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 RequiredPerforming 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-upopen 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-upopen 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 Protocolif 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 Protocolremove 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 Protocolplace 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 Notedeciding whether to pre-treat with bulk acid silica

(based on extract color)

pre-treatment needed pre-treatment not needed

AN-008 Protocol Noteexample 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