CAPE Technologies

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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
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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.
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Facilities Required Performing this method using
the CAPE Technologies DF1 Immunoassay Kit
requires a modest laboratory facility with
capacity for 4-8C 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.
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Equipment Required for Sample Preparation (not
available from CAPE Technologies see EL-002)
tabletop centrifuge with buckets for 40 mL vials
and 16 x 125 mm tubes
orbital platform or similar shaker for extraction
0.1 g balance
vortex mixer
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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
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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
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Supplies Required for Sample Preparation (see
AN-008 for full information)
SP3 Sample Preparation Kit (extraction materials
not shown)
CAPE Technologies Kits
SP2-ST Sample Preparation Starter Kit
SP2-RK rack for column procedure
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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 BJ) 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)
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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
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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
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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 mini-columns, bulk
acid silica for pretreatment (packaging may vary)
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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
place 40 mL extraction vial onto balance and
tare using wooden spatula, weigh out 5.0 g of
soil
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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
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AN-008 Protocol add 20 mL of 11 hexaneacetone
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
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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
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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 hexaneacetone 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
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AN-008 Protocol Note deciding whether to
pre-treat with bulk acid silica (based on extract
color)
pre-treatment needed pre-treatment not
needed
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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
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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
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Prepare coupled column system (method b)
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)
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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
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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
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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
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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)
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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
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Extract Cleanup
place carbon column and reservoir in rack and add
6 mL of 11 toluenehexane to reservoir
pressurize as before and allow solvent to flow
through to waste
release pressure to stop flow when level reaches
tip of reservoir
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Extract Cleanup
reverse carbon mini-column on same reservoir
add 12 mL toluene
pressurize as before and capture eluate in glass
evaporation tube
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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
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Method Summary (IN-DF1 step J1-J2)
warm EIA kit reagents
prepare EIA wash 1
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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
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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
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Method Summary (IN-DF1 step J6)
add standards (in keeper)
gently mix each tube immediately
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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
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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.
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Method Summary (IN-DF1 step J8)
tap rack inverted on paper towel to remove excess
wash solution
pour out contents and wash each tube 4x with 1 mL
of EIA wash 1 (0.01 Triton in water)
dump washes into waste basin
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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.
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Method Summary (IN-DF1 step J10)
tap rack inverted on paper towel to remove excess
wash water
pour out contents as before and wash each tube 4x
with 1 mL of bottled distilled water
dump washes into waste basin
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Method Summary (IN-DF1 step J11)
use Repeater pipettor to add 0.5 mL HRP-Substrate
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
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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