Office of Water

www.epa.gov January 2024

Method 1621
Determination of Adsorbable Organic Fluorine (AOF)
in Aqueous Matrices by Combustion Ion
Chromatography (CIC)

Method 1621 January 2024

 U.S. Environmental Protection Agency
Office of Water (4303T)
Office of Science and Technology
Engineering and Analysis Division
1200 Pennsylvania Avenue, NW
Washington, DC 20460

EPA 821-R-24-002

Method 1621 January 2024

 Method 1621
Determination of
Adsorbable Organic Fluorine (AOF) in Aqueous Matrices
by Combustion Ion Chromatography (CIC)

January 2024

Notice

This document represents the AOF method developed by the EPA Office of Water, Engineering and
Analysis Division (EAD). This method is not approved for Clean Water Act compliance
monitoring until it has been proposed and promulgated through rulemaking.

A multi-laboratory validation of the procedure has been completed and the report on the results of that
study is available.

Laboratories, regulatory authorities, and other interested parties are encouraged to review the method,
and where appropriate, utilize it for their own purposes, with the explicit understanding that this
method has not been promulgated.

Method 1621 January 2024

 Acknowledgements

This method was prepared under the direction of S. Bekah Burket and Adrian Hanley of the Engineering
and Analysis Division (EAD), Office of Science and Technology (OST), within EPA’s Office of Water
(OW), in collaboration with ASTM International.

EPA acknowledges the support of a number of organizations in the development and validation of this
draft method, including the developers of the original procedure, the members of EPA’s workgroup, the
laboratories that participated in the multi-laboratory method validation study, and EPA’s support
contractor staff at General Dynamics Information Technology, including Mirna Alpizar, Harry McCarty,
and Kenneth Miller. (A more detailed list of individuals and organizations can be found in References 13
and 14)

Disclaimer

Mention of trade names or commercial products does not constitute endorsement or recommendation for
use.

Contact

Please address questions, comments, or suggestions to:

CWA Methods Team, Engineering and Analysis Division (4303T)

Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue
Washington, DC 20460

https://www.epa.gov/cwa-methods
https://www.epa.gov/cwa-methods/forms/contact-us-about-cwa-analytical-methods

Method 1621 i January 2024

 Table of Contents

Acknowledgements ........................................................................................................................................ i
Disclaimer ...................................................................................................................................................... i
Contact .......................................................................................................................................................... i
1.0 Scope and Application ..................................................................................................................... 1
2.0 Summary of Method ........................................................................................................................ 2
3.0 Definitions ....................................................................................................................................... 2
4.0 Contamination and Interferences ..................................................................................................... 2
5.0 Safety ............................................................................................................................................... 4
6.0 Equipment and Supplies .................................................................................................................. 5
7.0 Reagents and Standards ................................................................................................................... 8
8.0 Sample Collection, Preservation, Storage, and Holding Times ..................................................... 11
9.0 Quality Control .............................................................................................................................. 12
10.0 Calibration and Standardization..................................................................................................... 16
11.0 Sample Preparation and Adsorption .............................................................................................. 19
12.0 Instrumental Analysis .................................................................................................................... 22
13.0 Performance Tests during Routine Operations .............................................................................. 24
14.0 Data Analysis and Calculations ..................................................................................................... 25
15.0 Method Performance...................................................................................................................... 27
16.0 Pollution Prevention....................................................................................................................... 27
17.0 Waste Management........................................................................................................................ 28
18.0 References ...................................................................................................................................... 28
19.0 Tables, Diagrams, Flowcharts, and Validation Data ..................................................................... 30
20.0 Figures ........................................................................................................................................... 32
21.0 Glossary ......................................................................................................................................... 33

List of Tables

Table 1. Example Retention Time and Pooled MDL and ML Values...................................................... 30

Table 2. Example Calibration Standard Solutions .................................................................................... 30
Table 3. IPR and OPR QC Acceptance Limits ......................................................................................... 30
Table 4. Example Performance Data from the MLVS for MS/MSD Samples Spiked with PFHxS ........ 31
Table 5. Summary of Quality Control ...................................................................................................... 31

Method 1621 ii January 2024

 Method 1621
Determination of Adsorbable Organic Fluorine (AOF) in Aqueous Matrices
by Combustion Ion Chromatography (CIC)

January 2024

1.0 Scope and Application

1.1 Method 1621 is for use in the Clean Water Act (CWA) to estimate the concentration of adsorbable
organic fluorine (AOF) in aqueous matrices by combustion ion chromatography (CIC).

1.2 The method measures organofluorine compounds from per- and polyfluoroalkyl substances (PFAS)
and non-PFAS fluorinated compounds such as pesticides and pharmaceuticals that can be retained
on at least 80 mg of granular activated carbon (GAC). The result is reported as the concentration of
fluoride (F-) in the sample.

1.3 Short-chain (less than 4 carbons) organofluorine compounds are poorly retained on GAC while
long-chain (more than 8 carbons) hydrophobic organofluorine compounds readily adsorb to
surfaces. These issues can cause low recoveries for these types of fluorinated compounds.

1.4 By their very nature, many components of PFAS present analytical challenges unique to this class
of analytes. For example, PFAS analytes readily adhere to the walls of the sample containers and
may stratify in the container.

1.5 Relative to the Clean Water Act and the methods approved for compliance monitoring at 40 CFR
Part 136, AOF is a “method-defined parameter” (MDP). An MDP is a parameter defined solely by
the method used to determine the analyte. In the case of AOF, Method 1621 estimates an aggregate
concentration of the organofluorine compounds in the sample that are retained on the sorbent.
Therefore, the EPA has limited the extent to which this method may be modified without prior EPA
review. The analyst may not use sorbents other than granular activated carbon, amounts of granular
activated carbon less than 80 mg per sample, or sample containers made from different materials.

1.6 The method development studies demonstrated that there were no negative effects on the adsorption
of organofluorine onto carbon when the sample contains organic carbon concentrations up to
140 mg/L. The nitrate wash employed in the method is capable of removing up to 8 mg/L of
inorganic fluorine that may be adsorbed in conjunction with organofluorines, reducing the positive
bias from inorganic fluorine. The method is capable of adsorbing AOF in samples with chloride
concentrations up to 500 mg/L without causing peak interference in the chromatogram. Due to the
ubiquitous occurrence of PFAS, unless strict cleaning protocols are followed, the method can be
subject to significant blank contamination. Nonetheless, the method can reliably quantitate
organofluorines at low part-per-billion levels.

1.7 For the reasons discussed in Sections 1.2 to 1.6, this method may be used to estimate the aggregate
contributions of the organofluorine compounds present in the sample. As such, data users are
advised that the numerical results generated by this method are not expected to be as accurate or
precise as those from targeted methods for PFAS. In addition, given the large number of potential
PFAS and other organofluorine compounds that may be present in environmental samples, the EPA
has adjusted some of the quality control and method performance testing approaches employed in
this procedure.

Method 1621 1 January 2024

1.8 The instrumental portion of this method is for use only by analysts experienced with CIC or under
the close supervision of such qualified personnel. Each laboratory that uses this method must
demonstrate the ability to generate acceptable results using the procedure in Section 9.2.

1.9 The pooled method detection limit (MDL) and Minimum Level (ML) for AOF determined during
multi-laboratory validation study (Reference 14) are provided in Table 1 as examples of method
performance.

2.0 Summary of Method

2.1 Aqueous samples are prepared and adsorbed using method-specific procedures. A sample aliquot
of approximately 100 mL is passed through two GAC columns, each containing 40 – 50 mg of
carbon.

2.2 The GAC columns are rinsed with sodium nitrate to remove inorganic fluoride, combusted at
≥1000 °C in an oxygen or oxygen/argon stream, and the gaseous hydrogen fluoride is absorbed into
reagent water.

2.3 The fluoride is separated by ion chromatography (IC) and identified by comparing sample fluoride
retention time to retention times for calibration standards acquired under identical IC conditions and
quantified using the external standard technique.

3.0 Definitions

Definitions are provided in the glossary at the end of this method.

4.0 Contamination and Interferences

4.1 Solvents, reagents, glassware or plasticware, and other sample processing hardware may yield
artifacts and elevated baselines causing misinterpretation of chromatograms. Specific selection of
reagents and solvents may be required. All items must be routinely demonstrated to be free from
interferences through the analysis of matrix blanks prepared with each batch.

4.2 Clean all equipment prior to, and after each use, to avoid fluoride cross-contamination. Typical
cleaning solvents used include water, methanol, and methanolic ammonium hydroxide (1%). The
residual AOF content of disposable plasticware and filters must be verified by batch/lot number.

4.2.1 Glass containers have been found to have high levels of fluoride contamination; therefore,
it is highly recommended that the use of glass labware be avoided for this method
whenever possible. If any glass labware is used in the preparation or storage of reagents,
the glassware must be cleaned by rinsing with methanolic ammonium hydroxide, followed
by rinsing with reagent water, followed by a methanol rinse, and air drying.

4.2.2 The combustion boats should be cleaned by soaking in methanol for at least one hour,
scrubbing with cotton swabs, rinsing with reagent water, and baking in a kiln or furnace
(Section 6.2.2) at 450 °C for 1 hour, then stored in a desiccator or stored submerged in
reagent water until use to prevent contamination from fluoride present in air particles. Prior
to use, baked combustion boats must be rinsed with methanol and dried. If the combustion
boats are stored longer than three days, then they must be baked again prior to use.

Method 1621 2 January 2024

 4.2.3 All removable parts of the adsorption unit must be cleaned before sample analyses begin,
between samples, and at the end of the analysis batch, using the cleaning solvent
combination suggested by the manufacturer, followed by rinsing with reagent water.

4.2.4 All reusable plasticware must be washed with methanol or methanolic ammonium
hydroxide, followed by a minimum of two reagent water rinses.

4.2.5 It is highly recommended that the laboratory record and track which adsorption unit ports
are used to process each sample. This may assist the laboratory in tracking possible
sources of contamination for individual samples.

4.2.6 The gas adsorption tube (a.k.a., the U-tube) may get clogged with carbon residue after
several combustions. A routine clean-up procedure should be put in place by the laboratory
to prevent any carbon residue from clogging the tube. Manufacturer’s instructions should
be followed to properly pack the gas adsorption tube to prevent solid materials from
blowing into the tube.

Extremely dirty adsorption tubes may need to be cleaned by soaking in a solution of one-
part 1 M sodium hydroxide and two-parts isopropanol, scrubbing with a paste of aluminum
oxide powder, followed by rinsing with reagent water and air drying. Other cleaning
procedures suggested by the manufacturer may be used.

4.3 All materials used in the analysis must be demonstrated to be reasonably free from contaminants by
running method blanks (Section 9.3) with each sample batch (samples started through the extraction
process on a given analytical batch to a maximum of 10 field samples).

4.3.1 The GAC columns can be a major source of fluoride contamination, both organic and
inorganic. The type of capping material used in the columns can contribute as much as
30% of the background fluoride level. The level of fluoride background can also vary
greatly between vendors. The analysis of method blanks provides important information
regarding the presence of such contamination.

4.3.2 Each lot number of GAC columns must be tested by analyzing method blanks to ensure that
contamination does not preclude AOF quantitation at the reporting limit. There may be
background variation within a lot number; therefore, at least four columns from each new
lot number must be tested prior to putting the lot number into use.

4.3.3 Baseline contamination of GAC observed during method development ranged from 0.1 to
6 µg F-/L, based on a 100-mL sample size. Smaller sample volumes will produce higher
method blank results due to normalization of the measured mass of fluoride to a smaller
volume.

4.3.4 Opened packages of GAC columns and combustion boats must be stored in a desiccator
when not in use to prevent possible contamination from laboratory air, and away from any
potential source of fluoride contamination. Failure to properly store GAC columns may
result in contamination of the carbon and high method blanks.

4.4 Matrix interferences may be caused by contaminants that are co-adsorbed from the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon the
nature of the aqueous sample. High levels of co-adsorbed non-fluorine-containing contaminants
can cause breakthrough of organofluorine analytes on the GAC.

Method 1621 3 January 2024

4.5 Samples containing total suspended solids above 100 mg/L may clog the GAC columns and
adsorption unit lines, which may prevent proper adsorption of the sample. These types of samples
will need to be treated following the instructions in Section 11.3.

4.6 Alcohols, aromatic substances and carboxylic acids may lead to negative interference. Organic
carbon above 140 mg/L may lead to negative interference by inhibiting quantitative adsorption of
halogen bound organic substances to the activated carbon. These effects may be assessed by
dilution of the initial sample or spiking the sample with a standard solution at a known
concentration.

4.7 Inorganic fluoride in the sample may lead to biased high results. The nitrate wash step in this
method removes up to 8 mg/L inorganic fluoride present in the sample. Samples containing more
than 8 mg/L of inorganic fluoride should be diluted prior to analysis (Section 11.2.3).

4.8 Chloride elutes closely with fluoride in the ion chromatography portion of the analysis, which may
cause an interference when chloride is present at concentrations greater than 500 mg/L.

4.9 At concentrations higher than 500 mg/L, chloride may also compete for adsorption sites in the
carbon and prevent fluoride from being adsorbed.

4.10 Some organofluorines readily adsorb to surfaces. This method does not mitigate for surface
adsorption because the use of solvents to rinse sample bottles, tubing, or other surfaces in the
adsorption unit also would elute AOF from the GAC. Thus, results may be biased low for
organofluorines that are prone to surface adsorption. Additionally, adsorption apparatus from
different manufacturers may not yield comparable results for long-chain and neutral PFAS
compounds due to differences in the numbers of surfaces (e.g., tubing, syringes, etc.) with which
the sample comes in contact during sample loading onto the GAC.

5.0 Safety

5.1 The toxicity or carcinogenicity of each chemical used in this method has not been precisely
determined; however, each compound should be treated as a potential health hazard. Exposure to
these compounds should be reduced to the lowest possible level.

5.1.1 Perfluorooctanoic acid has been described as likely to be carcinogenic to humans. Pure
standards should be handled by trained personnel, with suitable protection to skin and eyes,
and care should be taken not to breathe the vapors or ingest the materials.

5.1.2 It is recommended that the laboratory purchase dilute standard solutions of the analytes in
this method. However, if primary solutions are prepared, they must be prepared in a hood,
following universal safety measures.

5.2 The laboratory is responsible for maintaining a current awareness file of Occupational Safety and
Health Administration (OSHA) regulations regarding the safe handling of the chemicals specified
in this method. A reference file of safety data sheets (SDS) should also be made available to all
personnel involved in these analyses. It is also suggested that the laboratory perform personal
hygiene monitoring of each analyst who uses this method and that the results of this monitoring be
made available to the analyst. Additional information on laboratory safety can be found in
References 1-4. The references and bibliography at the end of Reference 3 are particularly
comprehensive in dealing with the general subject of laboratory safety.

Method 1621 4 January 2024

5.3 Samples suspected to contain PFAS or other organofluorine compounds are handled using
essentially the same techniques employed in handling infectious materials. Well-ventilated,
controlled access laboratories are required. Assistance in evaluating the health hazards of particular
laboratory conditions may be obtained from State Departments of Health or Labor, many of which
have an industrial health service. Each laboratory must develop a strict safety program for handling
these compounds.

5.3.1 Protective equipment – Disposable plastic gloves, apron or lab coat, safety glasses or mask,
and a glove box or fume hood. During analytical operations that may give rise to aerosols
or dusts, personnel should wear respirators equipped with activated carbon filters. Eye
protection (preferably full-face shields) must be worn while working with exposed samples
or pure analytical standards. Nitrile or polyethylene gloves are commonly used to reduce
exposure of the hands.

5.3.2 Personal hygiene – Hands and forearms should be washed thoroughly after each
manipulation and before breaks (coffee, lunch, and shift). Avoid the use of hand lotions.

5.3.3 Confinement – Isolated work areas posted with signs, segregated glassware and tools, and
plastic absorbent paper on bench tops will aid in confining contamination.

5.3.4 Waste Handling – Good technique includes minimizing contaminated waste. Plastic bag
liners should be used in waste cans.

5.4 Samples that may contain high concentrations of biohazards must be handled with PFAS- and
fluoride-free gloves and opened in a fume hood or biological safety cabinet to prevent exposure.
Laboratory staff should know and observe the safety procedures required in a microbiology
laboratory that handles pathogenic organisms when handling highly contaminated samples.

6.0 Equipment and Supplies

Note: Any brand names, suppliers, and part numbers listed below are for illustration purposes only and
no endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those specified here (also see 40 CFR 136.6). Meeting the performance
requirements of this method is the responsibility of the laboratory.

6.1 Sampling equipment for discrete or composite sampling

6.1.1 Sample bottles and caps – Liquid samples– Sample bottle, high-density polyethylene
(HDPE) or polypropylene, with linerless HDPE or polypropylene caps.

Note: Do not use fluoropolymer-lined caps for sample containers.

6.1.2 Compositing equipment – Automatic or manual compositing system incorporating

containers cleaned per bottle cleaning procedure above (Section 4.2.4). HDPE tubing must
be used. If the sampler uses a peristaltic pump, a minimum length of compressible silicone
rubber tubing may be used in the pump only. Before use, the tubing must be thoroughly
rinsed with methanol, followed by repeated rinsing with reagent water to minimize sample
contamination. An integrating flow meter is used to collect proportional composite
samples.

Method 1621 5 January 2024

6.2 Equipment for cleaning combustion boats

6.2.1 Laboratory sink with overhead fume hood

6.2.2 Kiln or muffle furnace – Capable of reaching 450 ºC within 2 hours and maintaining
450 - 500 ºC ± 10 ºC, with temperature controller and safety switch (Cress Manufacturing
Co., Santa Fe Springs, CA, B31H, X31TS; Thermo ScientificTM Cat# FB1315M, or
equivalent). For safety, the kiln or furnace should be vented outside the laboratory, or to a
trapping system.

6.3 Equipment for sample and standard preparation

6.3.1 PFAS- and fluoride-free gloves, nitrile or polyethylene

6.3.2 Laboratory fume hood (of sufficient size to contain the sample preparation equipment listed
below)

6.3.3 Glove box (optional)

6.3.4 Equipment for determining total suspended solids

6.3.4.1 Oven – Capable of maintaining a temperature of 103 - 105 ºC

6.3.4.2 Desiccator

6.3.4.3 Filtration equipment (e.g., vacuum filtration apparatus)

6.3.4.4 Filter, glass fiber, 47 mm, 1 µm (Millipore SigmaTM or equivalent)

6.3.5 Balances

6.3.5.1 Analytical – Capable of weighing 0.1 mg

6.3.5.2 Top loading – Capable of weighing 10 mg

6.3.6 pH Paper, range 0-14, 0.5-unit readability (Whatman® PanpehaTM or equivalent)

6.3.7 Centrifuge tubes, polypropylene, with polypropylene caps for storing water-based standards
(Fisher Cat# 14-959-49A or equivalent)

6.3.8 Vials for storing methanol-based PFAS standards

6.3.8.1 Polypropylene crimp/snap vials, 1 mL (Agilent Cat# 5182-0567 or equivalent)

6.3.8.2 Clear snap cap, polyethylene, 11 mm (Fisher Scientific Cat# 03-375-24E or

equivalent)

6.3.9 Quartz wool (Fisher Cat# AC451040500 or equivalent), used as a prefilter for samples with
>100 mg/L of total suspended solids.

6.3.10 Graduated cylinder, polypropylene, 100 mL (Fisher Cat# 08-575-6D or equivalent), used to
measure the reagent water used to prepare quality control (QC) samples.

Method 1621 6 January 2024

6.4 Syringes

6.4.1 Plastic, Luer-lock syringes (BD Cat# 303134 or equivalent) for manually rinsing and
drying of GAC if not able to be done automatically by the adsorption unit.

6.4.2 Micro-syringes able to measure 10 – 1000 µL, used for all methanol-based standards.

6.4.3 Variable volume pipets with disposable polypropylene or polyethylene tips, used for
preparing calibration standards and any water-based standards.

6.5 Adsorption Unit (Analytik Jena APU sim, Trace Elemental Instruments XPREP A-6, Mitsubishi
TXA-04, or equivalent)
The adsorption apparatus must be capable of loading up to 125 mL of aqueous sample at a flowrate
no higher than 3 mL/min on up to three adsorption columns in series. The apparatus must also be
capable of washing the columns with sodium nitrate. The apparatus must allow for either manual
or automated rinsing of the columns with at least 3 mL of reagent water and drying with air.

Certain adsorption units may have fluoropolymer transfer lines that cannot be removed. In these
cases, the preparation batch method blanks must be rotated among the ports to ascertain that there is
no contamination in the system.

6.6 Glass columns containing between 40 and 50 mg GAC with a particle size of 50 – 150 µm
(Metrohm Cat# SNG-ICT0008 or equivalent) and compatible GAC column holder assemblies.
Other brands of GAC may be used as long as the background levels are below one-third the
regulatory compliance limit and there is no visible carbon migration from the column. Each new
lot of GAC columns must be tested (Section 4.3.2) prior to using for sample analysis.

6.7 Steel rod to transfer GAC into combustion boats (Mandel Cat# NS-TX3SCR or equivalent)

6.8 CIC Instrument – Automated furnace system integrated to an IC. Other instrument configurations
may be possible.

6.8.1 Autosampler capable of introducing combustion boats with the GAC into the combustion
tube.

6.8.2 Ceramic combustion boats compatible with the combustion system that can accommodate
up to 100 mg of carbon.

6.8.3 Furnace capable of maintaining an inlet temperature of at least 900 °C and minimum outlet
temperature of at least 1000 °C. The furnace unit must be capable of maintaining a
constant flow of oxygen gas (e.g., 300 - 400 mL/min) during pyrohydrolytic combustion
per manufacturer’s recommendations. If the system uses argon plus oxygen, then the
instrument should be able to keep a constant flow of argon gas (e.g., ~ 200 mL/min).

6.8.4 Gas absorption unit with an absorption tube which can hold a minimum of 5 mL volume
and can maintain a constant volume of reagent water. The adsorption unit should be able to
rinse the tube and transfer lines with reagent water or other solution as per manufacturer’s
instructions before and after each sample combustion. The gas absorption unit should be
interfaced to the IC and be capable of injecting an aliquot of the final absorption solution
into the IC.

Method 1621 7 January 2024

 6.8.5 Combustion tube made of quartz, or a combination of quartz and ceramic, capable of
withstanding temperatures up to 1100 °C. The combustion tube may include quartz wool,
or other suitable medium, to provide appropriate sample mixing to ensure complete
combustion of the sample.

6.8.6 Water delivery system capable to introduce reagent water at a controlled rate to produce
pyrohydrolytic combustion.

6.9 IC System

6.9.1 IC Column (Metrohm Metrosep A Supp 5, 5 µm, 4x150 mm column, Cat# 6.1006.520;
DionexTM IonPacTM AS24, 7 µm, 2x250 mm, Cat# 064153, or an equivalent column
compatible with the IC system and that can meet the peak resolution requirements in
Section 10.2.1)

6.9.2 Guard column (Metrohm Metrosep A Supp 5, 5 µm, 4x50 mm, Cat# 6.1006.500; DionexTM
IonPacTM AS24, 7 µm, 2x50 mm, Cat# 064151, or an equivalent guard column compatible
with the IC system and column)

6.9.3 Pumping system capable of delivering eluent at a flow rate between 0.1 to 3.0 mL/min with
a precision of at least 2%, or as recommended for this determination by the manufacturer.

6.9.4 Continuous eluent generation cartridges or devices are permitted.

6.9.5 Conductivity detector, temperature controlled to 0.01 °C, capable of at least 1 to 1000
μS/cm on a linear scale.

6.9.6 Interfaced data system to acquire, store, reduce, and output chromatographic data. The
computer software should have the capability of processing data by recognizing an IC peak
within the given retention time window. The software must allow integration of the
fluoride peak area response within specified time limits. The software must be able to
construct linear regressions or quadratic calibration curves and calculate concentrations.

6.10 Desiccator (Bel-ArtTM Cat# F420310000 or equivalent), used to store opened packages of GAC
columns (see Section 11.3.5) and combustion boats. If storing combustion boats in a desiccator, the
GAC columns and combustion boats must be stored separately (i.e., in a different desiccator) to
prevent possible cross contamination.

7.0 Reagents and Standards

Unless otherwise indicated, all reagents shall conform to the specifications of the Committee on
Analytical Reagents of the American Chemical Society (ACS), where such specifications are available.
Other grades may be used, provided it is first determined that the reagent is of sufficiently high purity to
permit its use without lessening the quality of the determination.

7.1 Reagents

Reagents prepared by the laboratory must be stored in HDPE or polypropylene containers. Proper
cleaning procedures (Section 4.2) must be followed prior to using the containers.

Method 1621 8 January 2024

 7.1.1 Methanol – HPLC grade or better, 99.9% purity, verified by lot number before use. Store
at room temperature.

7.1.2 Ammonium hydroxide, 28-30% w/w purity (Fisher Cat# A669S-500 or equivalent)

7.1.2.1 Methanolic ammonium hydroxide, 1% – Add 3.3 mL ammonium hydroxide to

97 mL methanol, store at room temperature, replace after 1 month. Used for
rinsing plasticware, glassware, and equipment (Section 4.2).

7.1.3 Potassium hydroxide (KOH), fluoride free, (Fisher Cat# P250-500 or equivalent)

7.1.3.1 Potassium hydroxide, 0.5 M – Dissolve 2.81 g KOH in 100 mL of reagent water
(Section 7.1.8). Store at room temperature, replace after 6 months. Used to
adjust sample pH (Section 11.2.1).

7.1.4 Sodium nitrate (Sigma-Aldrich Cat# 221341 or equivalent)

7.1.4.1 Sodium nitrate, 2 M – Dissolve 170 g of NaNO3 (Section 7.1.4) in 1 L reagent

water (Section 7.1.8), store at room temperature in an amber container or
protected from light, replace after 1 year.

7.1.4.2 Sodium nitrate, 0.01 M – Dilute 5 mL of 2 M NaNO3 (Section 7.1.4.1) into 1 L

reagent water (Section 7.1.8), store at room temperature in an amber container or
protected from light and replace after 6 months.

7.1.5 Sodium thiosulfate (Alfa AesarTM Cat# A1762936 or equivalent). Used to dechlorinate
samples (Section 11.2.2), as needed.

7.1.6 Gases

7.1.6.1 Oxygen – 99.999%

7.1.6.2 Argon – 99.999% (if used by the instrument)

7.1.7 Eluents – Various eluents may be used, based on manufacturer’s recommendations,

provided they give the proper resolution for the component peak. The background tends to
be lower when using hydroxide-based eluents (e.g., KOH, NaOH) in place of carbonate-
based eluents. To lower the background when using carbonate-based eluents, an additional
carbonate removal device may be installed.

Note: The use of electrolytically generated hydroxide-based and carbonate-based eluents is

permitted with this method and strongly encouraged. Part numbers are available from
the instrument manufacturers. When using electrolytically prepared eluents only reagent
water needs to be added to the system.

7.1.7.1 Example Carbonate-based Eluent – Dissolve 0.168 g sodium bicarbonate

[1.0 mM] (Fisher Cat# S233-500 or equivalent) and 0.6784 g sodium carbonate
[3.2 mM] (Fisher Cat# S495-500 or equivalent) in 2 L reagent water (Section
7.1.8). Store at room temperature, shelf life 1 month.

7.1.7.2 Example Hydroxide-based Eluent – Dissolve 8.0 g (or 5.25 mL) of 50% w/w
sodium hydroxide (Fisher Cat# SS410-4 or equivalent) in 950 mL of reagent

Method 1621 9 January 2024

 water (Section 7.1.8). This makes a 100 mM solution. Store at room
temperature, shelf life 2 months.

7.1.8 Reagent water – Laboratory purified water, ASTM Type I or Type II (Reference 5). Test
prior to use for PFAS content by analyzing method blanks.

7.2 Diethyl-p-phenylenediamine (DPD) Test Kit (Fisher Cat# S72367 or equivalent) or chlorine test
strips (Hach Cat# 2745050 or equivalent), for determination of residual chlorine (Section 11.2.2).

7.3 Standard solutions – Prepare from materials of known purity and composition or purchase as
solutions or mixtures with certification to their purity, concentration, and authenticity. Observe the
safety precautions in Section 5.

Note: Standards for the PFAS compounds used in this method are available from Cambridge Isotope
Laboratories, Wellington Laboratories, and other suppliers. Listing of specific suppliers does not
constitute a recommendation or endorsement for use. All diluted solutions must be stored in
HDPE or polypropylene containers that have been thoroughly rinsed with methanol.

Purchase of commercial standard solutions or mixtures is highly recommended for this method;
however, when these are not available, preparation of stock solutions from neat materials may be
necessary. If the chemical purity is 98% or greater, the weight may be used without correction to
calculate the concentration of the standard. Dissolve an appropriate amount of assayed reference
material in the required solvent. For example, weigh 10 to 20 mg of an individual compound to
three significant figures in a 10-mL stoppered volumetric flask and fill to the mark with the
required solvent. Once the compound is completely dissolved, transfer the solution to a clean
container and cap.

When not being used, store standard solutions as recommended by the vendor. Place a mark on the
container at the level of the solution for standards prepared in methanol so that solvent loss by
evaporation can be detected. Replace the solution if solvent loss has occurred. The laboratory must
maintain records of the certificates for all standards for traceability purposes.

7.3.1 Sodium Fluoride Stock Standard, 1000 mg F-/L, 1 mL = 1 mg F- (Reagents Specialty

Chemicals and Solutions Cat# CF135100-120A or equivalent)

7.3.1.1 Fluoride Working Standard, 10 mg F-/L – Dilute 0.5 mL of fluoride standard

(Section 7.3.1) in 50 mL reagent water (Section 7.1.8).

7.3.2 Calibration standard solutions – At least 5 fluoride calibration concentrations spanning the
AOF concentration range of interest for linear regression, and 6 concentrations for a
second-order calibration model, are required to prepare the initial calibration curve (Section
10.1). The lowest level calibration standard must meet a signal-to-noise ratio of 10:1.
Prepare the calibration standards by diluting the fluoride working standard (Section 7.3.2)
in reagent water. A fluoride calibration range of approximately 2.0 µg F-/L to 100 µg F-/L
(assuming a 100-mL sample volume) may be used as a starting point for determining the
calibration range.

Low-level and mid-level calibration verification (CV) standards are analyzed at the
beginning of the analytical sequence and every 10 field samples, for the purpose of
calibration verification.

Method 1621 10 January 2024

 Note: Additional calibration standards at levels lower than the lowest calibration standard listed
in the method, may be added to accommodate a lower limit of quantitation if the instrument
sensitivity allows. Calibration standards at the high end of the calibration may be
eliminated if the linearity of the instrument is exceeded, or at the low end if those
calibration standards do not meet the S/N ratio criterion of 10:1, as long as the required
number of calibration points is met.

7.3.3 PFAS Standards – Sodium perfluoro-1-hexanesulfonate (PFHxS) (Wellington Laboratories

Cat# L-PFHxS or equivalent) or Potassium perfluoro-1-hexanesulfonate (Cambridge
Isotope Laboratories Cat# ULM-12310-1.2, or equivalent)

AOF is calculated as a concentration of fluoride ion; therefore, the fluoride concentration

for the PFAS standard used, from the salt form concentration, in the initial demonstration
of capability and for spiking samples must be calculated for the compound as follows:
𝑛𝑛𝐹𝐹 × 18.998
𝐶𝐶𝐹𝐹 = 𝐶𝐶𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 ×
𝑀𝑀𝑊𝑊𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃
where,
CF = Concentration of fluoride ion in PFAS (or non-PFAS) compound in µg/mL
CPFAS = Concentration of the PFAS (or non-PFAS) compound in µg/mL
MWPFAS = Molecular weight of the PFAS (or non-PFAS) compound in g/mol
nF = Number of fluorine atoms in the compound
18.998 = Atomic weight of fluorine, g/mol

7.3.4 Chloride Stock Standard, 1000 mg Cl-/L (Sigma-Aldrich Cat# 1198970500 or equivalent).
Used for peak resolution (Section 10.2).

7.4 Desiccant – W. A. Hammond DrieriteTM Cat# 21005, or equivalent

7.5 Adsorption Tube Cleaning Reagents

7.5.1 Sodium hydroxide, 1 M - Dissolve 20 g of NaOH (Fisher Cat# S318-100 or equivalent) in

500 mL reagent water (Section 7.1.8), store at room temperature, replace after 1 year.

7.5.2 Isopropanol (Fisher Cat# A416-1 or equivalent)

7.5.3 Aluminum oxide powder (Agilent Cat# 8660-0791 or equivalent)

8.0 Sample Collection, Preservation, Storage, and Holding Times

8.1 Collect samples in HDPE or polypropylene containers following conventional sampling practices
(Reference 6). All sample containers must have linerless HDPE or polypropylene caps. Other
sample collection techniques, or sample volumes may be used, if documented.

8.2 Aqueous samples

8.2.1 Collect approximately 100 mL of sample in an appropriately sized HDPE or polypropylene

bottle. Samples that flow freely are collected as grab samples, or in refrigerated bottles
using automated sampling equipment. For this test, headspace is allowable.

Method 1621 11 January 2024

 Note: Because this method consumes the entire volume of sample, aqueous samples must be
collected at least in triplicate to allow sufficient volume for the determination of total
suspended solids, inorganic fluoride, residual chlorine, and pH, as well as having sufficient
volume for matrix spikes or reanalysis. If a specific program requires the analysis of field
replicates and/or spiked samples (Section 9.5), additional containers must be collected.

Because the target analytes are known to bind to the interior surface of the sample
container, subsampling should be avoided whenever possible to avoid further loss of the
analytes to the walls of a new container. Therefore, if a specific program requires a
sample volume smaller than 100 mL for analysis, it is highly recommended to collect the
sample in an appropriately sized HDPE or polypropylene container.

8.2.2 Maintain all aqueous samples protected from light at or below 6 ºC from the time of
collection until shipped to the laboratory. Samples must be shipped as soon as practical
with sufficient ice to maintain the sample temperature at or below 6 ºC during transport and
be received by the laboratory within 48 hours of collection. The laboratory must confirm
that the cooler temperature is at or below 6 °C upon receipt. If, upon receipt, the cooler
temperature is above 6 ºC, the laboratory must contact the client for further guidance. Once
received by the laboratory, the samples must be stored at or below 6 ºC until sample
preparation.

8.3 Holding times - Aqueous samples should be analyzed as soon as possible; however, samples may
be held in the laboratory protected from light for up to 90 days when stored at or below 6 ºC
(Reference 7).

9.0 Quality Control

9.1 Each laboratory that uses this method is required to operate a formal quality assurance program
(Reference 8). The minimum requirements of this program consist of an initial demonstration of
laboratory capability, analysis of samples spiked with organofluorine compounds to evaluate and
document data quality, and analysis of standards and blanks as tests of continued performance.
Laboratory performance is compared to established performance criteria to determine if the results
of analyses meet the performance characteristics of the method.

9.1.1 The laboratory must perform an initial demonstration of the capability (IDC) to
demonstrate the ability to generate acceptable precision and recovery with this method.
This demonstration is given in Section 9.2.

9.1.2 In recognition of advances that are occurring in analytical technology, and to overcome
matrix interferences, the laboratory is permitted certain options to improve separations or
lower the costs of measurements. These options include changes in sample volumes and
ion chromatography columns. Alternative determinative techniques and changes that
degrade method performance are not allowed without prior review and approval, as stated
in Section 1.5.

Note: For additional flexibility to make modifications without prior EPA review, see 40 CFR Part
136.6.

9.1.2.1 Each time a modification is made to this method, the laboratory is required to
repeat the IDC procedure in Section 9.2. If calibration will be affected by the

Method 1621 12 January 2024

 change, the instrument must be recalibrated per Section 10. Once the
modification is demonstrated to produce results equivalent or superior to results
produced by this method as written, that modification may be used routinely
thereafter, so long as the other requirements in this method are met.

9.1.2.2 The laboratory is required to maintain records of any modifications made to this
method. These records include the following, at a minimum:

a) The names, titles, business addresses, and telephone numbers of the

analyst(s) that performed the analyses and modification, and of the quality
control officer that witnessed and will verify the analyses and
modifications.
b) A narrative stating reason(s) for the modifications (see Section 1.7).
c) Results from all QC tests comparing the modified method to this method,
including:

i. Calibration (Section 10.0)

ii. Calibration verification (Sections 10.4 and 13.1)
iii. Initial precision and recovery (Section 9.2.1)
iv. Analysis of blanks (Section 9.3)
v. Accuracy assessment (Section 9.7)
d) Data that will allow an independent reviewer to validate each
determination by tracing the instrument output (peak height, area, or other
signal) to the final result. These data are to include:

i. Sample numbers and other identifiers

ii. Adsorption dates
iii. Analysis dates and times
iv. Analysis sequence/run chronology
v. Sample weight or volume (Section 11.0)
vi. Dilution data (Section 11.0)
vii. Instrument
viii. Ion chromatography column (dimensions, etc.)
ix. Operating conditions (temperatures, temperature program, flow rates)
x. Chromatograms and other recordings of raw data
xi. Quantitation reports, data system outputs, and other data to link the raw
data to the results reported

9.1.2.3 Alternative IC columns and column systems – If a chromatography column other

than the one specified in this method is used, that column system must meet all
the requirements of this method.

Note: The use of alternative IC columns or eluents may result in different retention times.

9.1.3 Analyses of method blanks are required on an on-going basis to demonstrate the extent of
background contamination in any reagents or equipment used to prepare and analyze field
samples (Section 4.3). The procedures and criteria for analysis of a method blank are
described in Section 9.3.

Method 1621 13 January 2024

 9.1.4 The laboratory must, on an ongoing basis, demonstrate that the analytical system is in
control through calibration verification and the analysis of ongoing precision and recovery
(OPR) standards, spiked with PFHxS standard at mid-level, and method blanks. These
procedures are given in Section 13.0.

9.2 Initial Demonstration of Capability

9.2.1 Initial precision and recovery (IPR) – To establish the ability to generate acceptable
precision and recovery, the laboratory must perform the following operations for each
sample matrix type to which the method will be applied by that laboratory.

9.2.1.1 Adsorb and analyze four aliquots of reagent water, spiked with sufficient volume
of PFHxS solution (Section 7.3.3) to achieve a concentration at or above the mid-
point of the calibration curve. One method blank must be prepared with the IPR
batch. All sample processing steps that are to be used for processing samples
must be included in this test.

9.2.1.2 Using results of the set of four analyses, compute the average percent recovery
and the relative standard deviation (RSD) of the concentration.

9.2.1.3 For the IPR, the RSD must be ≤ 20% and the average percent recovery between
80 – 120%. If the RSD and average percent recovery meet the acceptance
criteria, system performance is acceptable, and analysis of blanks and samples
may begin. If, however, the RSD exceeds the precision limit or the average
percent recovery falls outside the range for recovery, system performance is
unacceptable. Correct the problem and repeat the test (Section 9.2).

9.2.2 Method detection limit (MDL) – Each laboratory must also establish an MDL for
organofluorine using the MDL procedure at 40 CFR Part 136, Appendix B. The MDL
determination should be performed using PFHxS as the spiked compound. The
concentration of each spiked MDL replicate is to be blank corrected using the average of
the MDL blanks prepared and analyzed together with the spiked MDLs in each batch. The
minimum level of quantification (ML) can be calculated by multiplying the MDL by 3.18
and rounding the result to the nearest 1, 2 or 5 x 10n, where n is zero or an integer (see the
Glossary for alternative derivations).

9.3 Method blank – One method blank must be prepared and analyzed with each sample batch (Section
4.3) to demonstrate freedom from contamination.

Because fluoride contamination in the GAC columns is a significant problem, maintaining a

historical record of method blank data is highly recommended. Use the equation below to calculate
the method blank AOF concentration if the data system software package does not perform the
calculation automatically.
Vabs
AOF (𝜇𝜇𝜇𝜇 𝐹𝐹 − ⁄𝐿𝐿) = �CMB × �
VMB
where,
AOF = Concentration of adsorbable organic fluorine in µg F-/L
CMB = Method blank measured concentration (sum of top and bottom GAC) reported without
volume corrections
Vabs = Total volume of absorption solution in mL of the sample prior to injection to IC
VMB = Volume in mL of method blank adsorbed

Method 1621 14 January 2024

 9.3.1 Analyze the method blank before the analysis of the OPR (Section 12.5).

9.3.2 If AOF is found in the blank 1) at a concentration greater than the ML for the analyte, 2) at
a concentration greater than one-third of the regulatory compliance limit, or 3) at a
concentration greater than one-tenth of the concentration in a sample in the preparation
batch, whichever is greatest, analysis of samples must be halted, and the problem corrected.
Other project-specific requirements may apply; therefore, the laboratory may adopt more
stringent acceptance limits for the method blank at their discretion. If the contamination is
traceable to the lot number of GAC columns, or to contamination of the adsorption unit
(e.g., a dirty column position), all affected samples must be re-adsorbed and reanalyzed,
provided that enough sample volume is available.

If after sample reanalysis, the new blank still shows contamination, the laboratory must
document and report the failures (e.g., as qualifiers on results), unless the failures are not
required to be reported as determined by the regulatory/control authority. Results
associated with blank contamination for an analyte regulated in a discharge cannot be used
to demonstrate regulatory compliance. QC failures do not relieve a discharger or permittee
of reporting timely results.

9.4 The specifications contained in this method can be met if the apparatus used is calibrated properly
and then maintained in a calibrated state. The standards used for initial calibration (Section 10.3)
and calibration verification (Sections 10.4, 13.1) may be prepared from the same source; however,
the use of a secondary source for calibration verification is highly recommended whenever
available. If standards from a different vendor are not available, a different lot number from the
same vendor can be considered a secondary source.

9.5 Depending on specific program requirements, field replicates may be collected to determine the
precision of the sampling technique, and spiked samples may be required to determine the accuracy
of the analysis. When spiked samples are required, sufficient volume should be provided for that
purpose.

9.6 An OPR, processed through the entire sample preparation procedure in Section 11.3, is required
with each adsorption batch. It is not possible to assess the recovery of every possible
organofluorine compound that may be detected with this technique. Therefore, PFHxS was chosen
as the OPR fortification analyte to represent organofluorine compounds because when added to
reagent water, it is well retained on GAC and does not significantly adsorb to surfaces. It is
recommended that the OPR be spiked at a concentration at the mid-level of the calibration curve.

9.7 Matrix spike (MS) and matrix spike duplicate (MSD) are required at a frequency of one set per
adsorption batch of no more than 20 field samples. It is recommended that MS/MSD aliquots be
spiked at a concentration at or above the mid-level of the calibration curve. Calculate the percent
recovery as follows:
CMS − Cn
%R = × DF × 100
Cs
where,
CMS = MS measured concentration (top and bottom GAC, blank corrected)
Cn = Concentration of the unfortified sample (top and bottom GAC, blank corrected)
Cs = Spiked concentration
DF = Dilution factor. If no dilution was performed, DF = 1.

Method 1621 15 January 2024

 Calculate the relative percent difference (RPD) as follows:

|MS − MSD|
RPD = × 100
(MS + MSD)/2
where,
MS = Percent recovery of the MS
MSD = Percent recovery of the MSD

10.0 Calibration and Standardization

10.1 Establishing instrument operating conditions

Establish IC operating conditions using the manufacturer’s instructions. The table below shows
instrument settings that were used during the method validation for two different CIC systems. The
columns specified below and are provided as guidance. The final adsorption solution volume is a
user-defined parameter and will differ among instruments. Check with the instrument manufacturer
for the appropriate volume of the final adsorption solution. Other CIC systems require different
instrument settings; however, the method performance criteria must still be met.

Metrohm Dionex
Example IC Parameters
Metrosep A Supp 5, 5 µm, Dionex IonPac AS24, 7 µm,
Separator column:
4 x 150 mm 2 x 250 mm
Metrosep A Supp 5 Guard 4.0, Dionex IonPac AS24, 7 µm,
Guard column:
5 µm, 50 x 4 mm 2 x 50 mm
Flow rate: 0.7 mL/min 0.3 mL/min
Injection volume: 1 mL 100 µL
Column temperature: 35 °C 30 °C
Detector cell temperature: 40 °C 35 °C
Example Combustion Parameters
Furnace inlet temperature: 1050 °C 900 °C
Furnace outlet temperature: 1050 °C 1000 °C (minimum)
Quartz tube with ceramic insert
Pyrolysis tube: Quartz
and quartz wool
Argon carrier flow: Only oxygen used 200 mL/min
Oxygen flow: 300 mL/min 400 mL/min
Humidified oxygen flow: 200 mL/min 100 mL/min
0.3 mL/min bled into front of
Water supply scale: 2
combustion tube by a dosing unit
Sample boat: Ceramic Ceramic
Adsorption solution: Reagent Water Reagent Water

10.2 Chromatographic conditions

10.2.1 The chromatographic conditions must be optimized for compound separation and
sensitivity. Fluoride must be adequately resolved chromatographically from the water dip
(if present). A resolution of at least 1.0 minute is necessary between the fluoride peak and
the water dip for proper peak integration. Hydroxide-based eluents do not produce a water
dip. Potential interferents include chloride concentrations over 500 mg/L, constituents in
the eluant, and other potential interferences found in the field samples that can elute very

Method 1621 16 January 2024

 close to the fluoride peak; therefore, the chromatographic conditions must be optimized to
give a resolution of at least 1.0 minute between the fluoride peak end and the beginning of
the chloride and other interfering peaks. A chloride standard (Section 7.3.4) with a
concentration above the mid-point of the calibration range must be injected together with a
mid-level fluoride standard to demonstrate the chloride and fluoride peaks are properly
resolved prior to initial calibration. The same optimized operating conditions must be used
for the analysis of all standards, blanks, IPR and OPR samples, and field samples.

The chromatographic conditions used during the method validation using two different
instruments and the columns specified in Section 10.1 are listed below. Different
instruments will require different operating conditions, especially when using different
eluents and different IC columns. Any modifications to IC conditions must still produce
conditions such that co-elution of the fluoride with the nearby chloride peak or other
interfering peaks is minimized to reduce the probability of peak misidentification or
positive bias.

Metrohm Dionex
Example IC Method Conditions
Time (min) mM KOH Curve
Initial 8.0 5
Isocratic at 0.7 ml/min with an eluent composed 3.50 8.0 5
of 3.2 mM Sodium Carbonate (Na2CO3) and 1 9.00 75 9
mM Sodium Bicarbonate (NaHCO3) 10.25 75 5
10.25 8.0 5
20.00 8.0 5
Example Autosampler Combustion Program
Position Wait time (sec) Speed (mm/sec) Position Wait time (sec) Speed (mm/sec)
200 mm 15 5 90 mm 60 10
End 540 5 End 600 10
Cool 300 Cool 60 40
Home N/A Home 120 20

10.2.2 Retention time calibration

10.2.2.1 Establish the retention time (RT) of fluoride by combusting the mid-level
standard from the ICAL in triplicate and analyze under the established IC
conditions.

10.2.2.2 Calculate the width of the RT window for fluoride at plus or minus three times
the standard deviation of the retention times determined in Section 10.2.2.1.
Note: Procedures for establishing RT windows from other sources may be employed if
they are clearly documented and provide acceptable performance. Such
performance may be evaluated using the results for the spiked QC samples
described in this method, such as OPR samples and matrix spike samples.

10.2.2.3 Once the RT window is established after calibration, the RT window for fluoride
is confirmed using the absolute retention time from the calibration verification
standard at the beginning of the analytical batch. For samples analyzed during
the same 24-hour period as an initial calibration, use the RT of the mid-point
standard of the initial calibration.

Method 1621 17 January 2024

 10.2.2.4 The RT windows must be re-established when a new IC column is installed and
after major instrument maintenance.

10.3 Initial calibration

Prior to the analysis of samples, each IC system must be calibrated with a minimum of 5 standard
concentrations if using a linear regression model, or with a minimum of 6 standards if using a
quadratic model (Section 7.3.2). The quadratic model should not be higher than 2nd order. The
calibration curve must not be forced through zero. The lowest concentration CAL standard should
be at or below the limit of quantitation (LOQ). This method calibrates and quantifies the
adsorbable organic fluorine concentration in a sample, using the external standard technique.
Standards are injected onto clean ceramic boats and go through combustion, but not through carbon
adsorption. By combusting the calibration standards instead of doing a direct injection into the IC,
the calibration compensates for any bias produced by loss of analyte that may happen during
combustion of field samples.

Note: The concentrations listed in Table 2 are those used in the validation studies and are provided as
guidance. Standards at other concentrations may be used, and wider calibration ranges with
standards either above or below the concentrations in Table 2 are allowed, provided that at least
5 contiguous standards are used (6 if using a quadratic model), with the lowest standard at or
below the LOQ and that all of the conditions in Sections 10.3.2 and 10.3.3 are met.

10.3.1 Initial calibration frequency

Each IC system must be calibrated after instrument and chromatographic conditions have
been established before analysis of samples can take place. The initial calibration must be
repeated whenever the laboratory takes corrective action that might change or affect the
initial calibration criteria (e.g., replacing IC column, replacing suppressor), or if the CV
acceptance criteria in Section 10.4 have not been met.

10.3.2 Initial calibration procedure

Prepare a minimum of 5 calibration standards (6 if using quadratic fit) using the working
fluoride standard (see Section 7.3.1.1). Analyze each calibration standard by injecting 200
µL into clean combustion boats. The calculated concentrations of the calibration standards
should be within 80 – 120% of the concentration true value. The RT for fluoride in the
ICAL must fall within the window established in Section 10.2.2.2.

10.3.3 Instrument linearity

Assess the calibration linearity based on the relative standard error (RSE) using the
equations below. The RSE must be ≤ 20%.
2
𝑥𝑥 ′ − 𝑥𝑥
� 𝑖𝑖 𝑥𝑥 𝑖𝑖 �
𝑛𝑛

𝑅𝑅𝑅𝑅𝑅𝑅 = 100 × �� 𝑖𝑖
𝑛𝑛 − 𝑝𝑝
𝑖𝑖=1
where,
xi = Nominal concentration (true value) of each calibration standard
x´I = Measured concentration of each calibration standard
n = Number of standard levels in the curve
p = Type of curve (2 = linear, 3 = quadratic)

Method 1621 18 January 2024

 10.3.4 Initial calibration corrective actions

If the instrument linearity or recovery criteria for initial calibration are not met, inspect the
system for problems and take corrective actions to achieve the criteria. This may require
the preparation and analysis of fresh calibration standards. All initial calibration criteria
must be met before any samples or required blanks are analyzed.

10.4 Calibration Verification

10.4.1 The calibration must be verified by analyzing CV standards prepared from a secondary
source of sodium fluoride. A low-level CV standard at the concentration of the lowest
calibration standard that is within the quantitation range must be analyzed at the beginning
of the analytical sequence. CV standards at a concentration in the middle of the
laboratory’s calibration range must be analyzed every 10 field samples, and at the end of
the analytical batch.

10.4.2 The RT for fluoride on the CV must fall within the window established in Section 10.2.2.

10.4.3 The percent recovery for the CV standards should be within 80 – 120%.

11.0 Sample Preparation and Adsorption

Samples containing high levels of suspended solids (e.g., visible particulates) can clog the GAC
columns and prevent proper sample adsorption. Therefore, for aqueous samples from unfamiliar
sources that contain particulates, total suspended solids must be determined using the procedure in
Section 11.1. Other analytical interferences are residual chlorine, sample pH, and inorganic
fluoride. To check for these interferences in samples from unfamiliar sources, follow the sample
pre-treatment steps in Section 11.2. One sample container must be selected to check for
interferences in Section 11.1 and 11.2. Any necessary adjustments to the sample based on results
from Section 11.2 must be done on the container reserved for AOF analysis (Section 11.3).

Note: Do not use any fluoropolymer-containing articles or task wipes in these extraction procedures.
Use only HDPE or polypropylene wash bottles and centrifuge tubes. Reagents and solvents for
cleaning syringes may be kept in glass containers.

11.1 Determination of Total Suspended Solids (TSS) – For aqueous liquids or multi-phase samples
consisting of mainly an aqueous phase. Choose one sample container to perform TSS analysis as
described below. Separate this container and do not use the leftover sample for AOF sample
processing in Section 11.3.

Note: The TSS procedure given here is gravimetric. Alternatively, other recognized procedures for
determining TSS in aqueous samples may be employed, including using a TSS probe.

11.1.1 Desiccate a glass-fiber filter (Section 6.3.4.4) and weigh to three significant figures.

11.1.2 Filter 20.0 ± 1.0 mL of well-mixed sample through the filter.

Method 1621 19 January 2024

 Note: The purpose of this analysis is simply to minimize the risk of clogging the GAC column
from samples with TSS content on the order of 100 mg/L (see Section 11.3). Therefore, the
volume of sample employed here was selected to identify samples high in TSS and still be
representative of the bulk sample collected. The TSS result is not used in the calculation of
the AOF result itself.

11.1.3 Dry the filter a minimum of 12 hours in the oven (Section 6.3.4.1) at a temperature between
103 – 105 ºC and cool in a desiccator (Section 6.3.4.2) for at least 1 hour. Take the filter
out of the desiccator and reweigh it.

11.1.4 Calculate TSS as follows:

(𝐴𝐴 − 𝐵𝐵) × 1000
𝑇𝑇𝑇𝑇𝑇𝑇 (𝑚𝑚𝑚𝑚⁄𝐿𝐿) =
𝐶𝐶
where,
A = Weight of filter + sample residue, mg
B = Initial weight of filter, mg
C = Volume of sample filtered, mL

11.2 Sample Pretreatment

11.2.1 pH Verification

Before adsorption, sample pH must be verified to be ≥ 5. Using the same sample container
used in Section 11.1, take an aliquot and check the sample pH with pH paper (Section 6.3.6
and Reference 9). If the pH of the sample is below 5, adjust the pH of the sample in the
container to be used for AOF analysis (Section 11.3) with 0.5 M potassium hydroxide
(Section 7.1.3.1). Lower molarities of KOH may be used; however, make sure that the
final volume of solution used to adjust the pH does not exceed 1 mL to prevent diluting the
sample.

11.2.2 Dechlorination

Test sample for the presence of chlorine using an aliquot from the sample container used in
Section 11.1 and either type of chlorine testing products in Section 7.2. If chlorine is
detected, dechlorinate the sample in the container that was designated for AOF analysis
(Section 11.3) using sodium thiosulfate at 5 mg of thiosulfate per every 1 mg/L of chlorine,
prior to sample adsorption.

11.2.3 Determination of Inorganic Fluoride

Determine the concentration of inorganic fluoride in the sample. This may be done by
directly injecting a diluted aliquot of well-mixed sample from the container used in Section
11.1 into the IC using the same instrument parameters and procedure as for the calibration
standards. A 1:10 sample dilution using reagent water (Section 7.1.8) is recommended to
prevent damage to the column and minimize the matrix effects for the chromatographic
peaks. Alternatively, other recognized procedures for determining fluoride in aqueous
samples may be employed, including ion-selective electrode procedures (sample distillation
is not required). Calculate the concentration of inorganic fluoride in mg/L.

Method 1621 20 January 2024

 Levels of inorganic fluoride > 8 mg F-/L may bias the AOF results high. If inorganic
fluoride concentration exceeds 8 mg F-/L, transfer a known volume aliquot of the sample
from the container designated to be used for AOF analysis, to a new PFAS-free HDPE or
polypropylene container, and dilute it with reagent water (final volume of sample must be
approximately 100 mL) to reduce the concentration of inorganic fluoride to just below
8 mg F-/L. The lowest dilution possible must be targeted to prevent overdiluting the
analyte of interest.

Note: Transferring a sample aliquot between the parent container and a secondary container for
the purpose of sample dilution may result in further loss of analyte due to the propensity of
organofluorines to adhere to surfaces.

11.3 Sample Processing

The procedure requires the preparation of the entire sample. Smaller sample volumes may be
analyzed for samples containing high levels of organofluorine; however, subsampling should be
avoided whenever possible. Typical sample size is approximately 100 mL.

Note: For samples containing > 100 mg/L of TSS (Section 11.1), a piece of quartz wool should be
placed inside an empty column ahead of the GAC columns and used as a prefilter to prevent the
GAC columns from clogging. The prefilter must be washed with the nitrate wash solution and
combusted in the same manner as the GAC columns. The concentration from the prefilter must
be added to the concentration of the GAC columns.

When a sample is processed using quartz wool as prefilter, a quartz wool blank is required to be
prepared with the batch because the prefilter material will add to the background fluoride levels
in the sample. A piece of quartz wool of similar size as that used for the sample, rinsed with 25
mL of 0.01 M sodium nitrate and 20 mL reagent water, must be combusted separately as a
prefilter blank. The total fluoride of the prefilter blank and the method blank must be subtracted
from the sample results.

11.3.1 Prepare one method blank and one OPR using reagent water in HDPE or polypropylene
bottles. Select a volume of reagent water that is typical of the samples in the batch. Spike
the OPR sample with PFHxS (Section 7.3.3) at the concentration of the mid-level
calibration point. If any of the samples in the preparation batch required dechlorination or
pH adjustment, separate method blanks and OPR with the maximum amount of sodium
thiosulfate and/or potassium hydroxide added to the samples must be prepared to check for
any possible contamination added by these chemicals.

11.3.2 Spike the MS and MSD samples directly in the original bottles at the mid-level calibration
point using PFHxS (Section 7.3.3). Adjust the spiking level and the sample dilution to
keep the expected results for these two aliquots within the calibration range of the
instrument. In the event that the field sample used for the MS/MSD requires dilution prior
to analysis due to high levels of inorganic fluoride, both the MS and MSD must be diluted
at the same dilution factor as the original sample after spiking.

11.3.3 Weigh each sample and MS/MSD bottle (with the lid) to 0.1 g. The final volume of the
aqueous sample analyzed is determined by weighing the full sample bottle and then the
empty sample bottle (Section 11.3.9).

Method 1621 21 January 2024

 11.3.4 Add 0.5 mL of 2 M sodium nitrate (Section 7.1.4.1) to each field sample, method blank,
OPR, MS, MSD, and any other QC sample containers. Mix by inverting the containers
3 – 4 times.

11.3.5 Place two GAC columns in tandem in the column holder apparatus of the adsorption unit.

Note: Opened packages of GAC columns must be kept tightly sealed and stored in a desiccator
(Section 6.10) to minimize possible contamination from laboratory air.

11.3.6 Load the sample onto the GAC columns at a flowrate of no more than 3 mL/min. The
typical volume of sample is 100 mL, but because the sample containers allow collection of
volumes up to 125 mL, the adsorption unit should be set up to consume the entire volume
of sample if possible. Monitor for leaks throughout the column holder assembly.

11.3.7 After sample loading is complete, wash both GAC columns and the quartz wool, if used,
with 25 mL of 0.01 M sodium nitrate (Section 7.1.4.2) to remove inorganic fluoride.

11.3.8 Rinse both columns and the quartz wool, if used, with approximately 20 mL of reagent
water to remove any residual nitrate. This rinse also helps to prolong the life of the
pyrolysis tube by decreasing the potential for devitrification. Dry the columns and quartz
wool, if used, using at least 3 mL of air using the syringe. Remove the GAC columns from
the column holders. If samples are not to be combusted immediately, store columns in a
desiccator at room temperature no longer than 5 days.

11.3.9 Determine the final volume of each field and QC sample used by weighing the empty
sample bottle (with the lid) to 0.1g and subtracting the final weight from the initial weight
(Sec. 11.3.3). In calculating the sample volume, assume a sample density of 1.0 g/mL.

11.3.10 Flush the sample transfer lines in the adsorption unit with 40 mL reagent water followed by
40 mL of a suitable solvent (e.g., methanol) and another 40 mL reagent water to ensure that
all surfaces in contact with samples are flushed. Use solvents or solvent combinations
recommended by the manufacturer to prevent harm to components of the adsorption unit.

12.0 Instrumental Analysis

Note: Because short-chain PFAS compounds are poorly retained on GAC, whenever the laboratory
analyzes samples from new sources or new clients, at least one sample must be analyzed by
separate combustions of the top and bottom columns. Likewise, projects involving mass balance
requests must be analyzed by separate combustions of the top and bottom columns. In these
cases, the laboratory must calculate and assess percent breakthrough to confirm that the GAC
column capacity has not been exceeded.

In addition, as noted in Section 6.6, whenever a new lot number of GAC columns is received, it
must be lot-tested for the presence of AOF, and all such testing must be performed with separate
combustions of the top and bottom columns and the percent breakthrough calculated.

However, when the laboratory consistently analyzes samples from the same sources for the
same clients, a single combustion of the top and bottom GAC columns is permitted.

Method 1621 22 January 2024

12.1 Instrument operating conditions (Section 10.1 and 10.2) must have been set, and an initial
calibration (Section 10.3) performed prior to analyses of any field samples. The same instrument
operating conditions used for the initial calibration must be used to analyze field and QC samples.

12.2 Equilibrate the IC system by pumping eluent through until a stable baseline is obtained. Because
fluoride from the air in the laboratory can accumulate in the combustion tube while the instrument
sits idle for any period of time, at least two empty combustion boats (a.k.a. boat blank checks) must
be combusted prior to analysis of any sample to clear fluoride from the system.

12.3 Perform a calibration verification check prior to sample analysis.

12.4 For each sample, transfer the carbon from both the top and bottom columns into a combustion boat
using the steel rod (see note above regarding separate combustions of top and bottom GAC
columns). When pushing the carbon into the combustion boats, push from the bottom of the
column to ensure that the transfer rod does not come in contact with any particulates that may have
collected on the capping material at the top of the carbon column. If doing separate combustions of
the columns, do not mix the order of the columns (top column vs. bottom column) so that the
percent breakthrough can be properly calculated. If quartz wool was used for prefiltration, it must
be combusted in a separate combustion boat. Place the combustion boats in the autosampler of the
CIC using clean forceps and combust following manufacturer’s instructions.

Note: The combustion boats must be cleaned by pre-baking them in a kiln or furnace at 450 ºC for at
least 1 hour prior to use. Clean combustion boats should be stored in a desiccator or submerged
in reagent water to reduce buildup of organofluorine on the boats from laboratory air and should
be used within three days of baking. Combustion boats must be baked again if stored longer than
three days. Prior to use, baked combustion boats must be rinsed with methanol and dried.

12.5 The analytical sequence for a batch of samples analyzed during the same time period is as follows.
Standards must be brought to room temperature prior to use.

1. Boat Blank Checks (at least 2)

2. Calibration Verification Standard (Low-level)
3. Method Blank
4. OPR
5. Samples (10 or fewer)
6. MS
7. MSD
8. Calibration Verification Standard (Mid-level)
9. Samples (10 or fewer)
10. Calibration Verification Standard (Mid-level)

If the results are acceptable, the results from the closing calibration verification analysis (#10
above) may be used as the opening analysis for the next analytical sequence, beginning with a new
method blank. If 24 hours have passed since the beginning of the analytical sequence, the next
analytical sequence must begin with a low-level CV.

12.6 If the fluoride response for any sample exceeds the calibration range, the sample must be re-
adsorbed using a lower sample volume, if there is sufficient sample volume available.

Method 1621 23 January 2024

13.0 Performance Tests During Routine Operations

The following performance tests must be successfully completed as part of each routine
instrumental analysis shift described in Section 12.5 above.

13.1 Calibration verification

Prior to the analysis of any samples, analyze a low-level calibration verification standard at the
beginning of the analytical sequence, and analyze a mid-level calibration verification standard after
every 10 field samples and at the end of the analytical sequence.

13.1.1 The calibration is verified if the percent recovery for the CV standards is within 80 - 120%.

13.1.2 The RT for fluoride on the CV must fall within the window established in Section 10.2.2.

13.1.3 If the CV criteria in 13.1.1 and 13.1.2 are not met, perform any necessary instrument
maintenance, and reanalyze the CV. If the CV still fails the criteria, recalibrate the
instrument according to Section 10.3.

13.2 Fluoride in the field samples and batch QC must elute within ± 0.2 minutes of the RT
corresponding to the fluoride peak from the beginning CV.

13.3 Method Blank – Analyze the method blank after the analysis of the CV and prior to the analysis of
samples. The method blank should not exceed 4 µg F-/L.

13.4 Ongoing precision and recovery (OPR)

13.4.1 After the CV and the method blank, analyze the OPR prior to analysis of samples from the
same batch to ensure the analytical process is under control.

13.4.2 Compute the percent recovery of the analyte.

𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 (µ𝑔𝑔 𝐹𝐹 − ⁄𝐿𝐿)

𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 (%) = × 100
𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 (µ𝑔𝑔 𝐹𝐹 − ⁄𝐿𝐿)

13.4.3 The OPR recovery must be within 70 – 130%. If the fluoride recovery meets the
acceptance criteria, system performance is acceptable, and analysis of samples may
proceed. However, if the concentration falls outside of the given range, the adsorption
processes are not being performed properly. In this event, correct the problem, re-prepare
the sample batch, and repeat the ongoing precision and recovery test if sample volume
allows.

13.5 Accuracy and Precision

13.5.1 Matrix Spikes – After the samples, prior to closing CV. The MS recovery must be within
50 – 150% recovery.

13.5.2 Duplication – Analyze the MSD after the MS. Calculate the relative percent difference of
the recoveries using the equation in Section 9.7. The RPD must be ≤ 30%.

Method 1621 24 January 2024

14.0 Data Analysis and Calculations

14.1 Qualitative determination and peak identification

Organofluorine is positively identified in a field or QC sample if the RT falls within the window
established in Sec. 10.2.2.2, centered on the RT from the opening CV.

14.2 Estimating sample concentration

As noted in Section 1.7, this procedure may be used to estimate the aggregate contributions of the
organofluorine compounds in the sample. Because of the common occurrence of background levels
of fluoride in the sorption media, the method subtracts the amount of fluoride observed in the
method blank from the fluoride in the sample to estimate the AOF attributable to the sample itself.
This type of background subtraction, while not permitted in many other EPA methods, is not unique
to this procedure, and is a fundamental aspect of the method-defined parameter that is AOF.
Moreover, the fluoride background levels in the GAC are likely to result in a method detection limit
driven by the MDLb rather than the spiked sample MDLs value.

Note: The blank subtraction process inherent in this method may yield negative concentrations for
samples that have low concentrations. See Section 14.3.2 for instructions on how to report such
results.

14.2.1 Single combustion: For single combustion analysis, calculate the AOF concentration as a
sum of the fluoride mass on the GAC columns and prefilter (if used), adjusted by
subtracting the method blank and prefilter blank (if used), using the data software package
for the CIC and the multipoint calibration. Alternatively, the equation below can be used to
calculate the AOF concentration if the data software package will not perform the
calculation. Do not use the daily calibration verification data to quantitate AOF in
samples.
(𝑀𝑀 + 𝑄𝑄𝑄𝑄𝑠𝑠 ) (𝐵𝐵 + 𝑄𝑄𝑄𝑄𝐵𝐵 )
𝐴𝐴𝐴𝐴𝐴𝐴 (µ𝑔𝑔𝐹𝐹 − /𝐿𝐿) = − × 𝐷𝐷𝐷𝐷
𝑉𝑉𝑠𝑠 𝑉𝑉𝐵𝐵
where,
M = Mass measured for field sample, µg F-
B = Mass measured for method blank, µg F-
QWs = Mass measured for sample quartz wool, if used, µg F-
QWB = Mass measured for blank quartz wool, if used, µg F-
Vs = Sample volume adsorbed onto column, L
VB = Method blank volume adsorbed onto column, L
DF = Dilution factor. If no dilution was performed, DF = 1

14.2.2 Separate combustions: Calculate the AOF concentration as a sum of the top and bottom
GAC columns (and prefilter(s) if used) masses, adjusted by subtracting the sum of the top
and bottom columns of the method blank, using the data software package for the CIC and
the multipoint calibration.

Note: When blank subtracting, the GAC columns of the samples should not be corrected
individually by subtracting the individual blank GAC column results (e.g., top sample GAC
– top blank GAC) because this may result in negative values if the bottom blank GAC
column has a higher concentration than the bottom sample GAC column.

Method 1621 25 January 2024

 Alternatively, the equation below can be used to calculate the AOF concentration if the data
software package will not perform the calculation. Do not use the daily calibration
verification data to quantitate AOF in samples. Adjust the final AOF concentrations to
reflect the actual sample volume used.
(𝑀𝑀1 + 𝑀𝑀2 + 𝑄𝑄𝑄𝑄𝑠𝑠 ) (𝐵𝐵1 + 𝐵𝐵2 + 𝑄𝑄𝑄𝑄𝐵𝐵 )
𝐴𝐴𝐴𝐴𝐴𝐴 (µ𝑔𝑔𝐹𝐹 − /𝐿𝐿) = − × 𝐷𝐷𝐷𝐷
𝑉𝑉𝑠𝑠 𝑉𝑉𝐵𝐵
where,
M1 = Mass measured for first column, µg F-
M2 = Mass measured for second column, µg F-
B1 = Mass measured for first column of the method blank, µg F-
B2 = Mass measured for second column of the method blank, µg F-
QWs = Mass measured for sample quartz wool, if used
QWB = Mass measured for blank quartz wool, if used
Vs = Sample volume adsorbed onto column, L
VB = Method blank volume adsorbed onto column, L
DF = Dilution factor. If no dilution was performed, DF=1

Calculate the percent breakthrough for each column pair, per sample, as follows:
(𝑀𝑀2 − 𝐵𝐵2 )
% 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵ℎ𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟ℎ = × 100
[(𝑀𝑀1 − 𝐵𝐵1 ) + (𝑀𝑀2 − 𝐵𝐵2 )]
where,
M1 = Mass measured for the first column, µg F-
M2 = Mass measured for the second column, µg F-
B1 = Mass measured for first column of the method blank, µg F-
B2 = Mass measured for second column of the method blank, µg F-

Note: Due to the poor retention of short-chain PFAS compounds, breakthrough >50% is typical for
samples composed mainly of short-chain PFAS compounds.

Percent breakthrough must not exceed 50%. If % breakthrough exceeds the limit, the sample
is exceeding the GAC capacity and must be reanalyzed at a lower volume, if sufficient
sample volume is available. In situations where the % breakthrough exceeds the limit, but the
blank-corrected sample result is less than 3x the laboratory’s MDL, reanalysis is not
necessary, and the data may be reported with an appropriate qualifier.

14.3 Reporting of analytical results

Because of the inherent blank subtraction involved in this method, the calculations in Section 14.2
may yield negative values. Therefore, the reporting practices below differ from those in most other
EPA methods.

14.3.1 Report results in µg F-/L. Other units may be used if required in a permit or for a project.
Report all QC data with the sample results.

14.3.2 Reporting Level

14.3.2.1 Report adsorbable organofluorine as fluoride in each field sample or QC standard

at or above the MDL determined in the laboratory to 2 significant figures.
Report any value below the ML and any negative value in a field sample as

Method 1621 26 January 2024

 “<ML,” where ML is the concentration of the analyte at the ML, or as required
by the regulatory/control authority or permit.

14.3.2.2 Although the samples are blank corrected, the blank results also must be reported
with the data. Report organofluorine as fluoride in a method blank at or above
the MDL to 2 significant figures. Report a result for the method blank below the
MDL as “<MDL” where the MDL is the concentration of the analyte at the
MDL, or as required by the regulatory/control authority or permit.

Note: Because the MDL value is calculated from the total concentration (the top and bottom GAC
columns), when performing separate combustions, the MDL should be compared to the
total AOF for the sample and method blank concentrations, as calculated in Sections 9.3
and 14.2, and not to the concentration of each individual GAC column.

14.3.3 Results from tests performed with an analytical system that is not in control (i.e., that does
not meet acceptance criteria for any QC tests in this method) must be documented and
reported (e.g., as a qualifier on results), unless the failure is not required to be reported as
determined by the regulatory/control authority. Results associated with a QC failure cannot
be used to demonstrate regulatory compliance. QC failures do not relieve a discharger or
permittee of reporting timely results. If the holding time would be exceeded for a
reanalysis of the sample, the regulatory/control authority should be consulted for
disposition.

15.0 Method Performance

Routine method performance is validated through analysis of aqueous reference samples, including
MS/MSD samples. Ongoing method performance is monitored through QC samples analyzed alongside
samples. The parameters monitored include percent recovery and blank concentrations.

This method was validated, and performance specifications were developed using data from the EPA’s
interlaboratory validation study (Reference 14). Table 1 provides the pooled MDL results from the multi-
laboratory validation study. Data from all the QC samples in that study were used to develop the QC
acceptance criteria in Table 3 (IPR/OPR) in all aqueous field samples. Table 4 provides results from the
multi-laboratory validation study involving nine laboratories are only provided as examples for this
method

Every laboratory performing analyses in support of Clean Water Act compliance monitoring must have an
effective quality management system in place (see Section 9.1). Such systems must include assessment
of all results against the various QC acceptance limits in a given analytical method, but also should
include procedures for longer-term internal evaluations of laboratory performance. The EPA expects that
responsible laboratories will perform such evaluations and develop and apply in-house acceptance
criteria, which by virtue of being from a single laboratory, will be narrower than the interim acceptance
criteria listed in the tables in this method.

16.0 Pollution Prevention

16.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity
of waste at the point of generation. Many opportunities for pollution prevention exist in laboratory
operation. The EPA has established a preferred hierarchy of environmental management
techniques that places pollution prevention as the management option of first choice. Whenever

Method 1621 27 January 2024

 feasible, laboratory personnel should use pollution prevention techniques to address waste
generation. When wastes cannot be reduced feasibly at the source, the EPA recommends recycling
as the next best option.

16.2 The compounds in this method are used in extremely small amounts and pose little threat to the
environment when managed properly. Standards should be prepared in volumes consistent with
laboratory use to minimize the disposal of excess volumes of expired standards.

16.3 For information about pollution prevention that may be applied to laboratories and research
institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction
(Reference 10).

17.0 Waste Management

17.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal
restrictions, and to protect the air, water, and land by minimizing and controlling all releases from
fume hoods and bench operations. Compliance is also required with any sewage discharge permits
and regulations. An overview of requirements can be found in Environmental Management Guide
for Small Laboratories (Reference 11).

17.2 Samples at pH < 2 or pH > 12, are hazardous and must be handled and disposed of as hazardous
waste or neutralized and disposed of in accordance with all federal, state, and local regulations. It
is the laboratory's responsibility to comply with all federal, state, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal
restrictions.

17.3 For further information on waste management, consult Less is Better-Laboratory Chemical
Management for Waste Reduction (Reference 10) and The Waste Management Manual for
Laboratory Personnel (Reference 12).

18.0 References

1. “Working with Carcinogens,” Department of Health, Education, & Welfare, Public Health
Service, Centers for Disease Control, NIOSH, Publication 77-206, August 1977, NTIS PB-
277256.

2. “OSHA Safety and Health Standards, General Industry,” OSHA 2206, 29 CFR 1910.

3. “Safety in Academic Chemistry Laboratories,” ACS Committee on Chemical Safety, 1979.

4. American Public Health Association, American Water Works Association, Water Environment
Federation. SM 1090 Laboratory Occupational Health and Safety. In: Lipps W C, Braun-
Howland E B, Baxter T E, eds. “Standard Methods for the Examination of Water and
Wastewater.” Approved by Standard Methods Committee 2010, published online August 2018;
revised 23 April 2021. https://www.standardmethods.org/doi/10.2105/SMWW.2882.011

5. ASTM-2018, ASTM D1193-06(2018), Standard Specification for Reagent Water, ASTM

International, West Conshohocken, PA, 2018, www.astm.org.

Method 1621 28 January 2024

 6. ASTM-2018. ASTM D3370-18, “Standard Practice for Sampling Water from Flowing Process
Streams,” ASTM International, West Conshohocken, PA, 2018, www.astm.org.

7. Willey, J., R. Anderson, A. Hanley, M. Mills, C. Hamilton, T. Thompson, and A. Leeson. 2021.
Report on the Single-Laboratory Validation of PFAS by Isotope Dilution LC-MS/MS. Strategic
Environmental Research and Development Program (SERDP) Project ER19-1409.

8. “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” USEPA

EMSL, Cincinnati, OH 45268, EPA 600/4-79-019, April 1979.

9. ASTM-2021. ASTM D-8294-21, Standard Practice for Estimating pH to Verify Status of

Aqueous Samples, ASTM International, West Conshohocken, PA, 2018, www.astm.org.

10. “Less is Better: Laboratory Chemical Management for Waste Reduction,” American Chemical
Society, 1993. Available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.

11. “Environmental Management Guide for Small Laboratories,” USEPA, Small Business
Division, Washington DC, EPA 233-B-00-001, May 2000.

12. “The Waste Management Manual for Laboratory Personnel,” American Chemical Society,
1990. Available from the American Chemical Society's Department of Government Relations
and Science Policy, 1155 16th Street NW, Washington, DC 20036.

13. “Report on the Single-laboratory Validation of Clean Water Act Method 1621 for Adsorbable
Organic Fluoride (AOF),” USEPA, Office of Science and Technology, Washington DC,
EPA-820-R-22-003, April 2022.

14. “Multi-laboratory Validation Study Report for Method 1621: Determination of Adsorbable
Organic Fluoride (AOF) in Aqueous Matrices by Combustion Ion Chromatography (CIC)”,
USEPA, Office of Science and Technology, Washington DC, EPA-821-R-24-003, January
2024.

Method 1621 29 January 2024

19.0 Tables, Diagrams, Flowcharts, and Validation Data

Table 1. Example Retention Time and Pooled MDL and ML Values

Organofluorine Fortified Pooled MDL ML
Analyte Retention Time (min)a Conc. (µg F-/L)b (µg F-/L)c (µg F-/L)d
Organofluorine 5.66 5.0 1.5 5.0
a Retention time based on Metrohm Metrosep A Supp 5 column and isocratic carbonate-bicarbonate elution and
provided as an example. Other column and elution reagents will give different retention times.
b Fluoride concentration used to determine MDLs. PFHxS used as source of organofluorine.

c Data for this table are derived from the multi-laboratory validation study using data from 10 laboratories. The pooled

MDL is an estimate of the sensitivity that should be achievable in a well-prepared laboratory. The actual MDL
generated in each laboratory should be used for data reporting and data quality assessments.
d The Minimum Level (ML) in this table is derived as a multiple of the pooled MDL value and other derivations may

apply. The actual ML generated in each laboratory should be used for data reporting and data quality assessments.

Table 2. Example Calibration Standard Solutions

Calibration Standards (µg F-/ L)
Analyte CS1 CS2 CS3 CS4 CS5 CS6 CS7
Fluoride 2.0 5.0 10 25 50 75 100
Other concentrations may be used and the calibration range may be extended as long as the RSE requirements are met.

Table 3. IPR and OPR QC Acceptance Limits

IPR
Mean OPR
Compound Recovery (%) RSD (%) Recovery (%)
PFHxS 80 – 120 20 70 - 130
Data for this table are derived from the multi-laboratory validation study and are
therefore the limits required for this method.

Method 1621 30 January 2024

 Table 4. Example Performance Data from the MLVS for MS/MSD Samples Spiked with
PFHxS1
# of Nominal Spike Recovery (%)
Sample Description Results Conc (µg F-/L) Mean Min Max RSD2 (%)
18 30 92.4 74.9 108.0 12.4
POTW Effluent 1
18 60 96.9 89.1 110.8 5.8
18 10 102.8 45.2 181.5 35.8
Dairy Effluent
18 30 95.7 70.0 113.7 11.2
18 30 85.7 72.8 99.8 9.5
Hospital Effluent
18 60 83.8 56.3 101.2 14.7
18 30 95.7 55.4 139.5 21.5
Metal Finisher Effluent
18 60 93.0 78.0 103.5 7.1
18 30 98.1 70.6 113.8 10.3
POTW Effluent 2
18 60 99.5 92.3 109.1 5.6
16 10 101.6 46.1 175.4 32.6
Bus Washing Station Effluent
18 30 94.4 63.2 112.3 15.3
18 10 101.3 71.9 146.5 15.8
Pharmaceutical Effluent
18 30 97.7 85.6 107.5 7.5
18 10 99.1 82.4 123.6 9.3
Other Industrial Effluent
17 30 99.7 91.2 114.2 5.7
18 30 97.0 79.9 122.5 10.7
POTW Effluent 3
18 60 98.4 85.4 117.2 8.2
1 Results are from the multi-laboratory validation study involving nine laboratories (see Reference 14) and are only
provided as examples for this method
2 Relative standard deviation of the recoveries from all MS/MSD samples across all laboratories

Table 5. Summary of Quality Control

Method Reference Requirement Specification and Frequency
Section 10.3 Initial Calibration (ICAL) Minimum 5 calibration standards for linear model
and 6 calibration standards for non-linear models.
Sections 10.2.2, 14.1 Retention Time (RT) window After ICAL and at the beginning of analytical
confirmation sequence
Section 10.4, 13.1 Calibration Verification (CV) At the beginning (low level) and every 10 samples
(mid-level)
Sections 9.1.3, 9.3, 13.3 Method Blank One per preparation batch
Section 13.4 Ongoing Precision and Recovery One per preparation batch
(OPR)
Sections 9.7, 11.3 Matrix Spike (MS/MSD) One set per preparation batch

Method 1621 31 January 2024

20.0 Figures

Chloride
Figure 1. Example of Ion Chromatograph for a Sample

Method 1621 32 January 2024

21.0 Glossary

These definitions and purposes are specific to this method but have been conformed to common usage to
the extent possible.

21.1 Units of weight and measure and their abbreviations

21.1.1 Symbols

ºC degrees Celsius
µg microgram
µL microliter
µm micrometer
< less than
≤ less than or equal
> greater than
≥ greater than or equal
% percent
± plus or minus

21.1.2 Alphabetical abbreviations

cm centimeter
g gram
L liter
M molar
mM millimolar
mg milligram
min minute
mL milliliter
mm millimeter
µg microgram
w/w percent weight by weight

21.2 Definitions and acronyms (in alphabetical order)

Adsorption batch – A set of up to 20 field samples and batch QC adsorbed onto GAC during the
same period of time (e.g., 8-hour shift) using the same lot of GAC, reagents, and standards.

Analysis batch – A set of samples analyzed on the CIC during a 24-hour period that is bracketed
by the appropriate method blank and CVs.

Adsorbable Organic Fluoride (AOF) – an aggregate measure of per- and polyfluoroalkyl

substances (PFAS) and non-PFAS fluorinated organic compounds (such as pesticides and
pharmaceuticals) that can be adsorbed from an aqueous sample on granular activated carbon and
determined by combustion ion chromatography. Because of the common occurrence of background
levels of fluoride in the sorption media, the method subtracts the amount of fluoride observed in the
method blank from the fluoride in the sample to estimate the AOF attributable to the sample itself.
AOF results are reported as the concentration of fluoride ion (F-) in the sample.

Calibration standard (CS) – A solution prepared from a primary standard and/or stock solutions
and used to calibrate the response of the instrument.

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 Calibration Verification standard (CV) – A standard prepared from a secondary source of
standard that is used to verify calibration. The method requires the analysis of low-level (e.g. CS-1)
and mid-level (e.g. CS-6) standards (See Table 2).

CFR – Code of Federal Regulations

CIC – Combustion ion chromatography

CWA – Clean Water Act

GAC – Granular activated carbon

IC – Ion chromatograph or ion chromatography

Initial Precision and Recovery (IPR) – four aliquots of a reference matrix spiked with the
analytes of interest and analyzed to establish the ability of the laboratory to generate acceptable
precision and recovery. An IPR is performed prior to the first time this method is used and any
time the method or instrumentation is modified.

Limit of Quantitation (LOQ) – The smallest concentration that produces a quantitative result with
known and recorded precision and bias. The LOQ shall be set at or above the concentration of the
lowest initial calibration standard (the lowest calibration standard must fall within the linear range).

Matrix Spike (MS) and Matrix Spike Duplicate (MSD) – A set of field samples to which a
known quantity of organofluorine compound is added in the laboratory prior to analysis. The
MS/MSD are processed in the same way as the regular samples to determine the contribution of
matrix interference and bias to the analytical method.

Method blank – An aliquot of reagent water that is treated exactly as a sample including exposure
to all glassware, equipment, solvents, and reagents that are used with samples. The method blank is
used to determine if analytes or interferences are present in the laboratory environment, the
reagents, or the apparatus.

Method-defined parameter (MDP) – A parameter defined solely by the method used to determine
the analyte.

Method Detection Limit (MDL) – The minimum measured concentration of a substance that can
be reported with 99% confidence that the measured analyte concentration is distinguishable from
method blank results (40 CFR 136, Appendix B).

Minimum level of quantitation (ML) – The lowest level at which the entire analytical system
must give a recognizable signal and acceptable calibration point for the analyte. The ML represents
the lowest concentration at which an analyte can be measured with a known level of confidence. It
may be equivalent to the concentration of the lowest calibration standard, assuming that all method-
specified sample weights, volumes, and cleanup procedures have been employed. Alternatively, the
ML may be established by multiplying the MDL (pooled or unpooled, as appropriate) by 3.18 and
rounding the result to the number nearest to 1, 2, or 5 x 10n, where n is zero or an integer (see 68
FR 11770).

MESA – Mining Enforcement and Safety Administration

Must – This action, activity, or procedural step is required.

Method 1621 34 January 2024

 NIOSH – The National Institute of Occupational Safety and Health

Ongoing Precision and Recovery (OPR) – a method blank spiked with known quantities of
analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
produced by the laboratory remain within the limits specified in this method for precision and
recovery.

Per- and Polyfluoroalkyl substances (PFAS) – A group of man-made fluorinated compounds that
are hydrophobic and lipophobic, manufactured and used in a variety of industries globally. These
compounds are persistent in the environment and have been found in animals, plants, as well as in
the human body.

Reagent water – Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.

Relative Standard Deviation (RSD) – The standard deviation multiplied by 100 and divided by
the mean.

Relative Standard Error (RSE) – The standard error of the mean divided by the mean and
multiplied by 100.

Retention time (RT) – the time it takes for an analyte to elute off the column

Should – This action, activity, or procedural step is suggested but not required.

Stock solution – A solution containing an analyte that is prepared using a reference material
traceable to EPA, NIST, or a source that will attest to the purity and authenticity of the reference
material.

Method 1621 35 January 2024