Joint International Conference on Long-term Experiments ...

MATERIALS AND METHOD
Apparatus
Clean all glassware with chromatic acid/sulfuric acid solutions, rinse thoroughly with
distilled water, oven-dry at 120 o C, and solvent-rinse before use to minimize interferences.
a) Meat chopper – Launders, Frary and Clark Co. or equivalent.
b) Digestion apparatus – 1) Heating mantle – 500 mL; 2) Friedrich condenser; 3)
Boiling flask
c) Separatory funnels
d) Flasks
e) Flash evaporator
f) Filter funnel
g) Sample clarification kit
h) Liquid chromatograph
i) Recording data processors
Reagents
a) Solvents – Ethanol, 190 proofs, distill before use; cyclohexane, distilled-in-glass;
acetonitrile, LC grade; double-distilled water.
b) NPAH standards – 5,7-Dimethylbenze acridine, dibenz acridine. Each standard
was purified by reverse phase LC, and the identity of each was verified by GC/MS
before use in this study.
c) Standard solutions – 1) Stock solutions – 0.1 mg/mL. Dissolve 1 mg of each
NPAH standard in 10mL ethanol. 2) Working standard solution dilute to volume with
ethanol.
d) Alumina – aluminum oxide 90, active basic, activity stage 1
e) Deactivated alumina – Prepared from alumina above
f) Solutions – 6N HCI, 10N KOH, ethanol-methylene chloride.
Extraction
Place 25 g homogenized smoked food into 500 mL boiling flask. Add 100 mL ethanol, 4 g
KOH, and boiling chips. Insert Friedrich condenser and reflux at rapid rate for 2 h. let digest
cool for 20 min, remove condenser, and filter contents of flask through ethanol-rinsed glass
wool pad into 1 L separatory funnel. Shake funnel vigorously for 2 min. let layers separate;
then draw lower aqueous layer into second 1L separatory funnel. Repeat extraction with 50
mL cyclohexane.
Add 20 mL 6N HCI and shake funnel for 2 min. let layers separate and draw off
bottom acidic layer into second 1L funnel. Repeat extraction in first funnel with 20 mL
portions of 6N HCI, and combine each extract with first extract in second funnel. Discard
cyclohexane layer remaining in first funnel.
Carefully add 50 mL 10 N KOH to acidic extracts while cooling funnel under cold tap
water. Add 50 mL cyclohexane and shake funnel for 2 min. Draw off lower aqueous layer
into second funnel and repeat extraction with 50 mL cyclohexane.
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