SOP for analyzing DISSOLVED AMMONIA ANALYSIS

SHANNON POINT MARINE CENTER

640

nm

rev. 2010 hyl

METHOD: Under alkaline conditions (pH 11 - 12), phenol reacts with ammonia (NH3) in the presence of alkaline hypochlorite (bleach) to form a blue-green colored complex (indophenols blue). Sodium nitroprusside (sodium nitroferricyanide) accelerates the formation, which is then measured spectrophotometrically at 640 nm. Reference: A Manual of Chemical and Biological Methods for Seawater Analysis (Parsons, Maita, & Lalli, 1984). Apparatus and Materials 100-ml volumetric flasks 50-ml volumetric flask micro pipets test tubes (13 x 100 mm) Spectronic 20 set up for 640 nm Phenol or Liquefied phenol (>89%) 95% v/v ethyl alcohol Sodium Nitroprusside Sodium citrate Sodium Hydroxide Sodium Hypochlorite (commercial bleach - no whitening agents or additives) Ammonium Sulphate (or Ammonium Chloride as Standard) Safety and Waste Handling Please follow all laboratory protocol for safety procedures and personal protective equipment (PPE) including gloves, lab coat, and safety glasses should be worn during the preparation and disposal of reagents. Some reagents preparation should be preceded under a fume hood. After analysis follow the waste disposal procedure, discard analyzed samples in the waste container and label the container. Operator must read and be familiar with the Material Safety Data Sheets (MSDS) for all chemicals used in this experiments, special Phenol, Sodium Nitroferricyanide and sodium hydroxide which is highly toxic. Please wear gloves and do analyses under fume hood. Reagents and standards Phenol solution (PHENOL IS HIGHLY TOXIC) H4 F2 R0 5g Phenol in 50mL 95% v/v ethyl alcohol (250 runs). Sodium nitroferricyanide solution (0.5% w/v) H3 F0 R1 . dissolve 0.25g Sodium Nitroferricyanide (sodium nitroprusside dihydrate), Na2[Fe(CN)5NO] 2H2O, in 50mL nanopure H2O. Store in amber glass bottle; stable at least 1 month (250 runs). Alkaline reagent Sodium Citrate: H1 F0 R0 NaOH: H3 F0 R1 dissolve 20g Sodium Citrate (Na3C6H5O7) and 1.0g NaOH in 100mL nanopure H2O (250 runs). Stable indefinitely. Sodium hypochlorite solution H3 F0 R0 use commercial Chlorox bleach (must be pure, no whitening agents or other additives, these will alter the chemistry and results drastically; This solution slowly decomposes once the seal on the bottle cap is broken - replace every 2 months).S

Oxidizing solution (125 samples) mix 50mL alkaline reagent and 12.5mL commercial Bleach (Chlorox) Keep stoppered when not in use & prepare fresh every day.

AMMONIA STANDARDS: Primary STDs both Analytical and Quality Control (1.5 mM) - 0.099105g Ammonium Sulphate, (NH4)2SO4 (F.W. 132.14), in 500mL nanopure H2O (or 0.0495525g in 250mL). Add 0.5mL (or 0.25mL) chloroform, store refrigerated. Stable many months. Secondary (Analytical) STDs: 2 STD Final conc. (M) 0.00 0.75 1.20 1.50 2.25 3.00 Use x L 1 STD dilute to 50mL H2O 0 25 40 50 75 100

Quality Control STD: 1.50 50

Note: Analytical and QC STDs must be made up the same day they are used.

Procedure
Let seawater samples come to room temperature before adding reagent. After reagents added, let it stand at room temperature for at least 1 hour (subdued light or at dark) for developing the color and the color is stable for 24 hours. ANALYSIS: Pipet 5mL aliquots - 3X DI H2O blanks, 3X analytical and quality control STDs, and 1X each sample into culture tubes. Under Hood, In sequence, Add 0.2mL phenol, vortex mix, 0.2mL Nitroferricynide, mix, 0.5mL oxidizing solution, mix. Let stand in the dark 1 hour. (The color is stable for 24 hrs) Vortex! Read extinction at 640 nm on spectrophotometer. Determine regression equation from blank and standard samples and estimate ammonia nitrogen in samples. Notes
Experience here have shown that, (1) wearing gloves and (2) acid rinses, followed by RO and Nanopure rinses, of all glassware just before use, keeps both blanks and variability low. Analytical standards left refrigerated overnight will degrade significantly. From McCarthys lab, ideas for keeping blanks low- add 5mL Deion H2O to culture tubes. Add solutions as above, vortexing after each, discard contents, rinse twice with DDW, add 5mL sample, vortex & discard. No need to cover tubes while in the dark, since ammonia cannot be complexed after the addition of oxidizing solution. Best to add reagents away from light, as brief exposure to light will yield high and variable blank. Everything washed in 10% HCl and rinsed multiple times with DIW. Wear gloves.