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CAPE Technologies
High Performance Dioxin/Furan Immunoassay Kit Application Note AN-008
Rapid Extraction, Cleanup, and EIA Analysis of Soils atLow 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 withbuckets for 40 mL vials and 16 x 125 mm
tubes
0.1 g balance
orbital platform or similar shaker
for extraction
vortex mixer
Equipment Required for Sample Preparation (not available from CAPE Technologies; see EL-002)
sample evaporation system using clean nitrogen or compressed air
small chemical fume hood
Equipment Required for EIA(not available from CAPE Technologies; see EL-001)
Repeater pipettor
Differential Photometer
positive displacement micropipettors
(adjustable 20 to 100 µL essential; 1-10 µL very
helpful)
Supplies Required for Sample Preparation
(see AN-008 for full information)
SP2-ST Sample Preparation Starter Kit
SP3 Sample Preparation Kit
(extraction materials not shown)
SP2-RK rack for column procedure
CAPE Technologies Kits
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)
reagent or bottled distilled water
waste dump basin or wash aspiration system
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
acid silica columns, carbon mini-columns, bulk acid silica for
pretreatment (packaging may vary)
40 mL extraction vials, wooden
spatulas, stainless steel mixing beads
place 40 mL extraction vial onto balance and tare;
using wooden spatula, weigh out 5.0 g of soil
AN-008 Protocol
if sample is visibly wet, pre-drying is needed
blot on absorbent paper and air dry
soil “pancake” after overnight air drying in hood, between layers of paper towel
blotting of wet soil samples using several layers of paper towel
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)
use one acid silica column per sample (include QA samples)
remove both end caps and
put column
into rack
add 10 mL hexane to top
of column
when hexane begins dripping from tip, rinse outside of tip with 1- 2 mL hexane
attach carbon mini-column with no air
bubbles
(very important to see AN-008 for details)
Prepare coupled column system (method 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
Prepare coupled column system (b)
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
complete sample loading onto carbon mini-column by adding 30 mL hexane wash (in 2 or 3
separate portions) and pressurize as before
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 )
AN-008 Protocol Note
example of pre-treatment with bulk acid silica
(coupled column systems just before end of hexane wash)
acid-neutral silica boundary should be difficult to see
(arrows)
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
pressurize as before and capture eluate in glass
evaporation tube
add 12 mL toluene
Extract Cleanup
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
Extract Cleanup/Solvent Exchange
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)
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
add 50-120 µL of methanol (depending on IN-DF1 Table 3 protocol) to each evaporation tube and mix 15 seconds using vortex mixer
Method Summary
(IN-DF1 step J7)
incubate 2-24 hrs at room temp.
transfer 50 µL sample to EIA tube (33-80% of sample in evaporation tube)
gently mix each tube
immediately
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 HRP-Substrate solution to each tube
blue color should be visible in at least some tubes
within the first few minutes
incubate 30 min at room temp.
Method Summary (IN-DF1 step J12-J13)
wipe each tube dry
add 0.5 mL Stop solution to each tube
read OD value in Differential
Photometer
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