PFAS isolation column exploration

Abstract

Provided herein are methods and systems for PFAS analysis using liquid chromatography techniques that employ a first column (e.g., an isolation column) and a second column (e.g., an analytical column), where at least one of the first column, the second column, or both the first column and the second column include a mixed pattern with anion exchange surface chemistry. The devices and methods provided herein can be used to improve the efficiency and / or sensitivity of a system that implements liquid chromatography for PFAS analysis. The methods of the present disclosure are particularly advantageous for trace level (e.g., ppm level or less) analysis of PFAS. In addition, the method of the present disclosure allows for improved separation of short and ultra-short chain PFASs.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 414,437, filed on October 7, 2022, entitled “PFAS Isolator Column Scouting,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to liquid chromatography methods for analyzing PFAS using a first column (e.g., a spacer column) and a second column (e.g., an analytical column). In particular, the present technology relates to using a spacer column to improve the performance of a system implementing liquid chromatography. The methods and systems of the present technology require the use of a mixed mode with anion exchange surface chemistry in at least one of the first column, the second column, or both the first column and the second column. Background Art

[0004] Per- and polyfluorinated alkyl substances (PFAS) are compounds of increasing importance to a variety of industrial sites and waterways. They are known to have very low degradability and to persist in the environment for a long time. This makes PFAS some of the most important environmental contaminants monitored globally. Their widespread use in a variety of commercial and industrial applications, including textiles, coatings, packaging materials, non-stick products and waterproof clothing, as well as their environmental persistence make them a truly global problem.

[0005] As more research focuses on PFAS exposure and effects, analytical techniques are also advancing to help research and monitoring efforts progress. Many attempts to quantify PFAS in the environment have been reported in the literature. Both targeted and non-targeted MS-based analytical strategies have been used to monitor PFAS levels in various matrices. Current analytical methods are often coupled to liquid chromatography systems to be able to separate PFAS from other compounds present in the sample matrix. However, PFAS are known to be used during the production of PTFE, a material used in tubing and other components of LC systems due to its inertness. The use of fluorinated coatings in LC flow paths and solvent storage bottles creates an environment where the mobile phase may be contaminated with PFAS and interfere with sample analysis. That is, PFAS from tubing, sample and solvent storage and injection, and from components of the LC system produce background levels of PFAS that interfere with or mask relevant information during MS analysis. Summary of the invention

[0006] Background PFAS contamination can interfere with low-level quantification requirements at health advisory levels, which are typically in the parts per trillion (ppt) range or lower, and give incorrect quantitative results in the samples being analyzed. Instrument-related background contamination can originate from LC components containing fluoropolymers (e.g., PTFE) that can leach into the mobile phase solution.

[0007] To fully or partially address the aforementioned problems and / or other problems that may have been observed by those skilled in the art, the present disclosure provides methods and / or systems, as described by way of example in the specific implementations set forth below.

[0008] Generally, the present technology relates to improving the performance of liquid chromatography systems used to analyze PFAS.

[0009] In one aspect, provided herein is a method for PFAS analysis using a liquid chromatography system employing a first column (e.g., an isolation column) and a second column (e.g., an analytical separation column). In some embodiments, the isolation column comprises a stationary phase comprising a mixed mode having an anion exchange surface chemistry. In some embodiments, the analytical separation column comprises a stationary phase comprising a mixed mode having an anion exchange surface chemistry. In some embodiments, both the isolation column and the analytical separation column (also referred to as the analytical column) comprise a stationary phase comprising a mixed mode having an anion exchange surface chemistry. In some embodiments, the surface chemistry of the stationary phases of the isolation column and the analytical column is independently selected from a mixed mode having an anion exchange surface chemistry and a reverse phase chemistry. In one embodiment, the reverse phase chemistry comprises C 18 Alkyl-bonded surface chemistry.

[0010] The methods and systems of the present technology utilize the surface chemistry of the stationary phase of the isolation column introduced in the liquid chromatography system to improve the chromatographic resolution of the analysis of PFAS peaks.

[0011] The methods and systems provided herein allow for reduction of PFAS background noise from the LC system and / or from the mobile phase, which results in increased sensitivity and accuracy of PFAS detection.

[0012] The methods provided herein effectively delay the elution of perfluorinated and polyfluorinated compounds (PFAS) released from pump parts, solvent lines and / or solvents, which can interfere with the detection and quantification of PFAS in samples (e.g., drinking water, food).

[0013] In one aspect, the present invention provides a method for analyzing fluorinated compounds using a liquid chromatography system including an isolation column and an analytical column, the method comprising: flowing a mobile phase through the isolation column; introducing a liquid sample into the liquid chromatography system through a sample injection port, wherein the liquid sample includes at least one type of fluorinated compound, and the sample injection port is located downstream of the isolation column; eluting the liquid sample through the analytical column so that at least one type of eluted fluorinated compound is separated as separated components; and flowing the separated eluted components to a detector, wherein the isolation column includes a stationary phase material having a mixed mode with an anion exchange surface chemistry.

[0014] Passing the mobile phase through the isolation column causes the background-related PFAS interferences to be retained throughout the isolation column and therefore do not co-elute with the PFAS from the sample during analysis.

[0015] In some embodiments, at least one type of fluorinated compound is a polyfluoroalkyl species.

[0016] In some embodiments, the number of carbon atoms present in the polyfluoroalkyl species is 2 to 18, 2 to 6, 3 to 18, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 4 to 6.

[0017] In some embodiments, the number of carbon atoms present in the polyfluoroalkyl species is greater than 3.

[0018] In some embodiments, the number of carbon atoms present in the polyfluoroalkyl species is greater than 2.

[0019] In some embodiments, at least one type of fluorinated compound includes one or more phosphate groups.

[0020] In some embodiments, the analytical column includes a stationary phase material having a mixed mode having an anion exchange surface chemistry.

[0021] In some embodiments, the analytical column comprises a stationary phase material having a reverse phase surface chemistry.

[0022] In some embodiments, the analytical column comprises a 18 Stationary phase materials with alkyl-bonded surface chemistry.

[0023] In some embodiments, the detector is used to perform mass spectrometry.

[0024] In some embodiments, the mass spectrometer comprises a tandem quadrupole mass spectrometer or a time-of-flight mass spectrometer.

[0025] In some embodiments, the inner surface of the spacer column is coated with an alkylsilyl coating. The alkylsilyl coating presented herein can prevent any interaction between the inner surface of the spacer column and the analyte (eg, PFAS-containing phosphate ester).

[0026] In some embodiments, the method further comprises quantifying at least one type of fluorinated compound after flowing the separated eluted components to a detector. In one embodiment, the concentration of at least one type of fluorinated compound in the liquid sample is less than 10 ng / L, less than 1 ng / L, less than 0.1 ng / L, less than 0.01 ng / L, and less than 0.001 ng / L.

[0027] On the other hand, the present invention provides a method for delaying the retention time of a contaminant in a liquid chromatography system including an isolation column and an analytical column, wherein the contaminant includes at least one type of first fluorinated compound, the method comprising: allowing a mobile phase to flow through the isolation column, wherein the contaminant is present in the system before the isolation column; introducing a liquid sample into the liquid chromatography system through a sample injection port, wherein the liquid sample includes at least one type of second fluorinated compound, and the sample injection port is located downstream of the isolation column; eluting the liquid sample through the analytical column so that at least one type of eluted fluorinated compound is separated as a separation component; and allowing the separated eluted components to flow to a detector so that at least one type of second fluorinated compound arrives at the detector before at least one type of first fluorinated compound, thereby delaying the retention time of the contaminant by at least 1 minute compared to the retention time of the at least one type of second fluorinated compound.

[0028] In some embodiments, the spacer column includes a stationary phase material comprising a mixed mode having anion exchange surface chemistry.

[0029] In some embodiments, the retention time of the contaminant is delayed by at least 5 minutes, at least 7 minutes, at least 8 minutes, or longer compared to the retention time of at least one type of second fluorinated compound.

[0030] In some embodiments, the first fluorinated compound and the second fluorinated compound are the same.

[0031] On the other hand, the present invention provides a method for analyzing fluorinated compounds using a liquid chromatography system including an isolation column and an analytical column, the method comprising: flowing a mobile phase through the isolation column; introducing a liquid sample into the liquid chromatography system through a sample injection port, wherein the liquid sample includes at least one type of fluorinated compound, and the sample injection port is located downstream of the isolation column; eluting the liquid sample through the analytical column so that at least one type of eluted fluorinated compound is separated as a separation component; and flowing the separated eluted components to a detector, wherein at least one column includes a stationary phase material having a mixed mode with an anion exchange surface chemistry.

[0032] The apparatus and methods of the present technology provide many advantages. In particular, the methods of the present disclosure can be used for quantitative (e.g., trace level detection) and qualitative analysis of long chain (>C6), short chain (C4-C6) and ultra-short chain PFAS (C2-C3). In addition, the inner surface (wetting surface) of the column (isolation column and / or analytical column) of the method of the present disclosure can be coated with an alkylsilyl coating that can prevent any interaction between the inner surface of the column and the PFAS eluted through the column. This allows sensitive and accurate analysis of PFAS-containing phosphates, which have a strong tendency to interact with the inner surface of the column (e.g., the inner surface of the metal). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present technology will be more fully understood through the following detailed description made in conjunction with the accompanying drawings, in which:

[0034] Figure 1A and Figure 1B Shown in the included C 18 Chromatogram of PFBA injected into an LC system with an isolation column. The analytical peak of PFBA was used as an internal standard in Figure 1A Shown in. Figure 1B The solvent blank chromatogram belongs to the mobile phase spiked with 0.025 ng / L PFBA.

[0035] Figure 1C and Figure 1D Shown in the included C 18 Chromatogram of PFPeA injected into the LC system with an isolation column. The analytical peak of PFPeA was used as an internal standard in Figure 1C Shown in. Figure 1D The solvent blank chromatogram belongs to the mobile phase spiked with 0.025 ng / L PFPeA.

[0036] Figure 2 Shown are the retention times of the analyte PFBA and interfering PFBA spiked into the mobile phase at a concentration of 0.025 ng / L injected in an LC system including a mixed mode isolation column.

[0037] Figure 3A , Figure 3B and Figure 3C The results show that when system-related PFAS contamination is present in the mobile phase (spiked with 0.025 ng / LPFAS), the conventional C 18 Retention time of various concentrations of PFBA (0.01 ng / mL, 0.1 ng / mL, 1 ng / mL) in the sample of the isolation column. Figure 3A The results obtained using a conventional isolation column for PFBA at a concentration of 0.01 ng / mL are shown; Figure 3B The results obtained for PFBA at a concentration of 0.1 ng / mL using a conventional isolation column are shown, and Figure 3C Results obtained using a conventional isolation column for PFBA at a concentration of 1 ng / mL are shown.

[0038] Figure 3D The use of C according to various embodiments of the present disclosure is shown 18 Retention time of PFBA in the sample on the mixed mode isolation column; the concentration of PFBA is 0.01 ng / mL. Figure 3E The use of C according to various embodiments of the present disclosure is shown 18 Retention time of PFBA in samples on a mixed-mode isolation column; the concentration of PFBA is 0.1 ng / mL. Figure 3F The use of C according to various embodiments of the present disclosure is shown 18 Retention time of PFBA in samples on a mixed-mode isolation column; the concentration of PFBA is 1 ng / mL.

[0039] Figure 4A , Figure 4B and Figure 4C The retention time of the PFOA sample (analyte peak of the internal standard) and the system-related PFOA contamination present in the mobile phase are shown. When the mixed-mode anion exchange isolation column according to the present disclosure is used to analyze the blank solvent spiked with 0.025 ng / LP FOA ( Figure 4A ), background PFOA peak (added to the mobile phase to artificially simulate the presence of PFOA interference, Figure 4C ) and the sample peak has a retention time difference of about 4 minutes. 18 When isolating the column, the background PFOA peak ( Figure 4C ) and sample peak ( Figure 4B ) have a retention time difference of less than 1 minute.

[0040] Figure 5A It shows that when the conventional C 18 Retention times of PFPrA and PFPrS analytes when the stationary phase is used as an analytical column for separation of PFPrA and PFPrS. Figure 5BShown are the retention times of PFPrA and PFPrS when an exemplary mixed mode with an anion exchange stationary phase is used as an analytical column for separation of PFPrA and PFPrS. Figure 5C Shown are the retention times of various PFAS of different chain lengths when an exemplary mixed mode with an anion exchange stationary phase is used as the analytical column.

[0041] Fig. 6A and Figure 6B Shown are the analytical peaks of perfluorooctanesulfonylaminoacetic acid (FOSAA) when separated using different stationary phases. Fig. 6A Shown is a chromatogram of FOASAA run in an analytical column with a conventional reverse phase stationary phase. Figure 6B Shown is a chromatogram of FOASAA run in an analytical column with an exemplary mixed-mode ion exchange stationary phase.

[0042] Fig. 7A and Figure 7B The retention time of trifluoroacetic acid (TFA), PFPrA, PFBA and PFPrS on a reverse phase column ( Fig. 7A ) and mixed mode Atlantis TM Premier BEH C 18 AX column( Figure 7B ) Due to the contamination problem of natural TFA analogs, isotope-labeled TFA analogs were used to represent TFA.

[0043] Fig. 8A and Figure 8B The use of a standard ammonium acetate gradient at constant pH ( Fig. 8A ) and ammonium hydroxide gradients with different pH ( Figure 8B ) , 46 PFAS were found in Atlantis TM Premier BEH TM C 18 Retention comparison on column AX.

[0044] Fig. 9A , Fig. 9B , Fig. 9C and Fig.9D Shows 13 Calibration curves of C2-TFA, PFPrA and PFPrS in the range of 5ng / L-200ng / L ( Figure 9A-9C ), and Waters_connect for the Quantitation Overview page, which highlights important American Society for Testing and Materials (ASTM) data quality guidelines such as residuals, calibration, quality control, and blanks ( Fig.9D). Due to the contamination problem of natural TFA analogs, isotope-labeled TFA analogs were used to represent TFA.

[0045] Fig. 10A and Fig. 10B It shows that in the absence of ( Fig. 10A ) and there are ( Fig. 10B ) Overlap of PFPrA retention in different types of samples (e.g., metal finish, leachate, and pulp and paper samples) with additional pH adjustment.

[0046] Fig.11A , Fig. 11B , Fig. 11C and Fig.11D Figure 2 shows the different types of PFAS in Atlantis TM PremierBEH TM C 18 Retention time stability on AX columns. Fig.11A Results of dNMeFOSE are provided; Fig. 11B Results for 13C8-PFOA are provided; Fig. 11C Results for 13C8-PFOS are provided; and Fig.11D Results for 13C3-HFPO-DA are provided.

[0047] Fig. 12A and Fig. 12B The performance of the method described herein is shown against an Environmental Resources Association (ERA) certified reference material, comparing the 18 Results obtained by running on both AX) and reverse phase columns.

[0048] Fig.13A A magnified chromatogram of a landfill leachate sample is shown, showing the C2-C6 carboxylates in the C 18 Elution on the AX column; and Fig. 13B A magnified chromatogram of a landfill leachate sample is shown, showing the elution of C2-C6 carboxylates on a reverse phase column. 18 Co-eluting peaks resolved on the AX column. TFA and PFHxA peaks are truncated due to magnification. DETAILED DESCRIPTION

[0049] Below is a more detailed description of various concepts and embodiments related to methods and systems for preventing and detecting PFAS interferences and ensuring accurate trace level analysis of PFAS in samples.

[0050] It should be understood that the various concepts introduced above and discussed in more detail below may be implemented in any of a variety of ways, as the disclosed concepts are not limited to any particular implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.

[0051] PFAS are a family of synthetic chemicals that have been produced since the late 1940s. PFAS contain multiple fluorine (F) atoms in place of hydrogen (H) atoms. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are two of the most widely recognized, environmentally relevant PFAS; despite the potential importance of thousands of PFAS, little is known about the toxicology of these compounds. The molecular structure of PFAS generally consists of: (a) a hydrophobic carbon chain (typically, but not limited to, 2-18 carbons in length) in which all (i.e., per-) or some (i.e., poly-) hydrogens are replaced by fluorine atoms such that they include no less than one fluoroalkyl moiety (C n F 2n+1 ); and (b) hydrophilic polar functional groups such as carboxylates, sulfonates, sulfonamides, phosphonates, and alcohols.

[0052] The small atomic size and strong electronegativity of fluorine provide PFAS with unique properties, such as very stable, strong acidity, high surface activity at very low concentrations, and / or water and oil repellency compared to their hydrocarbon counterparts. Therefore, many industries have used PFAS as processing aids and surfactants. Common consumer products that use PFAS include fire-fighting foams, metal coatings, non-stick cookware, medical devices, sample preparation and storage devices, professional clothing and textiles, and stain repellents.

[0053] The PFAS family includes more than 4,000 individual compounds and three subclasses; ultra-short-chain PFAS (C=2-3), short-chain PFAS (C=4-7), and long-chain PFAS (C>7), where C is the carbon number. Previous studies have shown that long-chain PFAS have higher bioaccumulation and bioaccumulation potential than ultra-short-chain and short-chain PFAS, which generally exhibit higher water solubility and greater mobility. Therefore, since the late 1990s, long-chain PFAS, especially perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), have received widespread attention from the scientific community and regulatory agencies as well as the public worldwide due to their bioaccumulation potential, persistence, toxicity, and ubiquitous presence in the environment.

[0054] Due to their widespread use, PFAS have been identified in industrial facilities, commercial household products, drinking water and wastewater, human food, and even living organisms. Exposure to PFAS, even at trace levels, can cause PFAS to accumulate in human blood because the carbon-fluorine bond is highly resistant to breakdown. As a result, PFAS are bioaccumulative in wildlife and humans because they typically remain in the body for extended periods of time. As a result, interest in testing for these compounds has grown rapidly.

[0055] Both targeted and non-targeted analytical methods have been used to monitor PFAS levels. Non-targeted analysis can be selected from (high resolution) mass spectrometry, combustion ion chromatography (CIC), particle induced gamma emission spectroscopy (PIGE), fluorine nuclear magnetic resonance (NMR), and combinations thereof. Targeted analysis can be selected from high resolution spectroscopy (FIRMS, e.g., quadrupole time of flight; Q-TOF) or tandem mass spectrometry (MS / MS).

[0056] The methods of the present technology including implementing LC systems can be used in conjunction with targeted and non-targeted analytical methods for on-site monitoring of PFAS levels. Specifically, the methods of the present disclosure are advantageous when used in conjunction with mass spectrometry techniques to quantitatively and / or qualitatively analyze PFAS in various samples.

[0057] However, PFAS are widely used in liquid-contacting parts of LC systems due to their inertness. Therefore, in a standard liquid chromatography (LC) (e.g., HPLC, UHPLC) setup, instrument-related PFAS interferences released from pump parts, solvent lines, and solvents can migrate through the system with the mobile phase and accumulate at the head of the analytical column between sample injections.

[0058] System-associated PFAS contamination can be affected by multiple factors, such as instrument brand and model, tubing and fitting materials, mobile phase solvent grade and storage conditions, and analytical method conditions.

[0059] When performing analyses at trace levels (eg, ppm levels or lower) and striving to achieve sub-ppt level detection of PFAS, PFAS contamination from LC components may prevent accurate identification and quantification of PFAS in a sample.

[0060] To eliminate this problem, the present disclosure provides a PFAS isolation column that delays PFAS associated with the system, preventing them from interfering with sample analysis. The isolation column is a universal solution that can be used in any type of LC system with any analytical column (fully porous or superficially porous with surface chemistry suitable for PFAS separation).

[0061] The methods of the present disclosure are effective in the analysis of target PFAS selected from, but not limited to, the fluorinated compounds listed in Table 1.

[0062] Table 1: Most prevalent PFAS found in the environment

[0063]

[0064]

[0065] The method of the present technology employs a PFAS isolation column that can eliminate the deleterious effects of background PFAS interferences from instrument-related sources by retaining the contaminants prior to the analytical column and eluting them only after the sample has been injected and the gradient elution has begun. The elution delay between the target PFAS in the sample and the background PFAS from the LC system is sufficient to separate them sufficiently to allow accurate quantification of the target compound in the sample.

[0066] In some embodiments, the elution delay between the target PFAS in the sample and the background PFAS from the LC system is at least 1 minute, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 30 minutes, or 60 minutes.

[0067] The PFAS isolation column (e.g., XBridge TM Premier BEH TM C 18 AX columns, commercially available from Waters Corporation, Milford MA) benefit from mixed mode with anion exchange surface chemistry to effectively delay PFAS background contamination originating from the instrument and therefore prevent co-elution with PFAS compounds present in the sample.

[0068] The mixed mode with anion exchange mixed mode surface chemistry of the stationary phase of the isolation column provides excellent retention for PFAS background (including long chain, short chain and ultra-short chain PFAS) to achieve baseline separation between background peaks and peaks from injected samples or standards. At the same time, the stationary phase of the isolation column of the present disclosure does not retain background analytes for too long (e.g., more than one hour), so after a short time (e.g., less than one hour) of running a high organic ratio mobile phase, although at the end of a gradient run, the isolation column is flushed and ready for the next analytical run.

[0069] As used herein, "mixed-mode chemistry" refers to chemical groups or moieties that produce more than one form of interaction between the stationary phase and the analyte to achieve analyte separation. Mixed-mode chemistry is a promising technology for obtaining novel PFAS separation selectivity. Specifically, the mixed mode with anion exchange chemistry of the present disclosure can be used to improve the retention of short-chain and ultra-short-chain PFAS.

[0070] The stationary phase materials of the present disclosure, including mixed mode with anion exchange surface chemistry, include one or more groups that can undergo any type of ionic interaction (eg, electrostatic interaction) with the analyte (eg, PFAS).

[0071] The term "anion exchange" refers to any functional group that can be converted into a charged group by protonation with an acid or deprotonation with a base.

[0072] In some embodiments, the hybrid mode with anion exchange chemistry includes one or more hydrophobic groups. In some embodiments, the hydrophobic groups of the hybrid mode with anion exchange surface chemistry of the present disclosure are C4 to C 30 In certain embodiments, the hydrophobic group is C 18 In other embodiments, the hydrophobic surface group is an aromatic, phenylalkyl, fluoroaromatic, phenylhexyl, pentafluorophenylalkyl, or chiral bonded phase. In one embodiment, the hydrophobic surface group is an embedded polar bonded phase.

[0073] The stationary phase materials disclosed herein can be in the form of particles, granular materials, monoliths, superficially porous materials, superficially porous particles, superficially porous monoliths, or superficially porous layers for use in open tubular column chromatography.

[0074] The stationary phase materials of the isolation columns and separation columns of the present disclosure can be independently selected from, for example, inorganic materials (e.g., silica, alumina, titania, zirconia), hybrid organic / inorganic materials, inorganic materials with a hybrid surface layer (e.g., silica, alumina, titania, zirconia), hybrid materials with an inorganic (e.g., silica, alumina, titania, zirconia) surface layer, or hybrid materials with different hybrid surface layers.

[0075] In some embodiments, the average pore size of the stationary phase material of the technique (e.g., the stationary phase of an isolation column or an analytical column) is about to about to about to or about to

[0076] In some embodiments, when the stationary phase is in particulate form, the average particle size of the stationary phase is about 0.3 μm-100 μm; about 0.5 μm-20 μm; 0.8 μm-10 μm; 1.0 μm-3.5 μm; or about 3.5 μm-5.0 μm. In one embodiment, the average particle size of the stationary phase is about 5.0 μm.

[0077] Example

[0078] Instrument-related PFAS interferences move through the LC system with the mobile phase and accumulate at the head of the analytical column between sample injections. However, an isolation column added to the mobile phase flow path immediately before the injection valve is an effective way to capture background PFAS contamination before it reaches the analytical column. If an isolation column according to the present technology is used to capture background PFAS (e.g., contamination caused by PFAS released from LC parts or PFAS present in the mobile phase), when the sample is injected into the LC system, only the PFAS in the sample will be concentrated at the head of the analytical column—because the background PFAS is captured. Then, during the gradient analysis, the PFAS from the sample will begin to move through the analytical column for analysis. The background PFAS trapped in the isolation column during equilibrium will be eluted and also pass through the analytical column. However, the elution time of the background PFAS will be later than that of the sample PFAS because they enter the analytical column after the sample is injected, due to the additional retention of the isolation column before reaching the injector. The net effect is that by using an isolation column, the instrument-related PFAS background contamination can be distinguished from the target PFAS in the sample.

[0079] The mobile phase composition used for analyzing PFAS solvents in the following examples included 2 mM ammonium acetate aqueous solution and 2 mM ammonium acetate methanol solution in gradient form (eg, composition variation by volume 95%-5%, composition variation 80%-1%).

[0080] Example 1: Effect of Conventional Isolation Columns on Retention of Background PFAS

[0081] Current or conventional isolation columns used in the art (such as C 18 Bonded columns) are not very effective in retaining background short-chain PFAS well. That is, the retention peaks of background short-chain PFAS are not delayed much compared to the retention peaks of analyte PFAS. To illustrate this situation, 18 The sample was spiked with 0.025 ng / LPFBA ( Figure 1A and Figure 1B ) and PFPeA( Figure 1C and Figure 1D ) as the mobile phase. Figure 1A and Figure 1C The retention time of the analytical peaks (shown as internal standards in Figure 1B and Figure 1D When compared to the background levels (i.e., those artificially generated by spiking into the mobile phase) in the system, it can be seen that the shorter chain PFBAs do not cause a delay. In addition, PFBA contamination in the system produces a background level ( Figure 1A and Figure 1B ). Figure 1C and Figure 1D Comparison of 50 μM MgCl2O3 and MgCl2O3 shows that the delay of the retention peak of PFPeA is less than 1 min, which may not be sufficient when analyzing trace levels of PFPeA.

[0082] Example 2: Effect of Mixed Mode Isolation Column on Retention of Background PFAS

[0083] To demonstrate the mixed-mode isolation column (Atlantis TM Premier C 18 Based on the applicability of Waters(R) AX, available from Waters Corporation, Milford, MA), the disclosed method was run with PFBA (perfluorobutyric acid) spiked into the aqueous mobile phase at a concentration of 0.025 ng / L to analyze PFBA at 1 ng / mL. Figure 2 The chromatogram in shows very good baseline separation of the analyte peaks injected onto the column, resulting in improved chromatographic resolution of the analytical peaks (e.g., peaks corresponding to target PFAS in the sample).

[0084] Example 3: Comparison of Mixed Mode Isolation Column and Conventional C for PFBA 18 Isolation column

[0085] To compare with the conventional C 18 The retention capacity of the isolation column and the mixed mode isolation column of the present disclosure, introduced into the LC system (Acquity TM Premier Solutions, available from Waters Corporation, Milford, MA) was spiked with 0.025 ng / L to analyze PFBA. Using the spiked mobile phase, three different concentrations of PFBA (0.01 ng / mL, 0.1 ng / mL, and 1 ng / mL) were injected into the system through the sample injection port (or valve). FIG. 3A to FIG. 3F The analytical peak of PFBA is shown when PFBA contaminant is present in the mobile phase. TM Premier C 18 AX, available from Waters Corporation, Milford, MA) showed well-resolved analytical peaks even at a concentration of 0.01 ng / mL ( Figure 3D ), but conventional C 18 Isolation column (BEH TM C 18 , available from Waters Corporation, Milford, MA) could not sufficiently delay the retention peak of PFBA present in the mobile phase ( Figure 3A , Figure 3B and Figure 3C ). Conventional C 18The column cannot delay the retention of PFBA sufficiently, which will cause huge background contamination problems in the case of analyzing lower level analytes (e.g., 0.01 ng / mL, 0.1 ng / mL). 18 When the column was used, the analyte peaks of 0.01 ng / mL and 0.1 ng / mL PFBA could not be well resolved for quantitative analysis.

[0086] Example 4: Comparison of Mixed Mode Isolation Column and Conventional C18 Isolation Column for PFOA

[0087] PFOA (perfluorooctanoic acid) was spiked into the aqueous mobile phase at a concentration of 0.025 ng / L. 13 The sample PFOA (labeled) was run separately from the blank mobile phase to observe the retention time of the analytical peak. In this example, the C as a conventional column was compared. 18 Column (BEH TM C 18 , available from Waters Corporation, Milford, MA) and a mixed-mode isolation column with anion exchange surface chemistry according to the present technology (Atlantis TM PremierC 18 AX, available from Waters Corporation, Milford, MA). Figure 4A , Figure 4B and Figure 4C The chromatogram in Figure 2 shows that when two different separation columns ( Figure 4A and Figure 4B ), the background PFOA peak (added to the mobile phase to artificially simulate the presence of PFOA interference, Figure 4C ) and the retention time difference between the sample peak. When analyzing longer chains (compared to, for example, PFBA), conventional column C 18 The retention capacity of C 18 The retention time difference between the background PFOA peak and the sample peak caused by the column is still less (less than 1 minute) than the retention time difference caused by the mixed mode column with anion exchange chemistry (about 4 minutes). That is, the separation column of the present disclosure with mixed mode having anion exchange chemistry is better than the conventional separation column. 18 The better the column, the better the resolution of the analytical peaks.

[0088] Example 5: Comparison of Retention of Ultra-Short Chain PFAS by Mixed Mode Isolation Column and Conventional C 18 Isolation column

[0089] Analysis of ultra-short chain PFAS (eg, less than 4 carbons) has been a challenge in the art because conventional columns cannot effectively retain ultra-short chain PFAS. Figure 5CShown is how the retention of PFAS increases with increasing chain length (carbon number).Two ultra-short chain PFAS (PFPrA and PFPrS) were tested using two different stationary phases. Figure 5A It is shown that when using conventional C18 stationary phase (BEH TM C 18 , available from Waters Corporation, Milford, MA) to separate PFPrA and PFPrS. Figure 5B The results show that when using a mixed mode with an anion exchange stationary phase (Atlantis TM Premier C 18 The retention times of PFPrA and PFPrS when PFPrA and PFPrS were separated by LC-MS / MS (Aqueous LC-MS, available from Waters Corporation, Milford, MA). The experimental parameters for this LC run are shown in Table 2 below. Figure 5B As can be seen in Figure 2, the retention times of PFPrA and PFPrS are greater than Figure 5A Retention times of PFPrA and PFPrS are shown. That is, mixed mode with anion exchange is more effective than conventional stationary phase in retaining ultra-short chain PFAS.

[0090] Experimental parameters

[0091]

[0092] Table 2: LC parameters

[0093] Time (min) Flow rate (ml / min) ％A ％B curve 0 0.3 95 5 6 1 0.3 75 25 6 6 0.3 50 50 6 13 0.3 15 85 6 14 0.3 5 95 6 17 0.3 5 95 6 18 0.3 95 5 6 22 0.3 95 5 6

[0094] Example 6: Analysis of PFAS-containing sulfonylaminoacetic acid using a mixed-mode ion exchange stationary phase

[0095] Perfluorooctanesulfonylaminoacetic acid (FOSAA) samples were run on an analytical column with a mixed-mode anion exchange stationary phase ( Figure 6B ) to evaluate its potential to retain PFAS-containing sulfonylaminoacetic acid. Specifically, a mixed mode (Atlantis TM Premier C 18 Ax, available from Waters Corporation, Milford, MA) analysis Figure 6B The inner surface of the column is coated with an alkylsilyl coating. Fig. 6A and Figure 6B The results are shown in Figure 2. TM C 18 ) Retention time of FOSAA eluted ( Fig. 6A) compared to the delayed retention time of FOSAA when using mixed-mode surface chemistry ( Figure 6B ).

[0096] Example 7: Analysis of water samples provided by the U.S. Environmental Protection Agency (EPA) Region 5

[0097] Water samples were collected at the following locations: landfill leachate, metal finish, wastewater effluent, wastewater influent, hospital discharge, bus wash station, power plant, pulp and paper mill, groundwater and surface water. Wastewater certified reference material from ERA (Project No. 404) was also analyzed along with the collected water samples to evaluate the performance of the method using certified materials.

[0098] Water samples were prepared according to the ASTM 8421 method (ASTMD8421-22). The entirety of each 5 mL water sample was used to avoid any compound loss in secondary sampling. Each sample was spiked with 160 ng / L isotopically labeled surrogate. 5 mL of 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. After filtration, 10 μL of acetic acid was added to each sample. Additional acetic acid was added to samples with pH>4 as needed. An aliquot of each sample was transferred to a polypropylene autosampler vial for use in the Xevo TM The analysis was performed on a TQAbsoluteMS (available from Waters Technologies Corporation) coupled to an ACQUITY HPLC system modified with a PFAS kit (available from Waters Technologies Corporation). TM Used in conjunction with Level I FTN BSM system.

[0099] Data Check

[0100] Using Atlantis TM Premier BEH TM C 18 Data generated by the AX columns are evaluated against the data quality guidelines outlined in ASTM 8421, which include the following:

[0101] 1. A minimum of 5-point linear calibration curve must be used.

[0102] 2. The deviation (% error) of the calibration standards and QC injections must be 30% of the expected concentration.

[0103] 3. The blank response must be less than 50% of the LLOQ injection response.

[0104] 4. The internal standard response must be within 30% of the batch median response.

[0105] 5. Internal standard and native retention times must be within 5% (+ / - 3 seconds) of the expected retention time.

[0106] 6. The ion ratio must be within 30% of the average reference peak.

[0107] LC conditions

[0108] LC System: ACQUITY Level I BSM with FTN

[0109] Vial: 700μL polypropylene screw-cap vial (p / n: 186005219)

[0110] Analytical column: Atlantis Premier BEH C18 AX 2.1 mm × 100 mm, 1.7 μm (p / n: 186009368)

[0111] Isolation column: Atlantis Premier BEH C18 AX 2.1mm×50mm, 2.5μm (p / n: 186009390)

[0112] Column temperature: 35°C

[0113] Sample temperature: 10°C

[0114] Injection volume: 30 μL

[0115] Flow rate: 0.3mL / min

[0116] Mobile phase A: 2 mM ammonium acetate in water

[0117] Mobile phase B: 0.1% (v / v) ammonium hydroxide in methanol

[0118] Table 3. Gradient table

[0119] Time (min) ％A ％B curve 0 99 1 initial 2 99 1 6 3 75 25 6 8 50 50 6 15 15 85 6 16 0 100 6 20 0 100 6 20.1 100 0 6 23.5 100 0 6 24 99 1 6

[0120] MS conditions

[0121] MS system: Xevo TM TQ Absolute

[0122] Ionization mode: ESI

[0123] Capillary voltage: 0.5kV

[0124] Source temperature: 100°C

[0125] Desolventization temperature: 350℃

[0126] Desolvent flow rate: 900L / hour

[0127] Cone flow rate: 150L / hour

[0128] Multiple Reaction Monitoring (MRM) Method

[0129] The complete MRM method details are provided in Table 4 below, where CE is the collision energy and CV is the cone voltage.

[0130] Table 4: MS method conditions for PFAS included in the analysis

[0131]

[0132]

[0133]

[0134]

[0135] like Fig. 7A As shown in the chromatogram in Figure 2, PFAS from the carboxylic acid family with chain lengths below C4 are not adequately retained on standard reversed-phase columns. TM Premier BEH TM C 18 For AX columns, the hydrophobicity of the CF chains is not the only retention mode for PFAS. Functional groups, such as -CO2 or -SO4, also play a role in retention, allowing for increased retention of ultra-short chain PFAS such as TFA and PFPrA, where the CF chains are not as hydrophobic as long chain PFAS. Figure 7B The chromatograms in Figure 2 show that using the gradient described in this article, ultra-short chain PFAS are TM Premier BEH TM C 18 Increased retention on column AX.

[0136] The standard PFAS gradient method utilizes aqueous and organic mobile phases with an additive (usually ammonium acetate) that maintains a constant pH throughout the gradient. This is appropriate for reversed-phase columns that retain compounds based solely on their hydrophobicity or polarity and separate compounds using increasing organic concentrations across the gradient. TM PremierBEH TM C 18In the case of AX columns, anion exchange selectivity is exploited by varying the pH over a gradient, which essentially activates (retention) and deactivates (elution) the ion exchange sites on the column. Thus, optimal resolving power is obtained when the organic composition and pH are varied over a gradient. Figure 8A-B Comparison of Atlantis PremierBEH C 18 Chromatographic resolution of 44 selected PFAS on AX column using a typical water / methanol solution with ammonium acetate gradient at constant pH ( Fig. 8A ) compared to the resolution when using a water / methanol solution with an ammonium hydroxide gradient (which increases the pH during the gradient run) ( Figure 8B ). In the ammonium acetate gradient, all 44 PFAS eluted within an approximately 3-minute window. Utilizing ammonium hydroxide as a mobile phase additive to create the pH gradient improved resolution within an approximately 13-minute elution window. Benefits of improved chromatographic resolution include improved mass spectral data quality by allowing more dwell time for each MRM function, as well as reduced chances of matrix interferences from co-eluting matrix compounds.

[0137] preparation 13 Calibration curves of C2-TFA, PFPrA and PFPrS in the range of 5ng / L-200ng / L, such as Figure 9A-9C As shown. Due to the contamination problem of natural TFA analogs, isotope-labeled TFA analogs are used to represent TFA. Fig.9D The waters_connect for the quantitation overview page is shown in FIG, which highlights important ASTM data quality guidelines such as residuals, calibration, quality control, and blanks.

[0138] Fig. 10A shows the overlap of PFPrA retention in different types of samples without additional pH adjustment; Fig. 10B PFPrA retention time with additional pH adjustment is shown. With all samples adjusted to the same nominal pH, Fig. 10B The results show that the retention time of the early eluting compound is more stable and falls within the 5% retention time tolerance. Additionally, the compound is stable throughout the remainder of the gradient and is well within the 5% retention time tolerance ( Fig.11A , Fig. 11B , Fig. 11C and Fig.11D ).

[0139] In addition to ensuring that 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 are shown in Figure 12, which also compares the quantitative results from the reversed phase column. The mixed mode and reversed phase data are very similar, indicating that the use of C18 There were no major matrix components coeluting with the AX column, leading to ion suppression or enhancement. Overall, the analysis of the wastewater CRM demonstrated that the mixed-mode column analysis method was accurate. Additionally, the mixed-mode column analysis was demonstrated to be comparable to the C-only 18 Reversed-phase columns are equally accurate.

[0140] In Atlantis TM PremierBEH TM C 18 After analyzing different sample types on the AX column, significant levels of TFA, PFPrA, and PFPrS were identified in the landfill leachate samples. BEH TM C 18 The analysis of samples using the Atlantis HPLC column included only PFPrA, which was poorly retained on the column. TM Premier BEH TM C 18 Due to the mixed-mode chemistry of the AX column, significant amounts of PFAS may have been missed in the analysis of this sample. Not only are ultra-short chain PFAS included using the mixed-mode column, but the remaining PFAS that are typically targeted in conventional testing methods can also be analyzed on the same column in the same single injection. Table 5 lists the quantitative amounts of each PFAS identified in the landfill leachate sample using both the reversed-phase column and the mixed-mode column, along with the calculated percent difference. For all PFAS identified, the percent difference between the two sets of quantitative results was within 15%, with the exception of PFBA, which had a percent difference of 33%. The higher percent difference for PFBA is believed to be due to the use of the C 18 The AX column resolves possible co-eluting peaks, which results in C 18 Overestimation of PFBA in landfill leachate samples on the column, e.g. Fig.13A C 18 The circled peak in the AX chromatogram ( Fig.13A ) appears to cause peak broadening / slight peak tailing of PFBA in reversed phase chromatograms ( Fig. 13B ).

[0141] Table 5: Use C 18 Quantification of 46 PFAS in landfill leachate samples using both AX mixed-mode and reversed-phase columns Compound

[0142]

[0143]

[0144] (ND) Not Detected, (NA) Not Applicable

[0145] By taking advantage of the hydrophobic and ionic properties of PFAS, Atlantis TM Premier BEH TM C 18 The AX column has been shown to successfully retain ultra-short chain PFAS, such as TFA and PFPrA, while maintaining the ability to analyze and quantify all other traditional long chain PFAS in a single injection. Atlantis TM Premier BEH TM The use of C18 AX columns allows laboratories to expand the PFAS panel on their current LC-MS / MS systems to analyze from ultra-short-chain to long-chain PFAS in a single injection.

[0146] The above embodiment shows the conventional inverting C 18 The superior properties of mixed-mode ion exchange stationary phases in separating and analyzing PFAS compared to conventional columns. In particular, the isolation columns of the present disclosure having mixed-mode ion exchange stationary phases result in a greater delay in the retention time of PFAS associated with background compared to conventional columns. Therefore, the contamination-free chromatograms of PFAS analytes allow the analysis of even trace levels of PFAS analytes.

Claims

1. A method for analyzing fluorinated compounds using a liquid chromatography system comprising an isolation column and an analytical column, the method comprising: a. allowing the mobile phase to flow through the isolation column; b. introducing a liquid sample into the liquid chromatography system through a sample injection port, wherein the liquid sample comprises at least one type of fluorinated compound, and the sample injection port is located downstream of the isolation column; c. eluting the liquid sample through the analytical column so that at least one type of the eluted fluorinated compound is separated as a separation component; as well as d. flowing the separated eluted components to a detector, wherein the spacer column comprises a stationary phase material comprising a mixed mode having an anion exchange surface chemistry.

2. The method of claim 1, wherein at least one type of fluorinated compound is a polyfluoroalkyl substance or a perfluoroalkyl substance.

3. The method according to claim 2, wherein the number of carbon atoms present in the polyfluoroalkyl substance is 3 to 5.

4. The method of claim 2, wherein the number of carbon atoms present in the polyfluoroalkyl substance is greater than 5.

5. The method of claim 1, wherein at least one type of fluorinated compound comprises one or more phosphonate groups.

6. The method of claim 1, wherein the analytical column comprises a stationary phase material comprising a mixed mode having an anion exchange surface chemistry.

7. The method of claim 1, wherein the analytical column comprises a stationary phase material comprising a reverse phase surface chemistry.

8. The method of claim 7, wherein the analytical column comprises a stationary phase material comprising C 18 Alkyl-bonded surface chemistry.

9. The method of claim 1, wherein the detector is used to perform mass spectrometry.

10. The method of claim 9, wherein the mass spectrometer comprises a tandem quadrupole mass spectrometer or a time-of-flight mass spectrometer.

11. The method of claim 1, wherein the inner surface of the spacer is coated with an alkylsilyl coating.

12. The method of claim 1, further comprising quantifying at least one type of fluorinated compound after flowing the separated eluted components to the detector.

13. The method of claim 1, wherein the concentration of at least one type of fluorinated compound in the liquid sample is less than 0.1 ng / L.

14. A method of delaying the retention time of a contaminant in a liquid chromatography system comprising a spacer column and an analytical column, wherein the contaminant comprises at least one type of first fluorinated compound, the method comprising: a. allowing the mobile phase to flow through the isolation column, wherein the contaminants are present in the system before the isolation column; b. introducing a liquid sample into the liquid chromatography system through a sample injection port, wherein the liquid sample comprises at least one type of a second fluorinated compound, and the sample injection port is located downstream of the isolation column; c. eluting the liquid sample through the analytical column so that at least one type of the eluted fluorinated compound is separated as a separation component; as well as d. flowing the separated eluted components to a detector such that at least one type of second fluorinated compound reaches the detector before the at least one type of first fluorinated compound, thereby delaying the retention time of the contaminant by at least 1 minute compared to the retention time of the at least one type of second fluorinated compound.

15. The method of claim 14, wherein the spacer column comprises a stationary phase material comprising a mixed mode having an anion exchange surface chemistry.

16. The method of claim 14, wherein the retention time of the contaminant is delayed by at least 5 minutes compared to the retention time of at least one type of second fluorinated compound.

17. A method for analyzing fluorinated compounds using a liquid chromatography system comprising a spacer column and an analytical column, the method comprising: a. allowing the mobile phase to flow through the isolation column; b. introducing a liquid sample into the liquid chromatography system through a sample injection port, wherein the liquid sample comprises at least one type of fluorinated compound, and the sample injection port is located downstream of the isolation column; c. eluting the liquid sample through the analytical column so that at least one type of the eluted fluorinated compound is separated as a separation component; as well as d. flowing the separated eluted components to a detector, wherein at least one column comprises a stationary phase material having a mixed mode with anion exchange surface chemistry.

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