5. Procedurea. Calibration curve (duplicates)1. Prepare standard solutions with the following nitrite concentrations: 0, 2.5, 5,10 µg/ml: Add the following volumes of nitrite working solution into 100 mlcalibration bottles: 0, 5, 10, 20 ml (using a 1-10 ml pipette) and complete thevolume with dH2O. (mark the bottles as: 0, 2.5µg/ml, 5µg/ml, 10µg/ml)2. Verify the calculations.3. Mix carefully.b. Protein precipitation (no replicates)1. Use an analytical balance to weigh a 10g processed meat sample into a 250mlErlenmeyer flask. Prepare a similar bottle without meat as blank.2. Add 5ml borax solution.3. Add 75ml dH2O using a plastic measuring cylinder and incubate in a boilingwater bath for 15 min. Use weights so the Erlenmeyer flasks stay in place.4. Let the Erlenmeyer flask cool down.5. Transfer the solution into a 100ml calibration bottle.6. Add 2ml Carrez II.7. Add 2ml Carrez I while mixing.8. Complete the volume with dH2O.9. Wait for precipitation to happen.10. Filtrate the supernatant (upper layer) using a No. 4 filter paper into a beaker.c. Color formation1. Use a plastic measuring cylinder to add 50ml dH2O into a 100ml calibrationbottle. Prepare technical duplicates for blanks and samples (12 bottles in total:8 for calibration curve, 2 for the sample, 2 for blank).2. For the samples and blanks: Transfer exactly 25 ml from the filtrate into thecalibration bottle using a volumetric pipette3. For the calibration curve standards: Transfer the standards into an empty vialor beaker, then transfer exactly 10 ml of the solution into a new 100 mlcalibration bottle using a 10 ml volumetric pipette*.*Note: By starting with the low concentration standard (working from low concentration tohigh) you can use the same beaker and pipette.54. Add 10 ml Sulfanilamide solution, mix and add 6ml of diluted HCl:H20 4:9solution. Wait for 5 minutes.5. Add 2 ml α-Naphthylethylenediamine solution, mix and complete the volumewith dH2O.6. Incubate in the dark for 10 minutes for color development (purple).7. Transfer 200µl of each solution into a 96-well plate, do the transfer 3 times foreach bottle in separate wells (technical triplicates).8. Read absorbance at 538nm.