CAPE Technologies
High Performance Dioxin/Furan
Immunoassay Kit Application Note AN-008
Rapid Extraction, Cleanup, and
EIA Analysis of Soils at
Low to Mid-pg/g Levels
Illustrated Guide to Lab Setup, Sample
Preparation, and EIA Protocol
Revision March 31, 2005
This is a training presentation only.
It is designed to illustrate the materials, supplies, and
procedures involved in the modification of EPA Method 4025
described in CAPE Technologies Application Note AN-008. It
does not provide comprehensive protocol information. For
detailed information on equipment, supplies, and protocol,
refer to the appropriate CAPE Technologies document:
AN-008 for sample preparation protocol
IN-DF1 for immunoassay kit protocol
EL-001 for equipment used for immunoassay
EL-002 for equipment used for sample preparation
The following pages contain references to specific sections
of these 4 documents so you can connect the illustration to
the detailed information from that document.
Facilities Required
Performing this method using the CAPE Technologies DF1
Immunoassay Kit requires a modest laboratory facility with
capacity for 4-8°C storage of EIA kits.
This facility can be a small mobile or stationary field
laboratory.
Electrical refrigeration, a sink, and running water are helpful,
but not absolutely required.
A fume hood is required for the sample preparation work,
which uses hexane, acetone, and toluene.
Equipment Required for Sample Preparation
(not available from CAPE Technologies; see EL-002)
tabletop
centrifuge with
buckets for 40
mL vials and
orbital platform
16 x 125 mm
or similar shaker
tubes
for extraction
vortex
mixer
0.1 g
balance
Equipment Required for Sample Preparation
(not available from CAPE Technologies; see EL-002)
small chemical
fume hood
sample evaporation system using
clean nitrogen or compressed air
Equipment Required for EIA
(not available from CAPE Technologies; see EL-001)
positive displacement
micropipettors
(adjustable 20 to 100 µL
essential; 1-10 µL very
helpful)
Repeater pipettor
Differential
Photometer
Supplies Required for Sample Preparation
(see AN-008 for full information)
CAPE Technologies Kits
SP2-ST
Sample
Preparation
Starter Kit
SP2-RK rack for
column procedure
SP3 Sample
Preparation Kit
(extraction materials
not shown)
Supplies Required for Sample Preparation
(not available from CAPE Technologies; see AN-008)
HPLC grade or better hexane,
acetone, and methanol
ultrapure toluene (such as B&J)
glass Pasteur pipets and bulbs
glass pipets or glass syringes for
pipetting solvents
waste capture basin for column
procedure
borosilicate glass tubes (≈20 mL,
such as 16 x 125 mm)
Supplies Required for EIA Analysis
(see IN-DF1 for full information)
CAPE Technologies DF1 EIA Kit
(DF1-60 shown)
waste dump basin or
wash aspiration system
reagent or bottled
distilled water
AN-008 Method Set-up
open SP2-ST Kit and remove materials
glass reservoirs
bag of stopper/stopcock
assemblies
bag of 10 and 20 mL
polypropylene syringes
Luer plugs
AN-008 Method Set-up
open SP3 Kit and remove materials
40 mL extraction
vials, wooden
spatulas, stainless
steel mixing beads
acid silica columns, carbon minicolumns, bulk acid silica for
pretreatment (packaging may vary)
AN-008 Protocol
if sample is visibly wet, pre-drying is needed
blot on absorbent paper and air dry
blotting of wet soil samples using
several layers of paper towel
soil “pancake” after overnight air drying
in hood, between layers of paper towel
place 40 mL extraction vial onto balance and tare;
using wooden spatula, weigh out 5.0 g of soil
AN-008 Protocol
weigh out 20 g of sodium sulfate and add to same
40 mL vial
optional: 5 g sand can also be added for better
dispersal of samples (especially clays)
add 3 mixing beads to extraction vial
AN-008 Protocol
add 20 mL of 1:1 hexane:acetone to extraction vial
cap vials tightly and lay on their sides in the SP3 Kit box
or other box with padding to hold them tightly in place
shake at 350 rpm for 2-4 hours
if soils are high in clay, examine after 15-30 min to be
sure soil lumps are adequately dispersed and vials are
not leaking
AN-008 Protocol
remove from shaker and centrifuge 5 to 15 minutes at
1000 x g or less
using Pasteur pipet, remove supernatant to storage vial
(optional: can be left as supernatant over soil pellet in
original extraction vial)
soil samples after extraction and centrifugation, including method blank at left
note wide range of possible appearances
AN-008 Protocol
place chosen volume of extract (see AN-008 for
guidance) into evaporation tube with approx. 0.25 - 0.5
mL of tetradecane or other hydrocarbon keeper (exact
volume is unimportant)
evaporate hexane:acetone completely (caution:
acetone interferes with the oxidative portion of the
cleanup and can lead to false positive results if not
completely removed)
dilute residue in approx. 5 mL hexane
If highly colored, pre-treat with bulk acid silica
AN-008 Protocol Note
deciding whether to pre-treat with bulk acid silica
(based on extract color)
pre-treatment needed
pre-treatment not needed
AN-008 Protocol Note
example of pre-treatment with
bulk acid silica
(done in 16 x 125 mm
evaporation tubes after acetone
removal and dilution with
hexane)
Is pretreatment sufficient?
The supernatant should be clear
(no particulates) and colorless or
very lightly colored
Prepare coupled column system (method b)
add 10 mL
hexane to top
of column
use one acid silica column per
sample (include QA samples)
remove
both end
caps and
put column
into rack
Prepare coupled column system (method b)
when hexane
begins dripping
from tip, rinse
outside of tip with
1- 2 mL hexane
attach carbon minicolumn with no air
bubbles
(very important to see
AN-008 for details)
Prepare coupled column system (b)
insert
stopper/stopcock
assembly into top of
acid silica column
pressurize to allow
hexane to flow
through column into
waste basin
release pressure to
stop flow when
hexane level reaches
1-2 cm above bed of
acid silica column
remove
stopper/stopcock
assembly
Extract Cleanup
transfer
hexane diluted
extract to top
of prepared
acid silica
column
if pre-treated
with bulk acid
silica, then
transfer both
hexane and
acid silica to
column
replace
stopper/
stopcock
assembly,
and
pressurize
as before
Extract Cleanup
release pressure to stop flow
when level reaches 1-2 cm above
top of acid silica column (or top
of bulk acid silica from
pretreatment- note columns at
right with dark acid silica on top )
complete sample loading onto
carbon mini-column by adding
30 mL hexane wash (in 2 or 3
separate portions) and
pressurize as before
AN-008
Protocol Note
example of pretreatment with
bulk acid silica
(coupled column
systems just
before end of
hexane wash)
acid-neutral silica
boundary should
be difficult to see
(arrows)
Extract Cleanup
at end of 30
mL wash,
release
pressure to
stop flow
when air
begins to
penetrate
bottom
(neutral
silica)
portion of
column
remove carbon
mini-column and
place on clean
reservoir from
SP2-ST Kit
discard used acid
silica column
Extract Cleanup
place carbon
column and
reservoir in
rack and add 6
mL of 1:1
toluene:hexane
to reservoir
pressurize as
before and allow
solvent to flow
through to waste
release pressure to
stop flow when level
reaches tip of reservoir
Extract Cleanup
reverse carbon
mini-column on
same reservoir
add 12 mL toluene
pressurize as before and
capture eluate in glass
evaporation tube
Extract Cleanup/Solvent Exchange
add keeper
(stock vial from
DF1 Kit, diluted
with methanol),
according to
IN-DF1 Table 3
place tubes
in sample
evaporation
system and
evaporate
toluene
Method Summary (IN-DF1 step J1-J2)
warm EIA
kit reagents
prepare EIA
wash 1
Method Summary
evaporate sample
tubes until only a
small amount of
viscous residue
remains
centrifuge sample
tubes 2 minutes at
1-2000 x g
a small amount of keeper/sample residue should be
visible in the bottom (the residue volume is only 20% of
the original keeper volume); the residue should be
homogeneous with no color or precipitate visible
Method Summary (IN-DF1 step J3-J5)
label EIA
tubes and
prepare
for EIA by
rinsing
with water
use Repeater
pipettor to add 0.5
mL Sample
Diluent to each
tube
Method Summary (IN-DF1 step J6)
add
standards
(in keeper)
gently mix
each tube
immediately
Method Summary
(AN-008 step F12)
add 50-120 µL
of methanol
(depending on
IN-DF1 Table 3
protocol) to
each
evaporation
tube and mix
15 seconds
using vortex
mixer
if protocol B, then
let tube stand for
30-60 sec so liquid
can drain back to
bottom of tube
examine tubes before
proceeding- no color
or precipitate should
be visible in liquid
Method Summary
(IN-DF1 step J7)
transfer 50 µL
sample to EIA
tube (33-80%
of sample in
evaporation
tube)
gently mix
each tube
immediately
incubate 2-24 hrs at
room temp.
Method Summary (IN-DF1 step J8)
pour out
contents and
wash each tube
4x with 1 mL of
EIA wash 1
(0.01% Triton in
water)
tap rack
inverted on
paper towel to
remove excess
wash solution
dump washes
into waste
basin
Method Summary (IN-DF1 step J9)
use Repeater
pipettor to add
0.5 mL
Competitor-HRP
Conjugate to
each tube
incubate 15
min at room
temp.
Method Summary (IN-DF1 step J10)
pour out
contents as
before and
wash each
tube 4x with 1
mL of bottled
distilled water
tap rack
inverted on
paper towel
to remove
excess wash
water
dump washes
into waste
basin
Method Summary (IN-DF1 step J11)
use
Repeater
pipettor to
add 0.5 mL
HRPSubstrate
solution to
each tube
incubate 30 min
at room temp.
blue color should be visible
in at least some tubes
within the first few minutes
Method Summary (IN-DF1 step J12-J13)
read OD value in
Differential
Photometer
add 0.5 mL
Stop
solution to
each tube
wipe each
tube dry