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Glyphosate and AMPA Analysis in Crops

Glyphosate and AMPA Analysis in Crops. A simple and reproducible extraction and clean-up for HPLC Post-column derivatization Saji George, Lazslo Torma, Wendy Rasmussen, Maria Ofitserova Ph.D., Michael Pickering Ph. D., Pickering Laboratories. Current Use of Glyphosate.

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Glyphosate and AMPA Analysis in Crops

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  1. Glyphosate and AMPA Analysis in Crops A simple and reproducible extraction and clean-up for HPLC Post-column derivatization Saji George, LazsloTorma, Wendy Rasmussen, Maria Ofitserova Ph.D., Michael Pickering Ph. D., Pickering Laboratories

  2. Current Use of Glyphosate • Glyphosate is a nonselective herbicide and is used to control a great variety of annual, biennial, and perennial grasses, sedges, broad leafed weeds and woody shrubs. • It is used in fruit orchards, vineyards, conifer plantations and many plantation crops(coffee. tea, bananas). • It is used on non-crop areas(road shoulders and right of way) to control weeds and in forestry, gardening and horticulture. • Genetically modified, glyphosate tolerant crops increase the use of glyphosate and possible misapplication.

  3. Commonly Used Methods for Glyphosate Analysis in Crops • Monsanto HPLC post-column method (J.Agric. Food Chem. 1986: 34, 955 – 960) • GC/MSD (J.AOAC International. 2001: (84), 823 – 846)

  4. Monsanto Method • Extraction • Sample extracted with chloroform and 0.1N HCl • Clean-up • Chelex column, Fe 2+ form • Anion exchange column • Concentration • Evaporated to dryness and redissolved in 2 mL DI water • Analysis • HPLC/PCD using OCl-/OPA and FL detector

  5. GC/MSD Method • Extraction – Water as solvent • Clean-up – methylene chloride, CAX • Matrix specific modification – for high water, fat or protein content • Concentration – evaporated to dryness and re-dissolved in CAX mobile phase • Manual Derivatization – heptaflurobutanol in trifluoroacetic anhydride • Analysis-GC/MSD

  6. HPLC/Post-Column Method • Extraction - water • Clean-up – methylene chloride, CAX • Concentration • Analysis – HPLC/PCD using OCl-/OPA and FL detector

  7. Reagents/Equipment • Sample preparation • Methylene chloride • Acidic modifier solution(mixture of KH2PO4,, H2O, Methanol, HCl) • CAX eluant (mixture of water, HCl and Methanol) • CAX column(Pickering or Bio-Rad) • LC • Potassium eluant (K200), potassium regenerant (RG019) • Cation exchange analytical column(Cat.#1954150) • Cation exchange guard column(Cat.# 1953020) • Post-column • Reagent 1 – 100uL of 5% sodium hypochlorite solution in 950 mL of hypochlorite diluent(GA116) • Reagent 2 – 100 mg of OPA(Cat.#O120) and 2 g Thiofluor(Cat.#3700-2000) in 950 mL of OPA diluent(GA104)

  8. Extraction • To 25g of a homogenous sample add enough water (after estimation of moisture content) to make the total volume of water 125 mL. Blend and Centrifuge. • Matrix specific modification • High water content – reduce sample amount to 12.5g • High protein content – add 100mL HCl to 20mL of extract, shake and centrifuge • High fat content – do the methylene chloride partition twice.

  9. Clean-up – methylene chloride partition • To 20 mL of aqueous extract add 15 mL methylene chloride, shake for 2-3 min and centrifuge. • To 4.5 mL of aq layer add 0.5 mL acidic modifier solution. Shake and centrifuge.

  10. CAX clean-up • Prepare CAX column • Transfer 1 mL of the extract and elute to top of column • Wash with two 0.7 mL portions of CAX mobile phase and discard the effluent • Elute analytes with 12 mL CAX mobile phase

  11. Concentration • Evaporate to dryness using rotary evaporator or a vacuum vortex-type evaporator or lyophylize • Re-dissolve in 2 mL of the CAX mobile phase

  12. Instrumentation • LC • Post-column derivatization unit • Fluorescence detector

  13. LC and Post-Column Conditions • LC conditions • Column temperature: 55oC • Injection volume: 100mL • Flow rate: 0.40 mL/min • Mobile phases: Potassium eluent (K200), Regenerent (RG019) • Post column conditions • Reactor volume: 0.5 mL • Reactor temperature: 36oC • Reagent 1: hypochlorite solution • Reagent 2: OPA solution • Flow rates: 0.3 mL/min • Detection: fluorescence; lex 330nm, lem 465 nm

  14. Two-reagent Post-column System

  15. Post-column Derivatization Reaction

  16. Calibration curve

  17. Alfalfa

  18. Alfalfa glyphosate AMPA 0.500 ppm blank 0.250ppm

  19. Broccoli

  20. Broccoli glyphosate AMPA 0.500 ppm blank 0.250 ppm

  21. Strawberry

  22. Strawberry glyphosate AMPA blank 0.500 ppm 0.250 ppm

  23. Pickering CAX Column

  24. Calibration curve

  25. Advantages of Proposed Method • Simple and faster extraction and clean-up • Availability of post-column systems • Time consuming derivatization for GC/MSD

  26. Acknowledgements • Saji George, Wendy Rasmussen, Maria Ofitserova and Michael Pickering – Pickering Laboratories • Montana State Department of Agriculture

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