1. Prepare reagents and standards as described below.
   
2. Turn on power switches to autosampler, diluter, pump, and system unit, as well as PC and monitor.
   
3. Inspect tubing for potential failure and proper connections between autosampler, dilutor and reagent pump and along the length of the channels of the system unit.
   
4. Pump reagents through all reagent lines and allow the system to equilibrate. Proceed with the calibration once a stable baseline has been established.
   
5. Fill sampler tube about halfway with the 10 mg N/L working standard. Program dilutor to prepare the following standards: 4, 2, 1, 0.5, 0.2, 0.1 and 0 g N/L.
   
6. Prepare a calibration curve. For soil extracts, delete the 10 mg N/L standard result from the calibration curve; this concentration is much higher than typically found in soil extracts. When satisfied with instrument performance, prepare for and proceed with sampling run.
   
7. Begin sample run with a 1 mg N/L standard from a different standard lot number than used for the working standard. Follow with a nitrite standard of 1 mg N/L to check efficiency of the conversion of nitrate to nitrite.
   
8. Compare standards made from different lots as a calibration check. Calculate efficiency of the nitrate channel by dividing the nitrate standard by the nitrite standard and expressed as percentage. Efficiency should be close to 100%. If not, then the cadmium column may need to be regenerated. See manual for procedure.
   
9. Proceed with samples. For soil extracts, prepare one sampler tube per sample; for soil leachate samples, prepare triplicate sampler tubes per sample. Input standard or sample identifier information and tube number into Omnion FIA program.
   
10. Every 20 sampler tubes or so, include a known standard to check instrument stability and calibration. End the run with another nitrite standard to verify conversion efficiency.
    
11. Report only values that fall between the lowest and highest calibration standards. Report values less that the 0.0 standard, including negative values close to zero, as zero; large negative values would indicate an issue with the instrument performance and warrant further investigation. Samples exceeding the highest standard should be diluted and reanalyzed.
    
12. At the end of the run, turn the knob above the cadmium reduction column on the nitrate channel to isolate the column from the rest of the tubing. This prevents water and air from entering and destroying the column. Transfer reagent lines to a beaker of deionized water and allow the system to rinse for 10-15 minutes.
    
13. Pull the reagent lines from the water and allow air to be pumped through tubing. Store system dry.
    
14. Shut off power switches to autosampler, diluter, reagent pump, and system unit, as well as PC and monitor.

Preparation of Reagents

Follow all lab safety protocols when handling chemicals, including protective eye and skin wear and appropriate lab clothing. Work in fume hood.

Nitrate/Nitrite Reagents:

Derived from: J. Harbridge. 15 August 2007. Nitrate in 2M KCl Soil Extracts, QuikChem Method 12-107-04-1-J. Lachat Instruments, Loveland CO. Note that this method calls for soils extracted in 2M KCl but we use 1M KCl for extraction.

Ammonium Chloride Buffer, pH=8.5 Buffer
• Ammonium Chloride, 80.0 g
• Disodium Ethylenediamine Tetraacetic Acid Dihydrate, 1.00 g
• Sodium Hydroxide pellets, 12.8 g approx.
• Nanopure deionized water

Partially fill a 1 L volumetric flask with ~900 mL Nanopure deionized water. Add first two dry chemicals and dissolve. Adjust to pH 8.5 with sodium hydroxide. Dilute to mark with deionized water and invert to mix.

Sulfanilamide Color Reagent
• 85% Phosphoric Acid, 100 mL
• Sulfanilamide, 40.0 g
• N-1-napthylethylenediamine dihydrochloride, 1.0 g
• Nanopure deionized water

Partially fill a 1 L volumetric flask with ~600 mL deionized water. Add phosphoric acid and dry chemicals. Stir to dissolve (~20 minutes). Dilute to mark with deionized water. Invert to mix and store in dark bottle. Discard when pink.

Ammonia Reagents:

Derived from: J. Harbridge. 15 August 2007. Ammonia (Salicylate) in 2M KCl Soil Extracts, QuikChem Method 12-107-06-2-F. Lachat Instruments, Loveland CO.

EDTA Solution (6.6%), pH 7.0
• Ethylenediamine Tetraacetic Acid Disodium Salt Dihydrate, 66.0 g
• Sodium Hydroxide pellets, approximately 6.1 g
• Nanopure deionized water

Partially fill a 1L graduated beaker with ~800 mL deionized water. Add magnetic stirrer and ETDA salt. Adjust pH from initial pH of ~5.2 to pH 7.0 by adding sodium hydroxide pellets (~25). The solution changes from cloudy to clear as the pH nears 7.0. Transfer to 1 L volumetric flask and dilute to mark with deionized water. Invert to mix. Transfer to 1 L storage bottle.

Buffer
• Sodium Hydroxide, 28.0 g
• Sodium Phosphate Dibasic Heptahydrate, 50.0 g
• Nanopure deionized water

Partially fill a 1 L volumetric flask with ~900 mL deionized water. Add dry chemicals. Dilute to mark with deionized water. Invert to mix.

Salicylate – Nitroprusside Color Reagent
• Sodium Salicylate (Salicylic Acid Sodium Salt), 150.0 g
• Sodium Nitroprusside (Sodium Nitroferricyanide Dihydrate), 1.0 g
• Nanopure deionized water

Partially fill a 1 L volumetric flask with ~800 mL deionized water. Add dry chemicals and dilute to mark with deionized water. Invert to mix. Store in light-proof bottle. Make fresh weekly.

Hypochlorite Reagent
• 5.25% Sodium Hypochlorite, 100 mL
• Nanopure deionized water

In a 200 mL volumetric flask, dilute sodium hypochlorite to mark with deionized water. Invert to mix. Make fresh daily.

Preparation of Calibration Standards

Ammonia and Nitrate Working Standard (10 mg N/ L each)
• Purchased Ammonia standard of 1000 mg N/L, 1 mL
• Purchased Nitrate standard of 1000 mg N/L, 1 mL
• 1M KCL or Nanopure deionized water

Partially fill a 100 mL volumetric flask with ~90 mL of 1M KCl, if samples are soil extracts, or nanopore water, if samples are soil leachate. Add 1 mL of each purchased standard. Dilute to mark with either 1M KCl or deionized water, depending on sample type. Invert to mix.

Nitrite Working Standard (10 mg N/ L each)
• Purchased Nitrite standard of 1000 mg N/L, 1 mL
• 1M KCL or Nanopure deionized water

Partially fill a 100 mL volumetric flask with ~90 mL of 1M KCl, if samples are soil extracts, or nanopore water, if samples are soil leachate. Add 1 mL of nitrite standard. Dilute to mark with either 1M KCl or deionized water, depending on sample type. Invert to mix.