Oil and Grease, SPE, 10300

Oil and Grease

USEPA1 Solid Phase Extraction Method
5 to 1000 mg/L HEM and SGT-HEM

DOC316.53.01186
Method 10300
Hexane Extractable Gravimetry

Scope and Application: For water and wastewater

1

Equivalent to USEPA Method 1664A, Solid Phase Extraction (SPE)

Test preparation

Before starting the test:

Follow the instructions for sample collection and acidification in Sample collection, preservation and storage.
Assemble the Xenosep SPE apparatus as shown in Figure 14. Be sure to put the waste collection tube in the large flask
before installing the funnel assembly. Put the pattern side of the SPE filter down in the SPE filter support (Figure 15).
Wash all glassware in hot water with detergent, rinse with tap and distilled water and rinse with acetone or n-hexane.
Rinse all glassware thoroughly with hexane to make sure the analyte is not retained on the apparatus. Inadequate rinsing will
result in low recovery.
Determine a blank value (1 liter of distilled or deionized water) with each new lot of reagents. If the blank result is greater
than 5 mg, resolve the source of error or remove interferences before performing this procedure.
If analyzing for SGT-HEM, rinse the inner cavity of the aluminum dish with a few milliliters of acetone and then with a few
milliliters of hexane to remove any potential artifacts.
Label the aluminum dish and heat in an oven for one hour at 105 C. Cool the dish in a dessicator for 30 minutes. Weigh the
dish on an analytical balance to the nearest 0.1 mg. Record this weight as "B" for HEM step 22 or SGT-HEM step 12.
Make sure that the aluminum dish is removed from the hot plate before the solution has completely evaporated. Heating the
solution too long will result in low recovery.
Use a vacuum pump that can generate a Free Air Flow of at least 1 CFM (3 CFM preferred). Insufficient SPE disk drying will
result in low recovery.

Collect the following items :

Description
Hydrochloric acid, 1:1

Quantity
6 mL

Hexane (n-hexane) in Teflon-FEP wash bottle

1 wash bottle

Methanol in PE wash bottle

1 wash bottle

Deionized water in PE wash bottle

1 wash bottle

SPE Starter Kit, EPA Method 1664A

1 kit

SPE Consumables Kit

1 kit

SPE Solvent Recovery Kit

1 kit

Aluminum weighing dish

Hot plate

Vacuum pump

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Collect the following items (continued):
Description

Quantity

pH paper, 014 pH units

Lab stand

Clamp, swivel

Desiccator

For SGT-HEM:
Silica gel

1 bottle

125-mL Erlenmeyer flask

100-mL volumetric flask (for HEM results over 1000 mg/L)

Magnetic stir plate

Aluminum weighing dish

Additional sodium sulfate column

See Consumables and replacement items for reorder information.

Figure 14 Solid phase extraction assembly

1.

Hot plate

7.

Eluter tube

2.

Aluminum dish

8.

Clamp, tubing

3.

Glass dome

9.

3-way valve

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4.

Solvent recovery assembly

10. 2-way valve

5.

Funnel assembly (see Figure 15)

11. To vacuum

6.

Sodium sulfate column

12. Waste collection tube with O-Ring

1.

Aluminum clamp

4.

SPE filterpattern side down

2.

Funnel

5.

Stainless steel support

3.

Coupler with O-Rings

6.

SPE starter holder

Figure 15 Funnel assembly

Solid phase extraction for HEM

1. Add approximately
10 mL of hexane to the
funnel. Wait 5 seconds.
Turn the vacuum on and
off to pull the solvent into
the waste collection tube.
Repeat with a second
10-mL portion of hexane.

2. Turn the vacuum on

for 1 minute to dry the
filter. Turn off the vacuum.

3. Add approximately
10 mL of methanol to the
funnel. Wait 5 seconds.
Turn the vacuum on and
off to pull the solvent into
the waste collection tube.

4. Remove the tube and

discard the solvent waste.
Refer to the MSDS and
local regulations for safe
disposal of the solvent.

Do not allow the filter to

become dry.

Make sure the valves are

set to pull vacuum from
the funnel holder and the
tubing clamp is open.

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Solid phase extraction for HEM (continued)

5. Add approximately
20 mL of deionized water
to the funnel. Wait 5
seconds. Turn the vacuum
on and off to pull the water
into the flask.
Do not allow the filter to
become dry.
If the filter becomes dry,
repeat steps 35.

9. Turn the valve to apply

vacuum to the eluter tube.
Close the tubing clamp on
the funnel holder tube.

6. Slowly pour the

acidified sample into the
funnel and turn on the
vacuum. Use deionized
water to rinse any debris
from the walls of the
funnel.

7. Leave the vacuum on

for 4 to 8 minutes to air dry
the filter. Turn off the
vacuum.
Do not leave the vacuum
on for more than
8 minutes.

See Sample collection,

preservation and storage
for sample acidification.

10. Add 10 mL of hexane

to the empty sample
bottle. Shake the bottle in
a horizontal, circular
motion for 10 seconds to
rinse the bottle.

11. Use a transfer pipet to

collect the hexane from
the shoulder of the sample
bottle. Slowly rinse the
walls of the funnel with the
hexane. Go around the
funnel at least 3 times.
Repeat steps 1011 two
more times.

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8. Put the funnel

assembly on the eluter
tube.

12. Turn the vacuum on

and off to pull the solvent
into the flat-sided flask.
Rinse the walls of the
funnel with approximately
10 mL of hexane. Wait 5
seconds. Turn the vacuum
on and off.

Oil and Grease

Solid phase extraction for HEM (continued)

13. Remove the flat-sided

flask from the eluter tube.

14. Pour the hexane into

a pre-weighed aluminum
dish.

15. Rinse the flat-sided

flask with approximately
5 mL of hexane. Add the
hexane to the dish.

16. Put the dish on the hot

plate and place the glass
cover over the dish.

17. Close the 3-way valve

and open the 2-way valve
to pull vacuum from the
solvent recovery
apparatus.

18. Turn on the vacuum.

Turn the hot plate on low
heat (3585 C). Heat for
approximately 2 minutes.

19. Examine the dish for

dry spots. As soon as
there is a dry spot remove
the dish from the hot plate.
Put the dish in a fume
hood until the rest of the
hexane has evaporated.

20. When the dish is dry,

put the dish in a desiccator
for 30 minutes.

21. After 30 minutes,

weigh the dish to the
nearest 0.1 mg. Repeat
steps 20 and 21 until the
weight loss is less than
0.5 mg from the previous
weight. Record the weight.

22. Calculate the test

results:

Example:

AB
---------------------------------------- 1000
sample volume

B= 6.2318 g

A= weight of dish with

residue (g) (step 21)
B= weight of dish (g)

A= 6.2394 g
sample volume = 0.950 L
6.2394 g 6.2318 g
----------------------------------------------------- 1000 =
0.950 L
8.0 mg/L HEM

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Solid phase extraction for SGT-HEM (HEM < 1000 mg/L)

1. Add hexane to the

residue in the aluminum
dish from the HEM test
until the dish is
approximately half full
(approximately 60 mL).

2. Heat slightly to
dissolve all of the residue.

3. Pour the mixture into a

125-mL Erlenmeyer flask.
Rinse the pan and funnel
several times with hexane
and add to the flask. Add
hexane to approximately
the 100-mL mark.

4. Add 3 ( 0.3) g of
silica gel for every 100 mg
of HEM or fraction thereof:
3 mg/L HEM
-------------------------------------- =
100
silica gel (g)
Example: if the HEM value
was 735 mg, round 7.35 g up
to the next whole gram
increment (8 g or 800 mg)
and add 3 x 800/100= 24 g of
silica gel.

Do not add more than 30 g

silica gel.

5. Add a stir bar to the

flask and stir for 5 minutes.

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6. Put a new sodium

sulfate column into the
eluter tube. Put the funnel
on the eluter tube.

7. Install a clean
flat-sided flask.

8. Adjust the valves to

pull vacuum from the
eluter tube.

Oil and Grease

Solid phase extraction for SGT-HEM (HEM < 1000 mg/L) (continued)

Go to the HEM
procedure.

9. Pour the solution from

the Erlenmeyer flask into
the funnel. Turn the
vacuum on and off to pull
the solution into the flatsided flask.

10. Rinse the Erlenmeyer

flask with 5 mL hexane.
Add the hexane to the
funnel. Turn the vacuum
on and off.

11. Complete steps 1321

under Solid phase
extraction for HEM to
evaporate the solvent.

12. Calculate the test

results:
AB
---------------------------------------- 1000
sample volume

A= weight of dish with

residue (g) (step 21)

Rinse the walls of the

funnel with approximately
5 mL of hexane. Turn the
vacuum on and off.

B= weight of dish (g)

Example:
A= 6.2360 g
B= 6.2320 g
sample volume = 0.950 L
6.2360 g 6.2320 g
----------------------------------------------------- 1000 =
0.950 L
4.2 mg/L SGT-HEM
Report result as 5 mg/L
SGT-HEM

Solid phase extraction for SGT-HEM (HEM > 1000 mg/L)

1. Add hexane to the

residue in the aluminum
dish from the HEM test
until the dish is
approximately half full
(approximately 60 mL).
Heat slightly to dissolve all
of the residue.

2. Pour the mixture into a

100-mL volumetric flask.
Rinse the pan and funnel
several times with hexane
and add to the flask.

3. Dilute to the mark with

hexane and mix well.

4. Calculate the volume

to remove for the silica gel
treatment:
100,000
V 1000 = ----------------------------HEM value

V1000 = volume that

contains 1000 mg HEM

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Solid phase extraction for SGT-HEM (HEM > 1000 mg/L) (continued)

Go to the SGT-HEM
(HEM < 1000 mg/L)
procedure.

5. Remove the amount

calculated in step 4 from
the volumetric flask and
add it to a 125-mL
Erlenmeyer flask. Add
hexane to approximately
the 100-mL mark.

6. Add 30 g of silica gel

to the flask.
If the volume added in
step 5 contained less than
1000 mg HEM, calculate
the amount of silica gel to
add based on the mg HEM
that was added to the
flask.
3 mg HEM in aliquot
---------------------------------------------------------- =
100
silica gel (g)

7. Complete steps 512

in the procedure for Solid
phase extraction for SGTHEM (HEM < 1000 mg/L).

8. Correct the test result

for the reduced volume
that was treated with the
silica gel:
100
W d -------------- = W c
V 1000

where:
Wd = result from step 12
V1000 = volume removed
for silica gel treatment
Wc = corrected SGT-HEM
result

Interferences
For samples that are known to contain extremely high levels of oil and grease, use a smaller
sample volume and correct for the volume difference to give the result as a 1-L sample.
High concentrations of particulates in the water sample can clog the SPE filter or retain high levels
of water, which can lower the extraction efficiency. Inorganic particulates are easier to filter than
organic particulates. The following techniques may help to filter samples containing high levels of
particulates:

Decantingallow the sample to settle and pour the top portion into the funnel first. Just before
dryness, add the rest of the sample. Remove any sediment from the bottle and add it to the
SPE filter. Use deionized water to rinse any sediment that remains in the bottle.

Prefilters or prefilter fibersplace the prefilter or prefilter fibers into the coupler before the
funnel is attached.

Drying agentsadd magnesium sulfate to the SPE filter or to the prefilter to remove water that
may be trapped in the particulates.

Filtration aidsadd materials such as sodium chloride, sand, diatomaceous earth or glass
beads to help speed the complete filtration of samples that contain organic particulates.

This method is not applicable to materials that volatilize at temperatures below approximately
85 C (185 F). Petroleum fuels from gasoline through #2 fuel oil may be partially lost in the solvent
removal operation. Some crude oils and heavy fuel oils contain a significant percentage of
materials that are not soluble in n-hexane. Recoveries of these materials may be low.

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Sample collection, preservation and storage

Collect 1 L (9501050 mL) of sample in a wide-mouth glass bottle. Do not rinse the bottle with
sample before collection. The sample must be at room temperature before the test.
Sample volume
Complete the steps that follow to measure the sample volume.
6. Use a laboratory pen to mark on the sample bottle the liquid level of the sample.
7. When the test is complete, fill the bottle to this mark with tap water.
8. Pour the tap water into a 1-L graduated cylinder and write down the volume. Use this volume
for the sample volume in the final step of the test procedure for HEM or SGT-HEM.
Acidification
The sample must be acidified with 1:1 hydrochloric acid (HCl) solution to a pH of 2 or less before
the test is started. To find the volume of HCl to use, collect a separate aliquot, add HCl until the pH
is less than 2 and add this volume of acid to each sample bottle before collection. Do not dip pH
paper, a pH electrode, a glass rod or other materials into the sample because oil and grease in the
sample may adhere to these items.
If the test can not be started for more than a few hours after collection, cool and store the sample
at 04 C (3239 F). Acidified samples can be stored for 28 days.

Accuracy check
For USEPA reporting, each laboratory must first measure an acceptable MDL (Minimum Detection
Limit) and IPR (Initial Precision and Recovery) before samples can be measured. Before
measuring the MDL or IPR, run laboratory reagent water blanks to eliminate interferences.
Standard solution preparation
Required items:

Stearic acid, 98% minimum

Hexadecane, 98% minimum

Acetone for Organic Residue Analysis, residue less than 1 mg/L

100-mL Class A volumetric flask

10.0-mL Class A volumetric pipet

15.0-mL Class A volumetric pipet

1. Put 200 ( 2) mg stearic acid and 200 ( 2) mg hexadecane into a 100-mL volumetric flask.
2. Add 7585 mL of acetone and shake vigorously until all material has dissolved.
3. Allow to equilibrate to room temperature and fill to volume with acetone. Mix well. The
concentration of this stock solution is 4000 mg/L HEM (2000 mg/L SGT-HEM).
4. Use a volumetric pipet to dilute the stock solution for use in MDL, IPR or OPR measurements:
MDL standard solution:
a. Add 15 mL of the stock solution into a clean 100-mL volumetric flask. Dilute to the mark
with acetone and mix well.
b. Pipet 10 mL for HEM (or 20 mL for SGT-HEM) into a 1-L volumetric flask. Dilute to the
mark with deionized water at pH < 2 and mix well. The concentration of this standard
solution is 6 mg/L HEM (or 6 mg/L SGT-HEM).

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IPR or OPR standard solution:
Add 10 mL of the stock solution into 1 liter of deionized water and mix well. The concentration
of this solution is 40 mg/L HEM (20 mg/L SGT-HEM).
Note: To verify the concentration of the IPR/OPR standard, use a pipet to add 10 mL of the standard
solution into a pre-weighed flask or dish. Allow the acetone to evaporate in a fume hood. When dry,
weigh the flask or dish. The weight of the residue should be 40 (1) mg.

EPA requirements for MDL and IPR

MDL: measure seven replicates of a 6 mg/L standard solution, find the standard deviation and
multiply the standard deviation by 3.143 (Students t test). The acceptable limits are:

HEM: 1.4 mg/L

SGT-HEM: 1.6 mg/L

IPR: follow the procedure for HEM and SGT-HEM (if necessary) four separate times using a
40 mg/L HEM (20 mg/L SGT-HEM) standard solution. Report the average percent recovery (x)
and the standard deviation for both HEM and SGT-HEM. The acceptable limits are:

HEM: Precision(s) 11%; Recovery (x) 83101%

SGT-HEM: Precision(s) 28%; Recovery (x) 83116%

A low result can be an indication of loss during quantitative transfers or loss during heating. A high
result can be an indication of incomplete drying or contamination. Run a blank before running
samples. If the blank result is more than 5 mg, identify and remove the source of contamination.
After acceptable values are obtained for the MDL and IPR, keep records for USEPA verification.

Oil and grease reporting to USEPA

Include the following data with the HEM and/or SGT-HEM results for each set of up to 20 samples
per discharge source.
1. Blank value: must be less than 5.0 mg/L for HEM and SGT-HEM.
Note: It may be necessary to use a standard that matches the regulatory concentration limit, is 1 to 5
times higher than the concentration of the sample (B) or is at the concentration of the Ongoing
Precision and Recovery, whichever is highest. Divide the concentration of the spike (T) by 2 for
SGT-HEM if using the 40 mg/L HEM (20 mg/L SGT-HEM) standard.

2. OPR (Ongoing Precision and Recovery): add 10 mL of the 40 mg/L HEM (20 mg/L SGTHEM) standard to a 1-liter sample and run the test. The acceptable limits for recovery are:

HEM: 78114%

SGT-HEM: 64132%

3. MS and MSD (matrix spike and matrix spike duplicate): measure the HEM and SGT-HEM
concentration of the sample (B). Spike two 1-L samples with 10 mL of the 40 mg/L HEM
(20 mg/L SGT-HEM) standard and measure the concentration after spiking (A).
Calculate the Percent Recovery (P) as follows:
100 ( A B )
P HEM (40 mg/L) = ----------------------------------T
100 ( A B )
P SGT HEM = ----------------------------------T2

where:
A = concentration of the unspiked sample
B = concentration of the spiked sample
T = concentration of the spike solution
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If the recovery for HEM and SGT-HEM is within the acceptable limits for OPR (step 2), then
calculate the Relative Percent Difference (RPD).
Conc MS Conc MSD
RPD = ---------------------------------------------------- 200
Conc MS + Conc MSD

If the RPD for HEM is 18 and for SGT-HEM 34, then continue with step 4. If the recovery is
lower than the RPD, an interference may be present. Identify and correct the interference,
then repeat MS and MSD.
After every five MS/MSD tests, calculate the average percent recovery (Pa) and standard
deviation of the percent recovery (sp). Record these numbers as Pa 2sp. Update the
accuracy assessment on a regular basis (e.g., after five to ten new accuracy measurments).
4. Balance calibration: measure a 2 mg and a 1000 mg class S weight on the analytical
balance before and after each analytical batch. If the values are not within 10% of the actual
weight, recalibrate the balance.

Summary of method
Oil and Grease & Total Petroleum Hydrocarbons (TPH) include any material that may be
recovered as a substance that is soluble in the n-hexane extractant. These include substances
such as relatively non-volatile hydrocarbons, vegetable oils, animal fats, waxes, soaps, greases
and related materials. When measuring oil and grease (HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then the n-hexane is evaporated. The residue left is
weighed to determine the concentration of oil and grease materials in mg/L.
When measuring Total Petroleum Hydrocarbons (SGT-HEM) gravimetrically, the substances are
extracted from the sample with n-hexane, then mixed with silica gel to absorb non-TPH
components. Then the n-hexane is evaporated. Like the HEM, the residue left is weighed to
determine the concentration of total petroleum hydrocarbons.
Definition of HEM and SGT-HEM
Oil and grease is the conventional term used to define pollutants of this nature. The newer term
n-Hexane Extractable Materials (HEM) indicates this method may be applied to materials other
than oils and greases.
Likewise, the term Total Petroleum Hydrocarbons (TPH) was traditionally used to classify aliphatic
hydrocarbon materials. The newer term Silica Gel Treated n-Hexane Extractable Material
(SGT-HEM), indicates that this method may be applied to materials other than aliphatic petroleum
hydrocarbons that are not adsorbed by silica gel.

Consumables and replacement items

Required reagents
Description

Quantity/Test

Unit

6 mL

500 mL

88449

100200 mL

1L

2510253

Methanol, ACS grade

10 mL

500 mL

1446449

Silica Gel, 100200 mesh (for SGT-HEM)

130 g

500 g

2665034

Hydrochloric Acid Standard Solution, 1:1 (6 N)

Hexane, for Organic Residue Analysis

Catalog number

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Required apparatus
Description

Quantity/Test

Unit

Catalog number

Balance, Analytical, 115 VAC 60 Hz.

each

2936801

Bottle, wash, 500-mL Teflon, FEP

each

2505100

Bottle, wash, 500-mL

3/pkg

2927204

Bottle, wide-mouth, 1-L

each

2951401

Clamp, swivel

each

2503400

Cylinder, graduated, 1-L

each

108153

Desiccator

each

2088800
1428400

Desiccator Plate

each

Flask, Erlenmeyer, 125-mL (for SGT-HEM)

each

50543

Hot Plate (Thermolyne), 120 VAC, 50 Hz

each

2344100

Marker, laboratory

each

2092000

Oven, drying, 120 VAC, 50 Hz

each

1428900

Pump, vacuum, 27 in. Hg, 1.3 CFM

each

2824800

SPE Consumables Kit

24/pkg

2943800

SPE Starter Kit, EPA Method 1664A

each

2943231

SPE Solvent Recovery Kit

each

2514300

Stirrer, magnetic, 120 VAC

each

2343600

Stir Bar, 22.2 x 7.9 mm

each

2095350

Support stand

each

2504900

Tongs, crucible, 9-inch

each

56900

Unit

Catalog number

Recommended standards
Description
Hexadecane, 99%
Stearic acid

100 mL

2664842

500 g

2664934

Unit

Catalog number

Optional reagents and apparatus

Description
Acetone, for Organic Residue Analysis
Flask, volumetric, Class A, 100-mL
Flask, volumetric, Class A, 1-L
pH Paper, 014 pH units

1L

2510153

each

1457442

each

1457453

100/pkg

2601300

Pipet filler, safety bulb

each

1465100

Pipet, serological, 10-mL

each

53238

Pipet, volumetric, Class A, 10 mL

each

1451538

Pipet, volumetric, Class A, 15 mL

each

1451539

Silica Gel with indicator (for desiccator)

454 g

1426901

FOR TECHNICAL ASSISTANCE, PRICE INFORMATION AND ORDERING:

In the U.S.A. Call toll-free 800-227-4224
Outside the U.S.A. Contact the HACH office or distributor serving you.
On the Worldwide Web www.hach.com; E-mail techhelp@hach.com

Hach Company, 2007, 2010, 2012. All rights reserved. Printed in the U.S.A.

HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932

Edition 7