Expanding the Range of PFAS in a Single Injection to Include Ultra Short Chains Using the Atlantis™ BEH™ C₁₈ AX Mixed Mode Column

As the legacy long chain PFAS were voluntarily phased out of production, they were replaced by short and ultra-short chain PFAS of similar chemistries. Chromatography methods using common reverse phase columns do not retain the ultra-short PFAS, so these are not usually included in quantitative methods. The mixed mode chemistry of PFAS molecules, both hydrophobic and ionic, should be retained on mixed mode reverse phase and anion exchange column chemistries such as the Atlantis Premier BEH C₁₈ AX Column. TFA, PFPrA, and PFPrS were sufficiently retained using the Atlantis Premier BEH C₁₈ AX Column and can be analyzed and quantified in the same method as the legacy long chain PFAS compounds. A wide variety of water samples were evaluated on the mixed mode column with TFA, PFPrA, and PFPrS detected in significant quantities in a landfill leachate sample. Use of the Atlantis Premier BEH C₁₈ AX Column now broadens the scope of PFAS that can be routinely detected and quantified in a single injection to include the ultra-short chain PFAS down to C2 carbon chain length.

Benefits

• A single method that broadens the scope of PFAS compounds to now include analysis of C2 through C14 chain length PFAS by utilizing the mixed mode (hydrophobic and ionic) nature of the compounds
• Improvements in chromatographic retention and peak shape of ultra-short and short chain PFAS on the Atlantis Premier BEH C₁₈ AX Column allows for fewer manual peak integrations
• Accurate quantitative results for 46 PFAS in authentic matrix samples, verified by an ERA wastewater matrix certified reference material in order to assess reporting reliability

When the environmental impact and health hazards of the legacy long-chain (≥C8) PFAS were realized, a shift in manufacturing was made to substitute their use with short chain (C4–C7) and ultra-short chain (≤C3) PFAS.¹ Initially it was believed that these substituted short and ultra-short chain PFAS would have less environmental and health impacts, but it has been shown that they are highly mobile in the environment, degrade to very persistent end products, and still pose significant health concerns.² In order to get a broader, more accurate representation of PFAS contamination, one may expect expanded coverage in regulatory methods, requiring methods that can easily and accurately quantify the ultra-short chain compounds. However, many of the current PFAS regulations and standard methods don’t include PFAS in the ultra-short chain category. A summary of the common global methods and regulations currently in place for PFAS are listed in Table 1 with the short and ultra-short chain PFAS that are included in each method. This summary demonstrates that it is uncommon for methods to include PFAS in chain length less than C4, other than the ASTM 8421 that includes perfluoropropionic acid (PFPrA). Even though the ultra-short chain PFAS are often not included in routine PFAS methods, it does not mean they are not found in common samples. For instance, a 2019 study by Bjornsdotter et al. identified ultra-short chain PFAS such as trifluroracetic acid (TFA) and perfluoropropanesulfonic acid (PFPrS) in Swedish water samples at 14,000 and 15,000 ng/L, respectively.³ Additionally, it has been shown that the ultra-short chain PFAS can account for a portion of the unknown total fluorine content of samples as concluded by a 2021 study by Aro et al. In this study a total of 10% of fluorine content in wastewater samples could be identified, amongst which 4% was identified as ultra-short chain PFAS.⁴ Excluding ultra-short chain PFAS from routine quantitative methods can lead to a significant underrepresentation of PFAS present in samples.

As PFAS use and manufacturing has evolved, one possible reason that the ultra-short chain PFAS may not be included in routine PFAS methods is because of the difficulties with chromatographing these small compounds. PFAS analysis is most commonly performed using a reverse phase column, most typically a C₁₈ chemistry. The hydrophobic C-F chain on PFAS molecules allows for the retention and separation mechanism to work under these conditions. However, as the C-F chain length decreases, the ability for hydrophobic retention on the C₁₈ chemistry decreases. Typical C₁₈ based methods have PFBA eluting fairly close to the void volume of the column, indicating that it is just barely retained. Besides the hydrophobic chemistry of the C-F chain, most PFAS also have a negatively charged ionic head group, which provides a secondary retention mechanism that could be used. The Atlantis Premier BEH C₁₈ AX Column combines both the reverse phase C₁₈ and anion exchange (AX) retention chemistries into one column. In this case, a mixed mode column can take advantage of both portions of the PFAS compounds. Reverse phase and anion exchange combined together is already commonly used for PFAS sample preparation when utilizing solid phase extraction (SPE) in the format of weak anion exchange (WAX) based cartridges. In this application of mixed mode chemistries, the anion exchange portion is important for retaining the short chain PFAS as they have a weaker reverse phase mechanism than the long chain PFAS.

In this work, the mixed mode chemistry column, Atlantis Premier BEH C₁₈ AX Column, was evaluated to determine how short and ultra-short chain PFAS could be retained on this column and if it could be used for routine PFAS analysis of ultra-short through long chain PFAS.

Sample Preparation

Water samples analyzed were provided by US EPA Region 5. Water samples were collected in the following locations: landfill leachate, a metal finisher, wastewater effluent, wastewater influent, hospital discharge, a bus washing station, a powerplant, pulp and paper factory, ground water, and surface water. A wastewater certified reference material from ERA (item number 404) was also analyzed alongside the collected water samples to evaluate the method performance using a certified material.

Water samples were prepared in accordance with the ASTM 8421 method.⁵ The entirety of each 5 mL water sample was used to avoid any compound loss from subsampling. Each sample was spiked with 160 ng/L of isotopically labeled surrogates. 5 mL methanol was then added to each water sample and vortexed. The entire 10 mL sample was syringe filtered using a 25 mm, 0.2 µm polypropylene syringe filter. Following filtration, 10 µL acetic acid was added to each sample. Additional acetic acid was added to samples that had a pH > 4, as needed. An aliquot of each sample was transferred to a polypropylene autosampler vial for analysis on the Xevo™ TQ Absolute MS coupled to an ACQUITY™ I Class FTN BSM System modified with a PFAS Kit.

Data Review

The data generated using the Atlantis Premier BEH C₁₈ AX Column was evaluated against the data quality guidelines outlined in ASTM 8421, which included the following:

1. A minimum 5-point linear calibration curve must be utilized.

2. Deviation (% error) of calibration standards and QC injections must be 30% of the expected concentration.

3. Blank response must be <50% of response of the LLOQ injection.

4. Internal standard response must be within 30% of the median response of the batch.

5. Internal standard and native retention time must be within 5% (+/- 3 sec) of the expected retention time.

6. Ion ratios must be within 30% of the mean reference peaks.

Prior to availability of a mixed mode chromatography column, the reverse phase mechanism (C₁₈) has been primarily used to perform PFAS analysis.⁶ PFAS from the carboxylic acid family with chain lengths below C4 are not sufficiently retained on standard reverse phase columns, as shown in the top chromatogram of Figure 1. In this example, TFA (C2) and PFPrA (C3) elute within the void region (T₀) of the column. This distorts peak shape, as seen with PFPrA, but also significantly increases the chance of matrix interference from the unretained matrix compounds that also elute in this region. With the mixed mode Atlantis Premier BEH C₁₈ AX Column having both reverse phase and anion exchange mechanisms, the hydrophobicity of the C-F chain isn’t the only mode of retention for PFAS. The functional group, such as -CO₂ or -SO₄, also plays a role in retention, allowing for increased retention of the ultra-short chain PFAS like TFA and PFPrA where the C-F chain is not as hydrophobic as the longer chain PFAS. The bottom chromatogram in Figure 1 demonstrates the increased retention of the ultra-short chain PFAS on the Atlantis Premier BEH C₁₈ AX Column utilizing the gradient described in this work. Although TFA was retained on the mixed mode column, there was still a large TFA contamination present within samples prepared in the laboratory, which is a shared use laboratory where other applications are using TFA as a mobile phase modifier. This makes TFA contamination in samples and standards difficult to control. Therefore, chromatograms shown in this application note use the isotope labelled TFA analog as the representative peak for TFA.

Standard PFAS gradient methods utilize aqueous and organic mobile phases that contain an additive, typically ammonium acetate, that remains at a consistent pH throughout the gradient. This is appropriate for reverse phase columns that retain compounds based solely on their hydrophobicity or polarity and utilize increased organic concentration over the gradient to separate compounds. In the case of a mixed mode column like the Atlantis Premier BEH C₁₈ AX Column, the anion exchange selectivity is utilized by varying pH over the gradient which essentially activates (retention) and deactivates (elution) the ion exchange sites on the column. Therefore, the best resolving power is gained when both organic composition and pH are varied over the gradient. An example of this can be seen in Figure 2 which compares the chromatographic resolution of a selection of 44 PFAS on the Atlantis Premier BEH C₁₈ AX Column using a typical water/methanol with ammonium acetate gradient at a constant pH (Figure 2A) to the resolution using a water/methanol with ammonium hydroxide gradient (Figure 2B) that increases pH over the gradient run. In the ammonium acetate gradient, all 44 PFAS elute within an approximate 3-minute window. Utilizing ammonium hydroxide as a mobile phase additive to create a pH gradient increases the resolution over an elution window of approximately 13 minutes. Benefits from the increased chromatographic resolution include increased mass spectral data quality by allowing more dwell time for each MRM function, as well as decreased chances of matrix interference from co-eluting matrix compounds.

Data quality guidelines required by ASTM 8421 were achieved upon final sample analysis on the mixed mode column in all samples, except for a few detections of TFA, PFPrA, and PFBA in the blank samples that are a result of solvent contamination. An overview in Figure 3 demonstrates the data quality guidelines were achieved during sample analysis of a wide range of sample types.

During initial injections of the samples, the earliest eluting compounds did experience some severe retention time shifting that did not fall within the 5% expected retention time tolerance, as demonstrated in the top chromatogram of Figure 4. Once the retention time shift was observed in the early eluting compounds, the pH of the samples was tested. Even though an initial 10 µL acetic acid was added to each sample after dilution and filtration, as called for in the ASTM 8421 method, there were still a few samples that had elevated pH values. The solution used to create the calibration curve samples was approximately pH 4. Most of the samples analyzed did fall around pH 4 level with the 10 µL acetic acid added. However, the metal finisher sample tested at pH 5.5, the leachate tested near pH 5, and the pulp and paper sample tested at pH 4.5. It can be observed in Figure 4, that these three samples had reduced retention in accordance with their pH. The highest pH sample (water from the metal finisher) had the least retention (or earliest retention time) followed by the landfill leachate water, and then the pulp and paper factory water. Knowing this, additional acetic acid was added to each sample to adjust the pH to 4 and the samples were re-analyzed. With all samples adjusted to the same nominal pH value, the bottom chromatogram of Figure 4 demonstrates the retention times of the early eluting compounds are much more stable and fall within the 5% retention time tolerance. Additionally, compounds throughout the rest of the gradient are stable and well within the 5% retention time tolerance (Figure 5). This highlights the importance of monitoring the pH of the final samples prior to injection on the mixed mode column. The sample pH may not prove to be as variable when preparing samples using SPE as this sample preparation technique should remove most of the matrix that may cause pH changes and the final samples should be more uniform.

In addition to ensuring the data fell within the quality control guidelines of the ASTM 8421 method, a certified reference material was analyzed and quantified in the same batch as the water samples. The results from this analysis can be observed in Figure 6 which also compares the quantified results from a reverse phase column. The mixed mode and reverse phase data are extremely comparable indicating that there are no major matrix components that co-elute using the C₁₈ AX Column and causing ion suppression or enhancement. Most compounds quantified within the certified range of the standard, with a few quantifying slightly higher on both columns. This can potentially be explained by the fact that the certified values are based on a sample size of 500 mL and the sample size was adjusted to 5 mL for this study to replicate the sample size of the collected water samples. Therefore, the certified range may not be as accurate for this smaller sample size. Overall, the analysis of the wastewater CRM showed the mixed mode column analysis method to be accurate. Additionally, the mixed mode column analysis proved to be as accurate as the C₁₈ reverse phase only column.

Upon analysis of the different sample types on the Atlantis Premier BEH C₁₈ AX Column, significant levels of TFA, PFPrA, and PFPrS were identified in the landfill leachate sample. Previous analysis of the samples using a reverse phase column (ACQUITY BEH C₁₈ Column) only included PFPrA, which was poorly retained on the column. Without the inclusion of the ultra-short chain PFAS, made possible by the mixed mode chemistry of the Atlantis Premier BEH C₁₈ AX Column, significant amounts of PFAS would have been missed in the analysis of this sample. Not only were the ultra-short chain PFAS included using the mixed mode column, but the rest of the PFAS normally targeted in routine testing methods could also be analyzed on the same column, in the same single injection. Table 2 lists the quantified amount of each PFAS identified in the landfill leachate sample using both the reverse phase and mixed mode columns as well as the calculated percent difference. The percentage difference between both sets of quantified results was within 15% for all PFAS identified except for PFBA, which had a percent difference of 33%. The higher percent difference for PFBA is assumed to be a result of possible co-eluting peaks that were resolved using the C₁₈ AX column, causing an over-estimation of PFBA in the landfill leachate sample on the C₁₈ column, as highlighted in Figure 7. The peaks circled in the C₁₈ AX chromatogram appear to cause peak broadening/slight peak tailing of PFBA in the reverse phase chromatogram.

By taking advantage of the hydrophobic and ionic nature of PFAS, the Atlantis Premier BEH C₁₈ AX Column was shown to successfully retain ultra-short chain PFAS, such as TFA and PFPrA, while maintaining the ability to analyze and quantify all other legacy, long chain PFAS in a single injection. A variety of water samples that differed quite drastically in composition were quantified according to the ASTM 8421 data quality guidelines. Results were also verified using an ERA certified wastewater reference material giving confidence to the use of the Atlantis Premier BEH C₁₈ AX Column for a wide range of PFAS analysis. The use of the Atlantis Premier BEH C₁₈ AX Column will allow laboratories to expand the suite of PFAS capable of analysis in a single injection from ultra-short chain through long chain PFAS on their current LC-MS/MS system.

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