SFC or IC data, rather than relying on standard RP-LC settings from established in-house workflows.
Looking across detections and detection frequencies, we also saw that highly polar analytes remain generally difficult to capture, with detection rates dropping as polarity increases. While this can partly stem from poor chromatographic retention in some methods, it is likely also influenced by decreased ionization efficiency at very high polarity.
Lastly, it should be kept in mind that there will always be particularly challenging analytes that are difficult to capture using generic approaches. For example, some ultra-polar anions may only be retainable using a dedicated IC method.
From a practical standpoint, what should laboratories take away from this comparison?
The answer inevitably depends on the research question and the type of laboratory. In routine or high-throughput laboratories, where time and cost are limiting factors, it is often unrealistic to analyze all samples using multiple different methods. Nevertheless, recent research highlights that so-called “standard” NTS approaches (mainly RP-LC) cover only a relatively limited fraction of the overall chemical space. As a result, important compound classes may remain undetected when relying on a single, conventional method. We therefore argue that, in many cases, the strategic inclusion of just one carefully chosen complementary method can substantially enhance compound coverage without disproportionately increasing analytical effort. Thus, whenever possible, we recommend the combination of a primary NTS workflow with one complementary approach that maximizes additional information gain relative to the required resources. In the end, most of the workload is usually spent on data analysis and manual identification effort rather than on pure analytical runtime.
Based on your experience across the 12 methods, do you feel anything is missing from the current analytical toolbox?
While instruments and columns are offered in a quite wide variety, comprehensive data analysis remains a persistent bottleneck both in terms of workload and comprehensiveness. From our perspective robust and error-free data processing is a major challenge when dealing with complementary chromatography-HRMS data. In our study, we observed that some established feature detection routines can exhibit a significant number of false negatives. This is likely due to the strong raw data differences we saw in the different methods and the fact that most algorithms were developed on RP-LC-HRMS data. For example, while SFC typically produces very narrow peaks, IC peaks can sometimes be broader than one minute, and chromatograms can be considerably noisier. Capturing this large variability within a single feature detection algorithm is inherently challenging. Most algorithms rely on several parameters that need to be optimized or at least reasonably adjusted for a given data type. This task can become overwhelming – and quickly impractical – when many analytical methods are used in parallel.
Future development of making peak detection more comprehensive and less dependent on small parameter changes hopefully overcomes some of those limitations. Another major challenge is the alignment of data from different chromatographic methods. Typically, alignment relies on a combination of mass and retention time, but this is not readily transferable when fundamentally different retention mechanisms are involved. Addressing these challenges will be essential for enabling truly integrative and scalable multi-method NTS workflows.
Are there any emerging technologies or methodological directions you’re particularly excited about that might expand coverage of the polar domain further?
In our view, exciting progress in the polar domain is currently coming not from entirely new instrument platforms, but from advances in stationary phase chemistry and from a broader, more systematic application of existing polar chromatographic techniques and better data analysis routines.
On the technology side, the development of novel column chemistries – including mixed-mode phases – is particularly exciting. For example, while it was a challenge for decades, now columns are offered that allow analysis of C1 up to C12 PFAS with one single method on a standard RP-LC system! Also, the accessibility of SFC systems which are making their way in more laboratories shows the adoption of alternatives to RP-LC.
At the same time, we believe that expanding coverage of the polar chemical domain will benefit just as much from increased and more creative application of these methods. Techniques such as SFC and IC are still underutilized in most research laboratories. Wider adoption and better understanding of retention mechanisms for polar compounds could therefore also have substantial impact on future discoveries of pollutants.
Finally, where do you see this work going next?
In my current project FluorineID, I am applying multi-method NTS with the goal to identify the unknown “PFAS dark matter” in different environmental samples. Numerous studies have shown that the commonly quantified PFAS and organofluorines often only explain a very little fraction (e.g., <10 percent) of the extractable organic fluorine (EOF). Even NTS studies could rarely close this fluorine mass balance. We think that this partly comes from exactly this “limited chemical space coverage” of most NTS methods. The chemical space of fluorinated compounds is huge with currently 7.3 million PFAS in PubChem. For example, highly polar PFAS or even inorganic ionic liquids (e.g., hexafluorophosphate) often remain undetected and are therefore usually not considered in mass balance approaches while they have a significant contribution to the EOF. Hopefully, an efficient method combination can help us to unravel this unknown fluorinated substance space. Further bottlenecks that need to be addressed in this context are the comprehensive identification and in particular the (semi)quantification of compounds without reference standards.
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