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CHROMATOGRAMS, COLUMNS & GUARDSANION-EXCHANGE COLUMNNITRITE & NITRATE ANALYSISAOAC Ofcial method 993.03 1 for the analysis of nitrate involves reduction using spongy Cadmium which is toxic andcarcinogenic. FDA improved on this method by using Vanadium(III) chloride and heat 2 for the post-column reduction ofnitrate to nitrite. Nitrite reacts with this modied Griess reagent to produce a red chromophore with maximal absorbanceat 535nm. Pickering Laboratories Inc. has further improved this method by substituting the corrosive hydrochloric acid withmethane sulfonic acid.METHODAnalytical ConditionsColumn: Anion exchange column4.6 x 150 mm,Catalog number 0785150Temperature: 50 °CSample Injection Volume: 10 µLLC Flow Rate: 1 mL/minMobile Phase: 20 mM sodium chlorate and10 mM dihydrogensodium phosphate0.120.10NitritePost-column ConditionsPost-column System: PinnacleReactor Volume: 0.5 mLReactor Temperature: 100 °CReagent Flow Rate: 0.1 mL/minReagent: (i) 1 % Vanadium(III) chloride in 20 % methanesulfonic acid(ii) 1 % m-Nitro aniline in 20 % methanesulfonic acid(iii) 1 % N-(1-Naphthyl)ethylenediaminedihydrochloride in 20 % methanesulfonic acidMix 50 mL of (i) and (ii), and 1.25 mL of (iii) anddilute to 250 mL using 20 % methanesulfonic acidDetection: UV/VIS, max= 535 nmSample PreparationTo 5 g of baby food in a 50 mL centrifuge tube, add 25 mLof 50-60 °C water (for vegetables) or 15 mM Sodium acetate(for fruits) and shake for 10 min. Add 12.5 mL of acetonitrileand make up the volume to 50 mL using water (for vegetables)and sodium acetate (for fruits). Centrifuge the mixturefor 15 mins at 5000 rpm. Filter the supernatant through a0.45 µ nylon lter and dilute to fall within the linear range.NOTESPost-column reagent solutions are stored in plastic containers.All solutions are fi ltered through 0.45 µ nylon fi lter before use.Nitrate/Nitrite standards should be checked prior to use for oxidation.Sample pH should be checked to determine the choice of extraction solutionsince acidic pH facilitates the conversion of nitrite to nitrate.45AU0.080.060.040.020.00Nitrate2.00 4.00 6.00 8.00 10.00 12.00 14.00min20 ppm Nitrite and Nitrate StandardACKNOWLEDGMENTSJohn A. Casanova, Food and DrugAdministration, 60 8 th Street, Atlanta,GA 30309REFERENCES1. AOAC- Offi cial Methods of Analysisof AOAC International (2000) 17 th Ed.,Section 50.1.11.Use of Griess Reagents ContainingVanadium (III) for the Post-columnDerivatization and SimultaneousDetermination of Nitrite and Nitrite inBaby Food, John A. Casanova, Lois K.Gross, Sarah E. McMullen and FrankSchenck, Food and Drug Administration,60 8 th Street, Atlanta, GA 30309.PRODUCT CATALOGPICKERING LABORATORIES10/11