AMMONIA & NITRATE/NITRITE

 

NITRATE/NITRITE

 

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.

 

 

AMMONIA

 

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. 

 

 

 

AUTOANALYZER REAGENTS

 

 

Nitrate/Nitrite

 

Ammonium Chloride Reagent

Ammonium Chloride (NH₄Cl)                      85 g

MQ Water                                                  1000 mL

Brij-35                                                0.5 mL

Ammonium hydroxide( NH₄OH)           6.5 mL (found in hood in acid washing room)

--Dissolve Ammonium chloride in 900 mL Milli-Q water.

Add 6.5 mL of ammonium hydroxide to solution.  Add 0.5 mL of Brij-

35 and dilute to 1 L.

 

Color Reagent

Sulfanilamide (C₆H₈N₂O₂S)                              10 g

Concentrated Phosphoric Acid (H₃PO₄)        100 mL

N-1-Naphthylethylenediamine Dihydro-

     chloride (C₁₂H₁₄N₂ 2HCl)                           0.5 g

Milli-Q water                                       1000 mL

Brij-35                                                   0.5 mL

 

1.  To approximately 900 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 1 liter.

4.  Add Brij-35.  Store in cold, dark place(walk-in cooler). 

          Stability:  one month

 

 

Ammonia

 

Complexing Reagent

Sodium Potassium Tartrate

          (KNaC₄H₄O₆·4H₂O                                33 g

Sodium Citrate [HOC(COONa)

          (CH₂COONa)₂·H₂O]                              24 g

Milli-Q water                                         1000 mL

Brij-35                                                    0.5 mL

 

1.  Dissolve 33 g of potassium sodium tartrate and 24 g of sodium citrate

          in 950 mL of milli-Q water.

2.  Add 2 mL of concentrated sulfuric acid.

3.  Dilute to one liter with milli-Q.

4.  Add 0.5 mL of Brij-35.

 

Alkaline Phenol

 

It Would Be Wise To Wear Gloves and Glasses When Preparing This Solution

 

Phenol(C₆H₅OH)                                                    88 mL

Sodium Hydroxide, 20% w/v (NaOH)             180 mL

Milli-Q Water                                         1000 mL

 

1.  Prepare 20% NaOH solution by adding 36 g of NaOH to 150 mL of MQ and bringing solution to 180 mL with MQ.  Cool.

2,  Dissolve 88 mL (or 83 g) of  phenol in 50 mL of milli-Q water.

3.  While cooling under tap water, cautiously add 180 mL of 20% NaOH

          in small increments.  Stir rapidly while adding the NaOH.

4.  Dilute to one liter with milli-Q water.

 

Sodium Hypochlorite--Stock

Any good commercially available household bleach having 5.25%

          chlorine may be used.

 

Sodium Hypochlorite--Working

Dilute 100 mL of stock sodium hypochlorite to 500 mL with milli-Q.

 

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.

 

 

 

Cadmium Reduction Column Preparation

--Use cadmium from Bran-Luebbe (Technicon Autoananlyzer

          Suppliers

1.  Rinse the powder once or twice with a small quantity of clean diethyl

          ether or acetone to remove grease and dirt.

2.  Rinse with milli-Q water.

3.  Wash with a 1 N HCl solution.  Repeat.

4.  Rinse with milli-Q water; cadmium should be silvery.

5.  Cover cleaned cadmium powder with 30 mL cupric sulfate solution.

          Swirl cadium to allow powdder to contact copper solution.

          Cadmium powder should turn black.  Excess copper will precipitate as

          rust colored particles.

6.  Pour off copper solution and repeat rinse with fresh copper sulfate

          solution.  Repeat this step four times for approximately 3 grams of Cd.

7.  Pour off copper sulfate and rinse with milli-Q water until all rust colored

          precipitate is washed off.  Cadmium should be black.  To protect the     cadmium from exposure to air, cover the cadmium granules with

          ammonium chloride solution.

8.  Attach a purple/purple (2.50 ml/min) pump tube to a small funnel.  Fill the

          tube with ammonium chloride, eliminating any air bubbles by tapping

          the tube.  Clamp off the bottom of the tube just below the purple

          band.

9.  Using an eye dropper, fill pump tube with prepared cadmium.  Be careful

            not to allow the cadmium contact with the air.

10.  Insert a glass wool strand into the top of the tube.  Connect the two ends

          of the tube being careful to keep air bubbles up away from the          column of cadmium.

11.  Place cadmium column in reagent stream on nitrate analysis module.  Be

            sure to have ammonium chloride running through the column,        not milli-Q water.

12.  Condition the column by running a 100 ppm NO₃ standard through

          the system for five minutes.  Don’t introduce the color reagent into the

          system until after the high nitrate standard is finished.  After the

          column has been conditioned with the 100 ppm NO₃, allow the

          ammonium chloride reagent to rinse through the lines for 20 minutes

          before beginning analysis.

13.  The cadmium column is now ready for use in nitrogen analysis.  It may

          also be stored in a container of ammonium chlride reagent.       

 

 

 

 

AMMONIA AND NITRATE COMBINED STANDARDS PREPARATION

 

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

 

 

ANALYTICAL PROCEDURE

 

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 module.  DO 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.

6.  ONLY FILTERED SAMPLES(ODD-NUMBERED) NEED TO BE

            RUN FOR AMMONIA/NITRATE.

 

 

 

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:    8/24/97 by James Thoyre