A Review of the Latest Separation Science Research in PFAS Analysis

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of synthetic chemicals that have gathered significant attention because of their persistence in the environment and potential health risks. Analytical methods for PFAS detection and quantification have been developed to address the complex nature of these compounds in various matrices such as water, soil, air, and biological samples. This review provides a brief yet comprehensive overview of the separation science methods utilized for PFAS analysis, including liquid chromatography (LC), gas chromatography (GC), ion chromatography (IC), capillary electrophoresis (CE), and supercritical fluid chromatography (SFC). Additionally, mass spectrometry (MS) detection techniques, sample preparation methodologies such as solid-phase extraction (SPE) and solid-phase microextraction (SPME) are discussed. Special emphasis is placed on the analytical challenges posed by the diversity of PFAS compounds and their occurrence in different environmental and biological contexts. This review aims to provide a summary of the most current analytical techniques and their applications in PFAS research, contributing to the ongoing efforts to monitor and mitigate PFAS contamination.

Per- and polyfluoroalkyl substances (PFAS) are a large and diverse class of anthropogenic chemicals characterized by their strong carbon-fluorine bonds, which impart exceptional chemical stability and resistance to degradation. These properties have led to their widespread use in industrial applications and consumer products, including firefighting foams, non-stick cookware, water-repellent fabrics, and food packaging. However, the same properties that make PFAS useful in various applications also contribute to their persistence in the environment, earning them the moniker “forever chemicals.”

The environmental and health concerns associated with PFAS have prompted extensive research into their detection, quantification, and remediation. Analytical methods for PFAS must contend with the complexity of environmental and biological matrices, the wide range of PFAS compounds, and the often low concentrations at which they are found. Accurate and sensitive detection methods are essential for monitoring PFAS contamination, assessing exposure risks, and evaluating the effectiveness of remediation efforts.

Separation science plays a pivotal role in PFAS analysis, with techniques such as liquid chromatography (LC), gas chromatography (GC), ion chromatography (IC), capillary electrophoresis (CE), and supercritical fluid chromatography (SFC) forming the backbone of many analytical protocols. These techniques are frequently coupled with mass spectrometry (MS) to enhance sensitivity and specificity. Sample preparation methods, including solid-phase extraction (SPE) and solid-phase microextraction (SPME), are crucial for isolating PFAS from complex matrices.

This review provides an examination of the various analytical methods employed in PFAS research, highlighting strengths, limitations, and applications. By addressing the challenges associated with PFAS analysis and exploring the latest advancements in separation science and detection technologies, this review aims to contribute to the development of more effective strategies for monitoring and managing PFAS contamination in the environment.

Liquid Chromatography-Mass Spectrometry (HPLC-MS and UHPLC-MS)

High-performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) are widely used for PFAS analysis because of their high resolution and sensitivity. These are often coupled with MS for specific analyte detection. The following are recent papers describing these analytical techniques; HPLC methods are described in references (1,2), and the UHPLC methods in references (3–5).

PFAS are persistent and mobile, accumulating in the environment and organisms, with harmful health effects. A recently published review focused on the advancements in analytical methodologies for PFAS detection over the past decade, emphasizing water as a major exposure source (1). The review article provided an overview of sample collection, storage, preservation, purification, chromatographic separation, and analysis, highlighting LC coupled with tandem MS (LC–MS/MS) and targeted analysis as the primary tools for PFAS determination. The review also identifies important challenges and issues related to PFAS analysis (1).

A rapid online solid-phase extraction liquid chromatography high-resolution mass spectrometry (online SPE-LC–HRMS) method was developed for analyzing 11 ultra-short and short-chain PFAS in surface water (2). The method’s optimization involved testing various chromatographic and SPE columns, as well as evaluating SPE loading conditions, filters, sample acidification, and mobile phases. This optimized method was applied to 44 river water samples from eastern Canada, including sites near airports with fire-training areas. The most frequently detected PFAS were trifluor acetic acid (TFA), perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), trifluoromethane sulfonic acid (TMS), and perfluorobutane sulfonic acid (PFBS) (2). Higher levels of certain PFAS were found near fire-training areas compared to urban rivers, while TFA, TMS, and 1:3 acid showed no significant elevation, likely because of atmospheric deposition or diffuse sources. Nontarget and suspect screening revealed additional ultra-short and short-chain PFAS, including perfluoroalkyl sulfonamides and perfluoroalkyl sulfonamide propanoic acids, detected for the first time in environmental surface waters (2).

Ultrahigh-pressure liquid chromatography (UHPLC), also known as ultrahigh-performance liquid chromatography, provides faster and more efficient separation compared to classical HPLC, with improved resolution and sensitivity. UHPLC methods are described in references (3–5).

PFAS are widespread pollutants that can enter the food chain, with fish, fruits, and eggs being major contributors to human exposure. A new method using isotope dilution and UHPLC coupled to HRMS was developed to determine 18 PFAS in eggs. Analysis of 132 samples (organic, barn, and caged eggs) showed PFAS levels near detection limits with no significant differences among the groups (3). The highest PFAS concentrations in eggs were used to estimate dietary exposure in different Italian populations, revealing that children are more exposed than adults. This data suggests that the recent tolerable weekly intake of 4.4 ng/kg body weight could be exceeded when considering cumulative dietary intake from various foods (3).

Wastewater treatment plants (WWTPs) struggle to remove PFAS, resulting in their presence in water and sewage sludge. Hydrothermal treatment offers an alternative by recovering energy and valuable products from sludge. One study analyzed 15 PFAS in sludge and hydrochar using sonication-solid phase extraction and LC–Orbital trap-high resolution-MS/MS, validating the method with good linearity, recoveries (48-126%), and low detection and quantification limits (4). Sludge samples from Ioannina, Greece, and hydrothermally treated samples showed PFHxA and PFOS as the most abundant PFAS. Hydrothermal treatment spiked with PFAS achieved removal efficiencies of 86.9%, 91.8%, and 95.7% at spiking levels of 10, 50, and 200 ng/g, respectively (4). The process almost completely removed perfluorocarboxylic acids (PFCAs) (except perfluorooctanoic acid [PFOA]), while perfluorosulfonic acids (PFSA) concentrations increased with intermediate formation. These findings highlight hydrothermal treatment’s potential in managing PFAS in sewage sludge, aiding further studies and scaling up hydrothermal carbonization technology for safer disposal (4).

PFAS are synthetic and resilient compounds found in many industrial and consumer products, widely distributed in the environment, humans, and wildlife. Detecting trace amounts of PFAS is challenging because of their complex pathways and detection limitations. A research paper aimed to develop methods for detecting and measuring PFAS in environmental samples using UHPLC paired with triple quadrupole MS (UHPLC-QqQ-MS/MS) (5). Three analytical methods were designed to identify 35 types of PFAS: the first method targeted 17 PFAS including PFCAs, perfluorosulfonic acids (PFSAs), and perfluoroethercarboxylic acids (PFECAs); the second identified five perfluoroalkyl sulfonamides (FASAs); the third focused on six (PFOA) isomers and seven perflurooctanesulfonic acid (perfluorooctane sulfonic acid [PFOS]) isomers. Each method involved optimizing sample preparation, UHPLC–MS/MS conditions, validation, and application to environmental water samples (5). These methods were also used in collaborative research at Auburn University to detect trace-level PFAS in water, sediment, fish, oysters, mussels, and algae. Each chapter of the dissertation study details the methodology, application, and results (5).

Gas Chromatography-Mass Spectrometry (GC-MS)

GC coupled with MS (GC–MS) is less common for PFAS analysis because of the thermal stability required for PFAS compounds. However, derivatization and other techniques can make PFAS amenable to GC analysis (6,7).

PFAS are used in food contact materials (FCMs) like polytetrafluoroethylene (PTFE)-based coatings for kitchenware and additives in paper and board. Because of growing environmental and health concerns, monitoring PFAS levels in FCMs and their migration into food is increasingly important. One study developed a method using thermal desorption–GC–MS (TD-GC–MS) to analyze PFAS. Besides commonly analyzed fluorotelomer alcohols (FTOHs), the study demonstrated that perfluorocarboxylic acids (PFCAs) and per- and polyfluoroether carboxylic acids (PFECAs), along with their thermolysis products, can be analyzed by GC–MS without derivatization (6). Group-specific selected ion monitoring (SIM) fragments allowed screening for perfluoroalkyl carboxylic acids (PFCAs) and fluorotelomer alcohols (FTOHs) via electron impact ionization (EI), with identity confirmation through EI scans and chemical ionization (CI) SIM measurements. The method achieved low pg range limits of detection (LODs) for PFCAs, FTOHs, and PFECAs, and it investigated the thermal degradation of PFCAs and PFECAs during TD-GC–MS measurement (6).

A recent study investigated novel and emerging PFAS in air, sediment, and waste- water from areas near fluorochemical-related industrial facilities (7). Using GC-based target and non-target analyses, the study characterized contamination profiles, finding fluorotelomer alcohols to be predominant, comprising 80% of neutral PFAS, followed by fluorotelomer acrylates. Air samples near a durable water repellent (DWR) facility showed the highest n-PFAS concentrations, significantly higher than other locations. Non-target analysis detected fluorotelomer iodides and methacrylates, indicating significant contamination. The study observed a shift from C8- to C6-based fluorochemicals, with shorter-chain PFAS prevalent in air and longer-chain PFAS in sediment, suggesting these media as secondary PFAS sources. Both untreated and treated industrial wastewater contained n-PFAS and their transformation products, enhancing understanding of PFAS distribution and transport across different environmental matrices (7).

Ion Chromatography-Mass Spectrometry (IC-MS)

IC coupled with MS (IC–MS) is effective for the separation and analysis of ionic PFAS, providing good sensitivity and selectivity for these molecular species (8,9).

Research published in 2024 has measured total fluorine in 45 Swedish consumer products suspected or known to contain fluorinated polymers. Products included cookware (70–550,000 ppm F), textiles (10–1,600 ppm F), electronics (20–2,100 ppm F), and personal care items (10–630,000 ppm F) (8). A validated pyrolysis-GC–MS (Py-GC–MS) method confirmed the organic nature of the fluorine and helped identify polytetrafluoroethylene (PTFE) in cookware, dental products, and electronics at concentrations as low as 0.1–0.2 wt% (8). The research also distinguished between three types of side-chain fluorinated polymers in textiles. Some products contained high levels of inorganic fluorine. This research quantifies fluorine in various consumer plastics, providing data relevant to recycling and insights into the use of Py-GC–MS for analyzing fluorinated polymers in consumer goods (8).

Emerging contaminants, particularly PFAS well-known as “forever chemicals,” pose significant environmental concerns. A 2024 study analyzed PFAS in complex liquid and solid environmental samples using combustion ion chromatography (CIC) to determine extractable organic fluorine (EOF) and adsorbable organic fluorine (AOF), aiming to provide a comprehensive view of PFAS contamination (9). The research evaluated the impact of pH, dissolved organic carbon, and suspended particles on AOF measurements and confirmed the effectiveness of a washing step for removing inorganic fluorine in samples with up to 8 mgF/L. CIC methods demonstrated good repeatability and reproducibility (9). AOF and EOF analyses accounted for 1% to 23% and 0.1% to 2% of total organic fluorine (TOF), respectively. PFAS compounds expressed as fluorine explained 0.2% to 11% (AOF) and 0.003% to 5% (EOF) of TOF. The study suggests that some fluorinated compounds might not be adsorbed, extractable, or could be lost during the analytical process, indicating that AOF and EOF are not entirely effective proxies for total PFAS assessment. However, these methods can still enhance understanding of PFAS sources and environmental behavior (9).

Capillary Electrophoresis (CE)

CE coupled with MS (CE–MS) offers high separation efficiency and can be useful for analyzing PFAS in many types of complex matrices (10).

PFAS, which are important environmental pollutants, require careful monitoring. Research published in 2020 demonstrated a method using nonaqueous capillary electrophoresis with conductivity detection to separate perfluoroheptanoic acid, perfluorooctanoic acid, and perfluorooctanesulfonate in under 5 min (10). The method utilized a background electrolyte consisting of 3-(N-morpholino)propanesulfonic acid and triethylamine in acetonitrile:methanol (50:50, v/v). It demonstrated high linearity (R² > 0.996) for analyte concentrations between 2 and 20 μM, with detection limits ranging from 0.30 to 0.75 μM. The method showed precision with relative standard deviations below 5.8%. When applied to wastewater samples via solid-phase extraction, detection limits reached 0.1–0.4 nM. The study also explored carbon aerogel-based and magnetic solid-phase extractions, suggesting the method could be adapted for portable, on-site analysis (10).

Supercritical Fluid Chromatography–Mass Spectrometry (SFC–MS)

SFC coupled with MS (SFC–MS) provides high resolution and sensitivity for PFAS analysis with reduced solvent usage (11).

A 2022 paper describes an ultrahigh performance supercritical fluid chromatography coupled with tandem mass spectrometry (UHPSFC–MS/MS) method developed to analyze 34 PFAS in food matrices (11). The method was optimized using a central composite design to refine parameters like column temperature, flow rate, co-solvent concentration, and back pressure. The final setup used a Torus 2-PIC column and ammonium acetate in the co-solvent, with optimal conditions of 38.7 °C column temperature, 8 mM ammonium acetate (AcoNH4), 1.9 mL/min flow rate, and 1654 psi adjustable bypass pressure regulator (ABPR). The method achieved limits of quantification below 0.2 ng/g for 97% of PFASs, meeting European Union (EU) standards. Applied to food samples from Algeria, the method detected perfluorooctanoic acid (PFOA) and perfluorobutanoic acid (PFBA) most frequently, with the highest concentrations found in fishery products (11).

Quadrupole Mass Spectrometry (MS)

Quadrupole time-of-flight MS (QTOF-MS) offers high resolution and accuracy for identifying and quantifying PFAS (12,13).

In 2024, a research paper assessed the migration of PFAS from various food packaging materials, including cardboard, recycled cardboard, biopolymer, paper, and Teflon trays. Migration tests used Tenax as a food simulant and optimized extraction with three consecutive ethanol extractions. UHPLC coupled with ion-mobility quadrupole-time-of-flight (QTOF) MS was used for analysis, improving PFAS identification (16). Eleven PFAS, including PFCAs, PFSAs, and perfluorooctanesulfonamidoacetic acids (FOSAAs), were detected in packaging from China at concentrations of 3.2 to 22.3 μg/kg, while no PFAS were found in packaging from Spain. All tested trays met safety standards, with PFAS levels below regulatory limits for food contact materials (16).

In 2023, one study developed a method to analyze PFAS in the eggs of Yellow-legged and Audouin’s gulls and the blood of Greater flamingos, using these birds as indicators of organic pollution. Samples were extracted with acetonitrile, cleaned with activated carbon, and analyzed using ultrahigh-pressure liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC–Q-TOF). The method included quantitative analysis of 25 PFAS using mass-labeled internal standards and untargeted screening with a high-resolution PFAS library to identify additional compounds. PFAS levels ranged from 0.45 to 55.2 ng/g in gull eggs and 0.75 to 125 ng/mL in flamingo blood, with PFOS, PFOA, and other PFAS being the primary detections. Additionally, perfluoro-p-ethylcyclohexylsulfonic acid (PFECHS) and 2-(perfluorohexyl)ethanol (6:2 FTOH) were identified. This method enhances PFAS analysis capabilities and supports using bird species for monitoring chemical pollution (17).

Triple-Quadrupole MS (QqQ-MS) provides high sensitivity and selectivity for targeted PFAS analysis (14,15).

A precise method was developed to measure specific PFAS in drinking water at parts per trillion levels. It combines solid-phase extraction (SPE) with liquid chromatography-tandem mass spectrometry (LC–MS/MS) for detection. The method was optimized and validated, demonstrating good calibration, low detection limits, and high precision. Isotopically labeled internal standards and surrogate standards were used for quantification and performance monitoring, respectively. The method achieved surrogate recoveries of 84-113%, detection limits of 1.0–3.0 ng/L, and minimum reporting limits of 5–10 ng/L, offering a valuable tool for assessing PFAS contamination in water (14).

One study developed an integrated QuEChERS method to quickly determine 22 PFAS in milk using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The method combines extraction and purification in one step, utilizing magnetic suction (Fe₃O₄-SiO₂) for solid-liquid separation instead of centrifugation. Key parameters such as extraction solvent and magnetic nanomaterial amounts were optimized. The method showed high precision (RSDs < 9.9%), low detection limits (0.004–0.079 μg/kg), and good recovery (71.7–116%). It offers advantages in sample pretreatment time, procedure efficiency, and reagent use, making it a highly sensitive and efficient technique for measuring PFAS residues in milk (15).

Sample Preparation Techniques for PFAS: SPE and SPME

Sample preparation methods useful for PFAS analysis are highlighted in this review (16–19).

SPE is commonly used for concentrating and purifying PFAS from water, soil, and biological samples before chromatographic analysis (16,17).

PFAS are harmful compounds found in
many products, and WWTPs may significantly contribute to PFAS pollution. This study developed a method to detect 16 PFAS in various environmental samples, including seawater, lake water, and wastewater (16). The process involved filtering, extracting, and purifying samples using weak-anion exchange SPE, followed by UHPLC with Orbital trap MS for analysis. The method showed strong linearity, good recovery rates, and low detection limits. Applied to water sources in Ioannina, Greece, it found perfluorooctanesulfonamide (PFOSA) and perfluoro-n-octanoic acid (PFOA) were the most common PFAS, with concentrations of up to 21 ng/L in secondary influent and 160 ng/L in hospital influent. Long-chain PFAS (C8–C13) made up to 38.5% of total PFAS in wastewaters. Secondary treatment removed approximately 60% of PFAS, increasing to 80% with tertiary treatment. PFAS levels in sea and lake samples were generally lower than in wastewater (16).

A precise method was developed to detect PFAS in drinking water at parts per trillion (ppt) levels. It uses SPE for concentration and LC–MS/MS for detection. The method was optimized and validated, showing acceptable calibration, minimum reporting limits (MRL), and limits of detection (LOD). Isotopically labeled internal standards were used for quantification, with surrogate standards monitoring performance. The method achieved recoveries of 84-113% for surrogates, with detection limits between 1.0–3.0 ng/L and MRLs of 5–10 ng/L, making it an efficient tool for assessing PFAS contamination in drinking water (17).

SPME is a solvent-free technique that can be used for the extraction of PFAS from a complex variety of sample types (18,19).

Research published in 2024 reported on the development of a rapid and sensitive method to quantify both legacy and emerging PFAS in environmental samples using SPME coupled with MS. A new SPME probe was created through in situ polymerization, optimized for better fluorine–fluorine interactions and electrostatic properties, achieving enrichment factors of 48–491. The SPME-MS system showed excellent linearity (r ≥ 0.9962) over a range of 0.001–1 μg/L and low detection limits of 0.1–13.0 ng/L. This method efficiently analyzed trace PFAS in tap water, river water, and wastewater, offering a simple and effective approach for environmental contamination assessment (18).

PFAS are fluorinated compounds known for their persistence, bioaccumulation, and toxicity. Because of their strong C-F bonds, they resist degradation, posing risks even at trace levels. Detecting PFAS requires highly sensitive and selective analytical methods. Traditional methods like solvent extraction with chromatography face challenges such as high detection limits, labor-intensive processes, limited selectivity, and high costs. Recent advancements in micro-extraction techniques, adhering to green chemistry principles, offer enhanced sensitivity and selectivity, are portable, and can be automated. This research reviews these microextraction and detection methods for PFAS, addressing current challenges and future trends (19).

General Sample Preparation Techniques for PFAS

A variety of sampling issues have been investigated relative to PFAS analysis and these are described in the following references (20–22).

PFAS are important industrial chemicals but present environmental and health risks because of their persistence and toxicity. Analyzing PFAS, especially in biomonitoring, typically involves liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Key challenges include low concentrations, small sample sizes, contamination, and matrix effects. Effective sample preparation is essential for enhancing sensitivity and selectivity, with SPE and protein precipitation (PPT) being commonly used techniques. This review assesses various sample preparation methods for PFAS in biological matrices, highlighting their advantages, difficulties, and future directions (20).

Research completed in 2023 presents a modified QuEChERS method for analyzing 24 (PFAS in various biological matrices. PFAS are persistent, toxic, and bioaccumulative, making their detection important across different environments. Traditional methods often target only a few PFAS compounds, emphasizing the need for a broader approach. The modified QuEChERS method enhances extraction and cleanup efficiency for PFAS in diverse matrices, including algae, plants, invertebrates, amphibians, and fish, by adjusting the extraction solvents and salts used (21).

A recent study investigated 66 materials for potential PFAS contamination during sample collection and shipping, using LC–MS/MS and particle-induced gamma-ray emission (PIGE) spectroscopy. Although none of the 22 materials tested had significant PFAS concentrations, 10 had trace levels of PFAS and 15 had high total fluorine content. No contamination pathways were identified. The study concludes that strict controls on materials without clear contamination risks are unnecessary, recommending future focus on materials in direct contact with samples that coudl realistically affect PFAS levels (22).

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About the Author

Jerome Workman, Jr. serves on the Editorial Advisory Board of Spectroscopy and is the Senior Technical Editor for LCGC and Spectroscopy. He is the co-host of the Analytically Speaking podcast and has published multiple reference text volumes, including the three-volume Academic Press Handbook of Organic Compounds, the five-volume The Concise Handbook of Analytical Spectroscopy, the 2nd edition of Practical Guide and Spectral Atlas for Interpretive Near-Infrared Spectroscopy, the 2nd edition of Chemometrics in Spectroscopy, and the 4th edition of The Handbook of Near-Infrared Analysis. Direct correspondence to: jworkman@mjhlifesciences.com