US Long-Term Ecological Research Network


This automated procedure for the determination of nitrate and nitrite utilizes the procedure whereby nitrate is reduced to nitrite by a copper-cadmium reductor column. The nitrite ion then reacts with sulfanilamide under acidic conditions to form a diazo compound. This compound then couples with N-1-napthylethylenediamine dihydrochloride to form a reddish-purple azo dye.

This automated procedure for the determination of ammonia utilizes the Berthelot Reaction, in which the formation of a blue colored compound believed to be closely related to indophenol occurs when the solution of an ammonium salt is added to sodium phenoxide, followed by the addition of sodium hypochlorite. A solution of potassium sodium tartrate and sodium citrate is added to the sample stream to eliminate the precipitation of the hydroxides of calcium and magnesium. 




Ammonium Chloride-EDTA Buffer Reagent
Ammonium Chloride (NH4Cl)                             85 g
MQ Water                                                  1000 mL
Disodium Ethylenediamine Tetraacetate (disodium EDTA)
(C10H14N2Na2O8·2H2O)                                 0.5 mL
Ammonium hydroxide( NH4OH)                      6.5 mL (found in hood in acid washing room)
TX10                                                            0.05 mL                                                                               
--Dissolve Ammonium chloride and EDTA in 900 mL Milli-Q water. Add Ammonium hydroxide and dilute to 1 L. Add TX10 to working reagent
Color Reagent
Sulfanilamide (C6H8N2O2S)                             20 g
Concentrated Phosphoric Acid (H3PO4)        50 mL
N-1-Naphthylethylenediamine Dihydro-
     chloride (C12H14N2 2HCl)                           1 g
Milli-Q water                                              500 mL
1. To approximately 400 mL of Milli-Q water add concentrated phosphoric acid and sulfanilamide. Dissolve completely. (Heat if necessary)
2. Add N-1-Naphthyethylenediamine dihydrochloride, and dissolve.
3. Dilute to 500 mL.Store in cold, dark place(brown bottle in the refridgerator). 


Complexing Reagent
Sodium Potassium Tartrate
          (KNaC4H4O6·4H2O                               33 g
Sodium Citrate [HOC(COONa)
          (CH2COONa)2·H2O]                              24 g
Sulfuric Acid                                                  2 mL
Milli-Q water                                              1000 mL
TX10                                                            0.5 mL
1. Completely dissolve 33 g of potassium sodium tartrate in 900 mL of Milli-Q water
2. Add 24 g of sodium citrate.
2. Add 2 mL of concentrated sulfuric acid.
3. Dilute to one liter with milli-Q.
4. Add TX10 to working reagent.
Alkaline Phenol

It Would Be Wise To Wear Gloves and Glasses When Preparing This Solution. Prepare in fume hood
Phenol, liquified (C6H5OH)                                    12 mL
10 N Sodium Hydroxide                                       85 mL
Milli-Q Water                                                      1000 mL
1. Add Sodium Hydroxide to 700 mL water. This is done in an icebath with a magnettic stir bar and stirrer
2. When the solution is cold, add the Phenol in small quantities, colling between each addition.
3. Dilute to one liter with milli-Q water.
4. Store in a brown bottle in the refridgerator
Sodium Hypochlorite--Stock
Any good commercially available household bleach having 6.00% chlorine may be used.
Sodium Hypochlorite--Working
Dilute 2.2 mL of stock sodium hypochlorite to 100 mL with milli-Q. This is prepared daily
Sodium nitroferriccyanide
Sodium Ferriccyanide                                   0.5 g
Milli-Q water                                            1000 mL
--Dissolve 0.5 g of sodium ferriccyanide in 900 mL of milli-Q and dilute to one liter.
Store solution in an amber bottle at room temperature
Cadmium Reduction Column Preparation
--The Astoria analytical cartridge uses an Open Tubular Cadmium Reactor coil
Reagents for OTCR Activation
1. Stock Ammonium Chloride/EDTA Buffer as prepared above
2. Cupric Sulfate Solution
    Cupric Sulfate CuSO4· 5H20                      10 g
    Milli-Q water                                       500 mL
    --Dissolve cupric sulfate in Milli-Q water
3. 1.0 N Hydrochloric Acid
1. Detach one end of the polyethylene tubing from the coil
2.Flush the OTCR with 10 cc of each reagent in the following order
a) Milli-Q water
b) 0.1 N Hydrochloric Acid
c)Milli-Q water
d) Cupric Sulfate Solution (flush this twice)
e) Milli-Q water
f) Stock Ammonium Chloride/EDTA Buffer


Ammonia Primary Stock Standard
          (Made up at 70,171.30 µg/L)
Ammonium Sulfate [(NH4)2SO4]                         0.3310 g
Milli-Q water                                                     1000 mL
Chloroform                                                             1 mL
1. In a one liter volumetric flask, completely dissolve 0.3310 g of ammonium sulfate in 1000 mL of milli-Q. 
2. Add one mL of chloroform as a preservative.
3. Put date of prep and concentration of the stock on bottle and store in brown bottle in the walk-in cooler.
Nitrate Primary Stock Standard
          (Made at 44011.63 µg/L)
Potassium nitrate (KNO3)                                  0.3176 g
Milli-Q Water                                                    1000 mL
1. Place potassium nitrate in a drying oven at 105 ºC for a minimum of one hour. Remove to dessicator.
2. Weigh 0.3176 g potassium nitrate out on balance. Completely dissolve in one liter of milli-Q.
3. Write prep date and concentration of solution on a brown 1 L bottle and store in the walk-in cooler.
Working Standard Solutions
--Working Standards are prepared on a balance in 125 mL S-bottles.
          Must be wet-capped bottles to keep out atmospheric nitrogen and ammonia.
Ammonia Primary Stock Solution                                 3.0 g
Nitrate Primary Stock Solution                                     1.5 g
Intermediate (Standard 20)
1. Place poly bottle on balance. Tare. Add 1.5 g of Nitrate primary stock solution. Record weight.
2. Add 3.0 g of ammonia primary stock solution and record weight.
3. Dilute to 100 g with milli-Q. Record final weight

Working Standards Preparation
Made from Intermediate(Std 20).
          Record weights after each addition to the poly bottle.
Standard 1              16 g of Intermediate. Dilute to 100 g.
Standard 2              10 g of Intermediate. Dilute to 100 g.
Standard 3              5.5 g of Intermediate. Dilute to 100 g.
Standard 4              2.5 g of Intermediate. Dilute to 100 g.
Standard 5              0.5 g on Intermediate. Dilute to 100 g.
Standard 10           10.0 g of Intermediate. Dilute to 40 g.
Blk                       100 mL.
--Calculate the standard concentrations as follows:
NO3= wt Intermediate*conc NO3 in Intermediate/ solution weight
NH4= wt Intermediate*conc NH4 in Intermediate/ solution weight
ICV Preparation
--Intermediates made from 1000 ppm Fisher Reference Standards.
NO3 Int                   0.5 g of 1000 ppm Ref. Dilute to 100 g.
NH4 Int                   0.75 g of 1000 ppm Ref. Dilute to 100 g.
NO3 ICV                   1.0 g of NO3 Int. Add acid and dilute to 100 g.
NH4 ICV                   1.0 g of NH4 Int. Add acid and dilute to 100 g.
--Calculate concentrations as follows:
          wt Int * 1000 ppm * 1000 ppb / wt solution

Start up
1. Switch to the NH4 module. Connect the tubing (make sure to switch the sample tube from ∑N to NO3) and change to the ammonia filters. Be sure to switch the air tube from the open-ended connector to the filtered air connector.
2. Turn on the Technicon (see start up in Dialy Operations Section).
          --must have full liter of ammonium chloride for a full day’s run.
3. Place the auto analyzer lines into the appropriate room temp reagents.
4. Run pump on high speed to get the pump through the lines. After the ammonium chloride is through the lines, place the cadmium column in the nitrate moduleDO NOT ALLOW AIR OR WATER TO GO THROUGH THE CADMIUM COLUMN!
5. Allow the baseline to stabalize for about an hour after the cadmium column is in place.
Run Set-Up
1. The ammonia/nitrate samples must be removed from the freezer in room 337. Thaw by spreading out circulating air around with small fan. Usually takes 1.5 hours before samples are ready. 
2. The samples may now be poured directly from the 20 mL sample vials into conical-bottom auto-analyzer cups.
Tray Set-Up
1. The conical-bottom cups must be filled with 1N HCl and aspirated.
2. Then the cups must be filled with water and aspirated. Repeat step 2.
          The second time it is critical to aspirated all the water droplets out
          of the bottom and sides of the cups.
3. When preparing the tray for analysis, sequence the samples and standards as follows: 
                   positions 1-5 for standards 1-5
                   position 6 is standard Blank
                   positions 7, 18, 29, and 40 are MQ
                   position 8 is ICV
                   positions 9-11 are TLBL’s(only at the start of a month)
4. Run a standard midway through each tray to monitor the cadmium column’s efficiency. 
5. All trays should contain 10% sample blanks(MQ) and 10% duplicates.
Instrument conditions
--Ammonia module: Filter on colorimeter is 630 nm. The standard calibration setting is typically around 0.0. Setting on the Houston Omniscribe are as follows:
                             chart speed: 5 cm/min with toggle switch on ÷10
                             recorder range: .01 V
--Nitrate module: Total nitrogen is analyzed as nitrate after digestion. Reduce sample volume introduced into the reaent stream by reducing the sample pump tubes from .32 ml/min to 0.1 ml/min. Otherwise, set up the autoanalyzer system as for dissolved nitrate plus nitrite analysis.
The filter on the colorimeter is 550 nm. The standard calibration setting is typically around 10.0. The Perkin-Elmer recorder settings are as follows:
                             chart speed: 5mm/min
                             recorder range: 10 mV
Last revised: 5/18/11 by Colleen Sylvester   
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