EPA Method 1664 Oil & Grease Analysis: Cutting n-Hexane Use by 40% with the Empore™ O&G SPE Disk
- Jul 30
- 10 min read
Authors: Michael Apsokardu, Xiaohui Zhang, Guotao Lu
Study summary: CDS Empore™ Oil and Grease (O&G) Solid Phase Extraction disks facilitate reliable sample preparation and provide excellent analyte recovery. This application note demonstrates the performance of the disk under EPA Method 1664 while reducing the total volume of n-hexane by 40% — from 80 mL to 50 mL per sample. Combined recovery of hexadecane and stearic acid from 1 L water samples was 85% with 8.0% RSD at the reduced volume, compared with 89% at 0.4% RSD at the original volume. Method blank background averaged 0.5 mg, consistently less than 2% of the extracted analyte mass.
TL;DR: EPA Method 1664 measures oil and grease as n-hexane extractable material — and n-hexane is expensive to buy, difficult to dispose of through waste streams, and found at high concentrations in landfills. A simple modification to the wash and elution steps of the Empore™ O&G disk procedure drops total n-hexane consumption from 80 mL to 50 mL per sample. Recovery falls only from 89% to 85% — still well above the 80% method threshold — and method blanks stay under 2% of extracted mass, confirming that the shorter wash does not leave the disk dirtier.
Study at a Glance
Introduction: Why Laboratories Want Less n-Hexane
EPA Method 1664 was originally designed as a performance-based method for the recovery of hexadecane from water samples by liquid–liquid extraction. Critically, the original method permits the use of alternative methods — such as solid phase extraction (SPE) — as long as all performance specifications are met. EPA Method 1664 was originally adapted for SPE by 3M™, the original manufacturer of Empore™ membranes. In this SPE form, performance is measured through the combined recovery of both hexadecane and stearic acid.
Elution of analytes from SPE sorbents is effectively accomplished using n-hexane. n-Hexane, however, is difficult to dispose of through waste streams and is frequently found in high concentrations in landfills. For a laboratory running oil and grease on a routine basis, solvent volume is simultaneously a purchasing line item, a waste disposal line item, and an environmental exposure. It is therefore desirable to minimize the volume needed for elution — to reduce not only environmental impact but also experimental costs — provided that analyte recovery is not compromised.
That last clause is the question this application note answers.
The obvious objection, tested directly: Cutting the wash volume raises a fair concern — does a shorter n-hexane wash leave residue on the disk that inflates the gravimetric result? Because Method 1664 is a total mass measurement rather than a compound-specific one, any background carried through the procedure is counted as oil and grease. Method blanks were therefore run under the reduced-volume conditions to quantify exactly this risk (see Results).
In this application note, a 1 L water sample was passed through a 47 mm Empore™ O&G disk and eluted with hexane under negative pressure. The extract was then dried to determine the extracted mass of hexadecane and stearic acid, and the effect of reduced eluent volumes was assessed. Validation data was determined from three replicate measurements (n=3) on the same lot of Oil and Grease disks.
Headline result:
Total n-hexane reduced from 80 mL to 50 mL per sample — a 40% reduction
Combined recovery: 89% at 80 mL (0.4% RSD) → 85% at 50 mL (8.0% RSD)
Both conditions clear the method's >80% acceptance threshold
Method blank background consistently < 2% of extracted analyte mass
Why the Empore™ Oil and Grease Disk for EPA Method 1664
Oil and grease is among the highest-volume tests in a municipal or industrial wastewater laboratory, which makes the per-sample economics of the extraction step matter more than in most methods. The Empore™ O&G disk format addresses this directly:
Performance-based compliance — SPE is an accepted alternative to liquid–liquid extraction under Method 1664, and the SPE adaptation of the method was originally developed by 3M™ on Empore™ media
Validated at reduced solvent volume — 85% combined recovery of hexadecane and stearic acid using only 50 mL of n-hexane per sample
Low background — method blanks averaged 0.5 mg, under 2% of extracted mass, which matters because Method 1664 is a gravimetric total-mass measurement
Batch-compatible — an all-glass filtration assembly is used, and a manifold is acceptable for running multiple extractions in parallel
Handles the full 1 L sample — filtration proceeds as quickly as the vacuum will allow, with no sample splitting required
Materials
SPE Media
Empore™ Oil and Grease SPE Disk, 47 mm — one disk per extraction, mounted in an all-glass filtration assembly
Chemicals
Stearic acid — Chem Service (West Chester, PA)
Hexadecane — Sigma-Aldrich (St. Louis, MO)
n-Hexane, methanol, acetone — HPLC grade, VWR International (Radnor, PA)
Sulfuric acid — VWR International (Radnor, PA)
Reagent water — treated in house using a Milli-Q Water Treatment System
Anhydrous sodium sulfate — granular, used as eluate drying agent
Standard Preparation
A stock solution was prepared by dissolving 200 mg each of stearic acid and hexadecane in 100 mL of acetone. Each 1 L water sample was prepared by first adjusting the pH to at least pH 2 with concentrated sulfuric acid, then adding 10 mL of the stock solution.
Why acidify before spiking? Stearic acid is a free fatty acid. Bringing the sample to pH 2 first keeps it in its protonated, neutral form so that it is retained by the non-polar sorbent rather than remaining ionised in the aqueous phase. Acidification prior to extraction is standard practice across the HEM methods.
Analysis Platform
Gravimetric. No chromatographic instrumentation is required. The n-hexane is evaporated from the collection vessel until a constant weight is reached; the collection vial is weighed, the weight compared to the tared weight, and the quantity of HEM — the oil and grease residue — calculated in mg/L.
Validated EPA Method 1664 SPE Procedure
The full procedure as performed, using the reduced 50 mL total n-hexane volume:
SPE Procedure (11-Step Protocol)
Assemble an all-glass filtration assembly using a 47 mm Empore™ O&G SPE disk. (Use of a manifold for multiple extractions is acceptable.)
Wash the apparatus and disk: add 20 mL of n-hexane to the reservoir. Pull a small amount through the disk with vacuum, turn off the vacuum, and allow the disk to soak for about three minutes. Pull the remaining solvent through and allow the disk to dry for five minutes.
Condition the disk: add approximately 30 mL of methanol to the reservoir. Pull a small amount through, let it soak for about one minute, then pull most of the remaining methanol through, leaving 3–5 mm of methanol on the disk surface.
Equilibrate: add 50 mL of reagent water and pull most of it through under vacuum, again leaving 3–5 mm of water on the disk surface.
Sample loading: add the water sample to the reservoir and, under vacuum, filter as quickly as the vacuum will allow. Drain as much water from the sample bottle as possible. Dry for five minutes.
Remove the filter assembly and insert a suitable sample tube for eluate collection.
Elution Step 1 (bottle rinse): add 10 mL of n-hexane to the sample bottle. Rinse the bottle thoroughly and transfer the solvent to the disk and filtration reservoir, rinsing all sides in the process.
Pull half of the solvent through the disk, then release the vacuum. Allow the remaining solvent to soak the disk for three minutes, then draw the remainder through under vacuum.
Elution Step 2 (glassware rinse): using a disposable pipette, rinse down the sides of the filtration glassware with 10 mL of n-hexane.
Drying: dry the combined eluent with 5–10 g of granular anhydrous sodium sulfate. Rinse the collection tube and the sodium sulfate each with a 5 mL portion of n-hexane and place the combined solvent into a concentrator tube.
Concentration: dry the extract to 1 mL under a gentle stream of nitrogen. The tube may be warmed gently.
Where the 50 mL Goes
Analysis
Evaporate the n-hexane from the collection vessel until a constant weight is reached. Weigh the collection vial, compare the weight to the tared weight, and calculate the quantity of HEM (oil and grease residue) present in units of mg/L.
Results and Discussion
Table 1 shows the combined recovery data of stearic acid and hexadecane from EPA Method 1664 using both 80 mL and 50 mL of n-hexane during the extraction process. The 80 mL figure is the volume used by 3M™ in the previous application report on Empore™ O&G disks. Table 1 also reports the extracted background mass from method blanks, which were performed exactly as described in the procedure but without the addition of the Method 1664 analytes to the water sample.
Table 1 — Combined Recovered Mass of Hexadecane and Stearic Acid
Table 1. Combined recovered mass (mg) of hexadecane and stearic acid in EPA 1664. n = 3 per condition, single lot of Oil and Grease disks.
Overall Recovery Performance
Reducing the total n-hexane volume produced comparable recoveries.
80 mL condition — 89% average recovery, 0.4% RSD
50 mL condition — 85% average recovery, 8.0% RSD
Recoveries greater than 80% are acceptable under the method, so both conditions pass
The 40% solvent reduction costs approximately four percentage points of recovery
These results indicate that reducing the total volume of n-hexane during the extraction process produces similar recoveries. The wider spread at 50 mL (8.0% versus 0.4% RSD) is driven by the third replicate at 31.0 mg; the first two replicates, at 36.2 and 34.9 mg, bracket the 80 mL average. Laboratories adopting the reduced-volume procedure should expect somewhat greater run-to-run variability than the near-identical triplicates seen at 80 mL, and should confirm precision against their own QC limits.
Method Blanks: Does a Shorter Wash Leave the Disk Dirtier?
This is the central objection to cutting the wash volume, so it was measured rather than assumed. Method blanks were performed as described in the procedure but without the addition of the EPA Method 1664 analytes stearic acid and hexadecane to the water sample.
The average background mass was 0.5 mg — consistently less than 2% of the total extracted mass. Reducing the wash volume of n-hexane did not produce significant amounts of background impurities.
The relative standard deviation on the blanks appears high at 21.5%, but this reflects the very small absolute masses involved: the three replicates were 0.6, 0.6 and 0.4 mg, so a 0.2 mg difference near the limit of gravimetric resolution dominates the statistic. In absolute terms, the spread across all three blanks is smaller than the rounding increment on the spiked samples.
Conclusion
A simple modification was made to EPA Method 1664 to reduce the total volume of n-hexane used during the wash and elution steps of extracting hexadecane and stearic acid from Empore™ Oil and Grease disks. The total volume of n-hexane was reduced by 40% without having an appreciable effect on the recovery of stearic acid and hexadecane:
Percent recovery with the reduced volume of n-hexane was 85% with 8.0% RSD
Recovery at the original 80 mL volume was 89% with 0.4% RSD
The recovered background mass was consistently less than 2% of the extracted stearic acid and hexadecane mass
Both conditions exceed the method's >80% acceptance threshold
The results indicate that Empore™ Oil and Grease Solid Phase Extraction disks are suitable for high recoveries while also reducing the total volume of n-hexane used during the extraction process — lowering both solvent purchasing cost and hazardous waste disposal burden for laboratories running HEM analysis routinely.
Scope note: The validation data presented herein was determined from three replicate measurements on the same lot of Oil and Grease disks. MDLs were not determined as part of this validation. Laboratories should establish method detection limits and demonstrate initial precision and recovery in their own facility as required by the method.
Frequently Asked Questions
What is EPA Method 1664 used for?
EPA Method 1664 is the U.S. EPA's performance-based method for measuring oil and grease in water as n-hexane extractable material (HEM). It was originally designed around the recovery of hexadecane from water samples by liquid–liquid extraction, and it explicitly permits alternative extraction techniques such as solid phase extraction provided the method's performance specifications are met.
What does HEM mean in oil and grease analysis?
HEM stands for n-hexane extractable material. Rather than identifying individual compounds, EPA Method 1664 reports the total mass of material that partitions into n-hexane and remains after the solvent is evaporated to constant weight, expressed in mg/L.
Can SPE disks be used instead of liquid–liquid extraction for EPA Method 1664?
Yes. Method 1664 is performance-based, meaning alternative procedures such as solid phase extraction are permitted as long as all performance specifications are met. The SPE adaptation of Method 1664 was originally developed by 3M™, the original manufacturer of Empore™ membranes.
How much n-hexane does the reduced-volume procedure use?
50 mL of n-hexane in total per sample: 20 mL to wash the disk and apparatus, 10 mL to rinse the sample bottle, 10 mL to rinse the filtration glassware, and two 5 mL portions to rinse the collection tube and the sodium sulfate. The original procedure used 80 mL.
Does reducing the n-hexane volume lower recovery?
Only marginally. Average combined recovery of hexadecane and stearic acid was 89% using 80 mL of n-hexane and 85% using 50 mL, based on three replicates each. Both results exceed the 80% acceptance threshold, so the 40% solvent reduction costs approximately four percentage points of recovery.
Does using less wash solvent increase background contamination?
No. Method blanks run with the reduced 50 mL volume gave an average background mass of 0.5 mg, consistently less than 2% of the total extracted analyte mass. Reducing the wash volume of n-hexane did not produce significant amounts of background impurity.
Why are both hexadecane and stearic acid used to validate the method?
The two analytes probe different retention behaviour. Hexadecane is a non-polar straight-chain hydrocarbon and stearic acid is a long-chain fatty acid, so a combined recovery figure demonstrates that the disk handles both the hydrocarbon and the polar-headgroup components typical of real oil and grease samples. Method 1664 performance for SPE is measured through this combined recovery.
Why must the water sample be acidified to pH 2?
Acidification converts free fatty acids such as stearic acid to their neutral, protonated form so they are retained on the non-polar sorbent rather than remaining ionised and passing through with the aqueous sample. In this study the pH was adjusted to at least pH 2 with concentrated sulfuric acid before the analytes were added.
References
3M. EPA Method 1664: N-Hexane Extractable Material Quantification; 2009.
U.S. Environmental Protection Agency. Method 1664B: N-Hexane Extractable Material and Silica Gel Treated n-Hexane Extractable Material by Extraction and Gravimetry; 2010.
U.S. Environmental Protection Agency. Air Emissions from Municipal Solid Waste Landfills — Background Information for Final Standards and Guidelines; 1991.
U.S. Environmental Protection Agency. Characterization of Municipal Solid Waste by Weight; 1992.
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