Pesticide Analytical Manual Vol. I SECTION 302 E1 EXTRACTION WITH ACETONE. LIQUID-L/QUID PARTITIONING WITH PETROLEUM ETHER//METHYLENE CHLORIDE References Luke, M.A., et al.(1975)/ Assoc Of Anal. Chem. 58, 1020-1026 Luke, M.A., et al.(1981)/. Assoc. Off. Anal. Chem. 64, 1187-1195 onfatty sample is blended with acetone and filtered. Most nonionic residues are acetone to methylenc cB acetone solution. Residues are transferred from aqueou aqueous layer after the first partitioning to aid transfer. Concentration step is repeated in the presence of petroleum ether to remove all traces of methylene chloride, then repeated again to produce final extract in acetone solution blender, high speed; explosion-proof Waring Blendor, I gt jar Buchner funnel (Buchner, porcelain, 12 cm diameter filter paper, Shark Skin, to fit Buchner long-stemmed funnel, glass, 4"diameter micro-Snyder column, graduated receiving flaskS, Kuderna-Danish concentrator(K-D),500 mL, with Snyder column, two-ball separatory funnel(separator), I L acetone, distilled from all-glass apparatus boiling chips, 20-30 mesh carborundum glass wool, Pyrex, see Section 204 for handling directions methylene chloride, distilled from all-glass apparatus petroleum ether, distilled from all-glass apparatus sodium chloride, reagent grade sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for handling directions Recons Prewash filter paper taminants Weigh 100 g chopped or blended sample into blender jar, add 200 mL acetone, and blend 2 min at high speed Filter with suction through 12 cm Buchner fitted with Shark Skin paper collect extract in 500 mL suction flask. Filtration is normally complete in <I min Continuation of vacuum for excessive period can reduce volume of extract and error in calculation Place 80 mL sample extract in I L separator, and add 100 ml petroleum ether and 100 mL methylene chloride. Shake vigorously l min Transfer lower aqueous layer to second I L separator
Transmittal No. 2000-1 (10/1999) Form FDA 2905a (6/92) 302–7 Pesticide Analytical Manual Vol. I SECTION 302 E1 EXTRACTION WITH ACETONE, LIQUID-LIQUID PARTITIONING WITH PETROLEUM ETHER/METHYLENE CHLORIDE References Luke, M.A., et al. (1975) J. Assoc. Off. Anal. Chem. 58, 1020-1026 Luke, M.A., et al. (1981) J. Assoc. Off. Anal. Chem. 64, 1187-1195 Principles Nonfatty sample is blended with acetone and filtered. Most nonionic residues are extracted into aqueous acetone solution. Residues are transferred from aqueous acetone to methylene chloride/petroleum ether by partitioning, with salt added to aqueous layer after the first partitioning to aid transfer. Concentration step is repeated in the presence of petroleum ether to remove all traces of methylene chloride, then repeated again to produce final extract in acetone solution. Apparatus blender, high speed; explosion-proof Waring Blendor, 1 qt jar Büchner funnel (Büchner), porcelain, 12 cm diameter filter paper, Shark Skin®, to fit Büchner long-stemmed funnel, glass, 4" diameter Kuderna-Danish concentrator (K-D), 500 mL, with Snyder column, two-ball micro-Snyder column, graduated receiving flask separatory funnel (separator), 1 L Reagents acetone, distilled from all-glass apparatus boiling chips, 20-30 mesh carborundum glass wool, Pyrex, see Section 204 for handling directions methylene chloride, distilled from all-glass apparatus petroleum ether, distilled from all-glass apparatus sodium chloride, reagent grade sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for handling directions Directions • Prewash filter paper with acetone to remove contaminants. • Weigh 100 g chopped or blended sample into blender jar, add 200 mL acetone, and blend 2 min at high speed. • Filter with suction through 12 cm Büchner fitted with Shark Skin® paper; collect extract in 500 mL suction flask. Filtration is normally complete in <1 min. Continuation of vacuum for excessive period can reduce volume of extract and cause error in calculation. • Place 80 mL sample extract in 1 L separator, and add 100 mL petroleum ether and 100 mL methylene chloride. Shake vigorously 1 min. • Transfer lower aqueous layer to second 1 L separator
SECTION 302 Pesticide Analytical Manual Vol I Dry upper layer of first separator by passing through about 1.5"sodium sulfate supported on washed glass wool in 4" funnel, collecting in K-D. (If extract will be cleaned up directly with C3, charcoal/Celite column, collect In vacuum rotary evaporator flas To separator with aqueous phase, add 7 g sodium chloride and shake vigorously 30 sec until most of the sodium chloride is dissolved Add 100 mL methylene chloride, shake l min, and dry lower organic phase through same sodium sulfate Extract aqueous phase with additional 100 mL methylene chloride and dry as above. Rinse sodium sulfate with about 50 ml methylene chloride (Ifextract will be cleaned up directly with C, Pxo follows.) described there instead of evaporating in K-D as Add boiling chips to K-D and concentrate solvent in K-D; start evaporation slowly by placing only receiver tube into steam. After 100-150 mL has evaporated, concentrator may be exposed to more steam. When liquid level in hot concentrator tube is about 2 mL, add 100 mL petroleum ether through Snyder column and reconcentrate to about 2 mL Add 50 mL petroleum ether and repeat concentration step. Add 20 mL acetone, and any of the concentration steps. Adjust volume of extract to suitable definite volume with Calculate equivalent sample weight in final solution mg sample equivalent 100 uL final extract 200+W-10 mL final volume where. 100 =g sample analyzed 80=mL filtered extract taken for liquid-liquid partitioning 200=mL acetone blended with 100 g sample W=amount(mL) of water present in sample(Section 201; if data are not available for particular raw agricultural commodity, use 85%) 10=adjustment for water/acetone volume contraction lur S, when sample contains 85% water(85 mL/100 g) and final extract Ime is 7 mL, each uL contains 4. 15 mg sample equivalent 200+85-107 uL final extract · Extract may itable, as is, for determination by glC with selective detectors(e.g, DG2, DG3). If co-extractives interfere with determination or aave rely affect chromatography, n up extract with Cl C2, or C5 prior to determinatio Clean up extract with Cl or C5 prior to determination by electron capture (DGl, DG7, etc. )or flame ionization detectors(DG6) Clean up extract with CB or C4 prior to determination by DLI for N-methylcarbamates
SECTION 302 Pesticide Analytical Manual Vol. I 302–8 Transmittal No. 2000-1 (10/1999) Form FDA 2905a (6/92) • Dry upper layer of first separator by passing through about 1.5" sodium sulfate supported on washed glass wool in 4" funnel, collecting in K-D. (If extract will be cleaned up directly with C3, charcoal/Celite column, collect in vacuum rotary evaporator flask.) • To separator with aqueous phase, add 7 g sodium chloride and shake vigorously 30 sec until most of the sodium chloride is dissolved. • Add 100 mL methylene chloride, shake 1 min, and dry lower organic phase through same sodium sulfate. • Extract aqueous phase with additional 100 mL methylene chloride and dry as above. Rinse sodium sulfate with about 50 mL methylene chloride. (If extract will be cleaned up directly with C3, proceed to concentration step described there instead of evaporating in K-D as follows.) • Add boiling chips to K-D and concentrate solvent in K-D; start evaporation slowly by placing only receiver tube into steam. After 100-150 mL has evaporated, concentrator may be exposed to more steam. When liquid level in hot concentrator tube is about 2 mL, add 100 mL petroleum ether through Snyder column and reconcentrate to about 2 mL. Add 50 mL petroleum ether and repeat concentration step. Add 20 mL acetone, and reconcentrate to about 2 mL. Do not allow solution to go to dryness during any of the concentration steps. Adjust volume of extract to suitable definite volume with acetone. • Calculate equivalent sample weight in final solution: mg sample equivalent µL final extract = 100 × 80 200 + W – 10 1 mL final volume × where: 100 = g sample analyzed 80 = mL filtered extract taken for liquid-liquid partitioning 200 = mL acetone blended with 100 g sample W = amount (mL) of water present in sample (Section 201; if data are not available for particular raw agricultural commodity, use 85%) 10 = adjustment for water/acetone volume contraction. Thus, when sample contains 85% water (85 mL/100 g) and final extract volume is 7 mL, each µL contains: 4.15 mg sample equivalent µL final extract 100 × 80 200 + 85 – 10 1 7 × = • Extract may be suitable, as is, for determination by GLC with selective detectors (e.g., DG2, DG3). If co-extractives interfere with determination or adversely affect chromatography, clean up extract with C1, C2, or C5 prior to determination. • Clean up extract with C1 or C5 prior to determination by electron capture (DG1, DG7, etc.) or flame ionization detectors (DG6). Clean up extract with C3 or C4 prior to determination by DL1 for N-methylcarbamates
Pesticide Analytical Manual Vol. I SECTION 302 E2 EXTRACTON WITH ACETONE. REMOVAL OF WATER WITH 40G HYDROMATRX References Luke, M.A., et al.(1975)/ Assoc Of Anal. Chem. 58, 1020-1026 Luke, M.A., et aL. (1981)/. Assoc. Of. Anal. Chem. 64, 1187-1195 Hopper, M.L.(1988)/. Assoc. Off. Anal. Chem. 71, 731-734 Principles Nonfatty sample is blended with acetone and filtered. Most nonionic residues are extracted from nonfatty foods into aqueous acetone solution. Water is removed from aqueous acetone solution by passing it through a column of specially treated diatomaceous earth(Hydromatrix) Residues are eluted from column with methyl- ene chloride. Up to 18.3 mL water, from 40 mL aqueous acetone extractant, is adsorbed by the column, which is re-usable Apparatus Deed; explosion-proof Waring Blendor, I qt jar Buchner funnel(Buchner), porcelain, 12 cm diameter filter paper, Shark Skin, to fit Buchner chromatographic column, 25 mm id X 500 mm, Teflon stopcock long-stemmed funnel, glass, 4"diameter powder funnel, glass, 4"diameter Kuderna-Danish concentrator(K-D), 500 mL, with Snyder column, two-ball micro-Snyder column, graduated receiving flask sieve. No. 30 Reagents acetone, distilled from all-glass apparatus buffer solution: 0. 1 M(18.6g/L)potassium phosphate monobasic(KH, PO) In water Hydromatrix material(pelletized diatomaceous earth), Part No. 0019-8003 Analytichem International, Harbor City, CA; also available through Varian methylene chloride, distilled from all-glass apparatus potassium phosphate monobasic, certified ACS grade wire gauze, 40 mesh stainless steel Prepare hydromatrix column Cut two pieces stainless steel gauze into circles of diameter slightly arger than chromatographic column id Place one circle in bottom of Place 50 g Hydromatrix material on No 30 sieve and sieve thoroughly to remove fines 3o2-9
Transmittal No. 2000-1 (10/1999) Form FDA 2905a (6/92) 302–9 Pesticide Analytical Manual Vol. I SECTION 302 E2 EXTRACTION WITH ACETONE, REMOVAL OF WATER WITH 40 G HYDROMATRIX References Luke, M.A., et al. (1975) J. Assoc. Off. Anal. Chem. 58, 1020-1026 Luke, M.A., et al. (1981) J. Assoc. Off. Anal. Chem. 64, 1187-1195 Hopper, M.L. (1988) J. Assoc. Off. Anal. Chem. 71, 731-734 Principles Nonfatty sample is blended with acetone and filtered. Most nonionic residues are extracted from nonfatty foods into aqueous acetone solution. Water is removed from aqueous acetone solution by passing it through a column of specially treated diatomaceous earth (Hydromatrix). Residues are eluted from column with methylene chloride. Up to 13.3 mL water, from 40 mL aqueous acetone extractant, is adsorbed by the column, which is re-usable. Apparatus blender, high speed; explosion-proof Waring Blendor, 1 qt jar Büchner funnel (Büchner), porcelain, 12 cm diameter filter paper, Shark Skin®, to fit Büchner chromatographic column, 25 mm id × 500 mm, Teflon stopcock long-stemmed funnel, glass, 4" diameter powder funnel, glass, 4" diameter Kuderna-Danish concentrator (K-D), 500 mL, with Snyder column, two-ball micro-Snyder column, graduated receiving flask sieve, No. 30 Reagents acetone, distilled from all-glass apparatus buffer solution: 0.1 M (13.6 g/L) potassium phosphate monobasic (KH2 PO4 ) in water Hydromatrix material (pelletized diatomaceous earth), Part No. 0019-8003, Analytichem International, Harbor City, CA; also available through Varian methylene chloride, distilled from all-glass apparatus potassium phosphate monobasic, certified ACS grade wire gauze, 40 mesh stainless steel Directions • Prepare Hydromatrix column: – Cut two pieces stainless steel gauze into circles of diameter slightly larger than chromatographic column id. Place one circle in bottom of column. – Place 50 g Hydromatrix material on No. 30 sieve and sieve thoroughly to remove fines
SECTION 302 Pesticide Analytical Manual Vol I Pour 40 g sieved Hydromatrix material into column with aid of powder funnel. Tap end of column lightly on benchtop to settle material. Place second stainless steel gauze circle on top of material in column With stopcock fully open, wash column with 150 mL buffer solution After buffer solution has passed into column and flow has slowed to 3-5 mL/min, wash column with 300 mL acetone Adjust flow to 50-60 mL/ min after first 100 mL acetone has eluted Wash column with 300 mL methylene chloride. Re-adjust flow to 50-60 mL/ min after first 100 mL methylene chloride has eluted Prewash filter paper with acetone to remove artifacts Weigh 100 g chopped or blended sample into blender jar, add 200 mL acetone, and blend 2 min at high speed Filter with suction through 12 cm Buchner fitted with Shark Skin paper; collect extract in 500 mL suction flask Filtration is normally complete in<l min Continuation of vacuum for excessive period can reduce volume of extract and cause error in calculation Prewash Hydromatrix column with 200 mL acetone followed by 200 mL methylene chloride immediately before each use. Discard wash solvents Place K-D under column. (If extract will be cleaned up directly with C3 charcoal/Celite column, collect in vacuum rotary evaporator flask. Trans fer 40 mL filtered acetone extract to top of column. Let extract pass into column until flow rate has slowed to <l mL/min Let column equilibrate 3 min at <l mL/ min Add 50 mL methylene chloride to column. After that has passed into column, add another 50 mL methylene chloride. After that has passed into column, add another 200 mL methylene chloride Collect eluate until flow rate has decreased to slow drip(about l mL/min) Total elution time is 6-8 min (If extract will be cleaned up directly with C, proceed to concentration step described there instead of evaporating in K-D as follows. Add boiling chips to K-D and concentrate solvent in K-D; start evaporation slowly by placing only receiver tube into steam. After 100-150 mL has in hot concentrator tube is about 2 mL, add 100 mL petroleum ethertel evaporated, concentrator may be exposed to more steam. When liquid through snyder column and re trate to about 2 mL. Add 50 mL troleum ether and repeat concentration step. Add 20 mL acetone, and reconcentrate to about 2 mL Do not allow solution to go to dryness during any of the concentration steps. Adjust volume of extract to suitable definite volume with acetone If extract will be cleaned up directly with Cl, Florisil column, it is not necessary to reconcentrate repeatedly (as above) to remove all traces of methylene chloride. Instead, add boiling chips and concentrate solvent in K-D to <5 mL without allowing K-D to cool, add 50 mL acetone through Snyder column, and reconcentrate to suitable definite volume; allow to 302-10 230
SECTION 302 Pesticide Analytical Manual Vol. I 302–10 Transmittal No. 2000-1 (10/1999) Form FDA 2905a (6/92) – Pour 40 g sieved Hydromatrix material into column with aid of powder funnel. Tap end of column lightly on benchtop to settle material. Place second stainless steel gauze circle on top of material in column. – With stopcock fully open, wash column with 150 mL buffer solution. – After buffer solution has passed into column and flow has slowed to 3-5 mL/min, wash column with 300 mL acetone. Adjust flow to 50-60 mL/ min after first 100 mL acetone has eluted. – Wash column with 300 mL methylene chloride. Re-adjust flow to 50-60 mL/min after first 100 mL methylene chloride has eluted. • Prewash filter paper with acetone to remove artifacts. • Weigh 100 g chopped or blended sample into blender jar, add 200 mL acetone, and blend 2 min at high speed. • Filter with suction through 12 cm Büchner fitted with Shark Skin® paper; collect extract in 500 mL suction flask. Filtration is normally complete in <1 min. Continuation of vacuum for excessive period can reduce volume of extract and cause error in calculation. • Prewash Hydromatrix column with 200 mL acetone followed by 200 mL methylene chloride immediately before each use. Discard wash solvents. • Place K-D under column. (If extract will be cleaned up directly with C3, charcoal/Celite column, collect in vacuum rotary evaporator flask.) Trans fer 40 mL filtered acetone extract to top of column. Let extract pass into column until flow rate has slowed to <1 mL/min. Let column equilibrate 3 min at <1 mL/min. • Add 50 mL methylene chloride to column. After that has passed into column, add another 50 mL methylene chloride. After that has passed into column, add another 200 mL methylene chloride. • Collect eluate until flow rate has decreased to slow drip (about 1 mL/min). Total elution time is 6-8 min. (If extract will be cleaned up directly with C3, proceed to concentration step described there instead of evaporating in K-D as follows.) • Add boiling chips to K-D and concentrate solvent in K-D; start evaporation slowly by placing only receiver tube into steam. After 100-150 mL has evaporated, concentrator may be exposed to more steam. When liquid level in hot concentrator tube is about 2 mL, add 100 mL petroleum ether through Snyder column and reconcentrate to about 2 mL. Add 50 mL petroleum ether and repeat concentration step. Add 20 mL acetone, and reconcentrate to about 2 mL. Do not allow solution to go to dryness during any of the concentration steps. Adjust volume of extract to suitable definite volume with acetone. • If extract will be cleaned up directly with C1, Florisil column, it is not necessary to reconcentrate repeatedly (as above) to remove all traces of methylene chloride. Instead, add boiling chips and concentrate solvent in K-D to <5 mL. Without allowing K-D to cool, add 50 mL acetone through Snyder column, and reconcentrate to suitable definite volume; allow to cool
Pesticide Analytical Manual Vol. I SECTION 302 Calculate equivalent sample weight in final solution: mg sample equivalent =100 uL final extract 200+W-10 mL final volume 100 =g sample analyzed 40=mL filtered extract taken for Hydromatrix partitioning 200=mL acetone blended with 100 g sample W=amount(mL) of water present in sample (Section 201; if data ar not available for particular raw agricultural commodity, use 83%0/ 10= adjustment for water/acetone volume contraction Thus, when sample contains 85% water(85 mL/100 g)and final extract olume is 5 mL, each uL contains: 2.9 mg sample equivalent 100× 200+85-105 L final extract Extract may be suitable, as is, for determination by glC with selective detectors(e.g, DG2, DG3). If co-extractives interfere with determination or adversely affect chromatography, clean up extract with Cl, C2, or C5 Clean up extract with Cl or C5 prior to determination by electron capture(DGl, DG7, etc. )or flame ionization detectors(DG6) Clean up extract with C3 or C4 prior to determination by DLl for N-methyl carbamates Re-use Hydromatrix column without further rinsing, unless any adsorbed color elutes from column(after about 20 uses). When this oc column as follow Do not change stopcock setting Flow rate will change due to different solvent densities, but this is of no consequence Wash column with 200 mL acetone, followed by sufficient volume (200-300 mL) buffer solution to remove any color left on column Once color has been removed. elute with 300 mL acetone followed by 200 mL methylene chloride. Column is now ready for re-use. 302-11
Transmittal No. 2000-1 (10/1999) Form FDA 2905a (6/92) 302–11 Pesticide Analytical Manual Vol. I SECTION 302 • Calculate equivalent sample weight in final solution: mg sample equivalent µL final extract = 100 × 40 200 + W – 10 1 mL final volume × where: 100 = g sample analyzed 40 = mL filtered extract taken for Hydromatrix partitioning 200 = mL acetone blended with 100 g sample W = amount (mL) of water present in sample (Section 201; if data are not available for particular raw agricultural commodity, use 85%) 10 = adjustment for water/acetone volume contraction. Thus, when sample contains 85% water (85 mL/100 g) and final extract volume is 5 mL, each µL contains: 2.9 mg sample equivalent µL final extract 100 × 40 200 + 85 – 10 1 5 × = • Extract may be suitable, as is, for determination by GLC with selective detectors (e.g., DG2, DG3). If co-extractives interfere with determination or adversely affect chromatography, clean up extract with C1, C2, or C5 prior to determination. • Clean up extract with C1 or C5 prior to determination by electron capture (DG1, DG7, etc.) or flame ionization detectors (DG6). Clean up extract with C3 or C4 prior to determination by DL1 for N-methylcarbamates. • Re-use Hydromatrix column without further rinsing, unless any adsorbed color elutes from column (after about 20 uses). When this occurs, restore column as follows: – Do not change stopcock setting. Flow rate will change due to different solvent densities, but this is of no consequence. – Wash column with 200 mL acetone, followed by sufficient volume (200-300 mL) buffer solution to remove any color left on column. Once color has been removed, elute with 300 mL acetone followed by 200 mL methylene chloride. Column is now ready for re-use