US Long-Term Ecological Research Network

This procedure determines the total nitrogen (TN) and total phosphorous(TP) in filtered and unfiltered samples.The material that cannot pass a .45 micron filter is considered particulate nitrogen or phosphorous, and is determined by the difference between the unfiltered and the filtered values. Water samples and standards are autoclaved in an alkaline persulfate digestion solution. The digestion begins at a pH of 12, necessary to break nitrogen bonds, and , during the course of the autoclaving, the pH drops to 2.3 (H2SO4 forming from the K2S2O8), the lower pH required to break organic phosphorous bonds.

In an alkaline environment, the persulfate oxidizes the organic nitrogen and ammonia to nitrate, and, in addition to the inorganic nitrate present, represents Total Nitrogen.(Some nitrogen compounds with triple and double bonds may not be affected by the digestion.)
As the digestion proceeds to acidic conditions, the persulfate oxidizes phosphorous compounds to ortho-phosphate.
After digestion, samples and standards are analyzed using a Technicon Autoanalyzer II. The autosampler splits the sample, and runs it through the ortho-phosphate and nitrate detection modules of the autoanalyzer simultaneously.
In the nitrate module, a copper-cadmium reductor column reduces the nitrate to nitrite. The nitrite ion then reacts with sulfanilamide under acidic conditions to form a diazo compound. This compound then couples with a color reagent to form a reddish-purple dye proportional to the nitrite concentration.
In the TP determination, the ortho-phosphate (PO4) reacts with ammonium molybdate in the presence of H2SO4 to form a phosphomolybdenum complex. Antimony potassium tartrate and ascorbic acid reduce the complex, forming a blue color which is proportional to the TP concentration.
                                                Digestion Reagent
Sodium Hydroxide (NaOH)                       12.0 g
Potassium persulfate (K2S2O8)*                30.5 g
Milli-Q water q.s.                                   1000 ml
1. Dissolve sodium hydroxide in 970 ml of fresh Milli-Q water. 
2. Dissolve 30.5g twice recrystallized potassium persulfate in
          NaOH solution. Bring to 1 L with MQ.
*Twice recrystallized potassium persulfate. Baker ® low nitrogen potassium persulfate is used, but needs to be recrystallized twice to remove nitrogen contamination from the reagent.
Potassium persulfate recrystallization
 1.     Dissolve 100 g of potassium persulfate in approximately 600 ml of Milli-Q previously heated to 60˚ C. Use a medium sized stir bar and a 1000 mL flask.
 2.     Filter the solution rapidly through a sintered glass funnel.
 3.     Rinse the 1000 mL flask.
 4.     Poor filtrate back into the flask used to heat the potassium persulfate solution.
 5.     Cool solution to about 4˚ C by placing the flask in ice water. Whirl the flask continuously to prevent the solution from freezing.
 6.     Filter the 4˚ C solution and wash with 1 or 2 squeezes of ice cold Milli-Q, save the white solid.
 7.     Discard the filtrate from the sidearm flask.
 8.     Rinse the flask used to cool the solution with Milli-Q
 9.     Fill the flask with 450ml of Milli-Q and heat to 60˚ C.
10.     Add the crystals from step 5 and mix into solution.
11.     Repeat steps 4 and 5. The white granules on top of the filter are crystals!
12.     Dry crystals in vacuo over anhydrous calcium chloride. Rapid drying in a good vacuum and thus at a low temperature is essential as this will minimize the sulfuric acid formation on the crystals.      
The yield is about 80%. The effect is illustrated by the blank obtained in the standard procedure-- .178 for the original reagent, .020 after one recrystallization and .010 µmole of N after two recrystallizations.


                                      Autoanalyzer Reagents
Phosphorous reagents
Stock Solution A, 4.9 N Sulfuric acid:
Concentrated sulfuric acid (H2SO4)                136 ml
Milli-Q water                                               1000 ml
--Add acid to 800 ml of water. Cool and dilute to 1 L.
Stock Solution B, Ammonium molybdate:
Ammonium Molybdate [(NH4)6Mo7O24H2O]      20 g *don’t use MCB
Milli-Q water                                                   500 ml
--Dissolve ammonium molybdate in 900 ml of Milli-Q water and dilute to 1 L. Store at 4° C.
Stock Solution C, Ascorbic acid:
Ascorbic acid (C6H8O6)                                         9 g
Milli-Q water                                                   500 ml
--Dissolve ascorbic acid in 400 ml of Milli-Q water and dilute to 500 ml. Store at 4° C. Keep well stoppered. Prepare fresh monthly or as needed. 
Stock Solution D, Antimony potassium tartrate:
Antimony potassium tartrate
[(K(SbO)C4H4O61/2H2O]                                       1.5 g
Milli-Q water                                                        500 ml
--Dissolve antimony potassium tartrate in 800 ml of Milli-Q water and dilute to 1 L. Store at 4° C.
∑P Combined Color Reagent*:
Stock Solution A                                              100 ml
Stock Solution B                                                30 ml
Stock Solution C                                                60 ml
Stock Solution D                                                 10 ml
Aerosol 22                                                              0.2 ml
*Use the combined color reagent for autoanalyzer analysis. Combine the solutions in order, mixing after each addition. Prepare fresh daily.
Total Nitrogen Reagents (as NO3)
Ammonium Chloride
Ammonium chloride (NH4Cl)                        85 g
Milli-Q water q.s.                                          1000 ml
Brij-35                                                              0.5 ml
Ammonium hydroxide (NH4OH)                          6.5 ml
--Dissolve ammonium chloride in 900 mL Milli-Q water. Add 6.5 ml 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 dihydrochloride
(C12H14N22HCl)                                                    0.5 g
Milli-Q water, q.s.                                         1000 ml
Brij-35                                                             0.5 ml
  1. To approximately 900 ml of Milli-Q water add concentrated        phosphoric acid.
  2.  Once solution is thoroughly mixed add sulfanilamide. Dissolve completely.     (Heat if necessary)
3. Add N-1 naphthyethylenediamine dihydrochloride, and dissolve. Dilute to 1 L.
4. Add Brij-35. Store in a cold, dark place. Stability: one month.
2% Copper Sulfate Solution
Cupric sulfate [(CuSO4)5H2O]                             10 g
Milli-Q water                                                   500 ml
Dissolve cupric sulfate in 400 ml Milli-Q water. Dilute to 500 ml.

Cadmium Reduction Column Preparation
Use cadmium from Bran-Luebbe-- Technicon Autoanalyzer 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; Cd should be silvery.
5. Cover cleaned cadmium powder with 30 ml cupric sulfate solution. Swirl cadmium to allow powder to contact copper solution. Cadmium powder should turn black. Excess copper will precipitate as rust colored particles.
6. Pour off copper sulfate 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 to contact 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 system for 5 minutes. Don’t introduce 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 analysis.
13. The cadmium column is now ready for use in nitrogen analysis or may be stored in a container of ammonium chloride reagent.
Phosphorous and Nitrate Combined Standards Preparation
Phosphorous Primary Stock Standard
          (Made at 49993.45 µg/L)
Potassium phosphate, monobasic (KH2PO4)...................0.2197 g
Milli-Q water                                                          1000 ml
1. Place potassium phosphate in drying oven at 105 °C for a      minimum of one hour. Remove to dessicator.
2. Weigh phosphate on analytical balance. Dissolve in 800 ml of           milli-Q water. Dilute to 1 L. Store in cooler.
Nitrate Primary Stock Standard
          (Made at 44011.63 µg/L)
Potassium nitrate (KNO3)                                  ..........0.3176
Milli-Q water                                                          1000 ml
1. Place potassium nitrate in drying oven at 105 °C for a minimum of    one hour. Remove to dessicator.
2. Weigh potassium nitrate on analytical balance. Dissolve in 800 ml     milli-Q water. Dilute to 1 L. Store in cooler.
Working Stock and Working Standards are prepared on a balance in 125 ml poly bottles. Diluent is 1% (vol/vol)HCl.
1% (vol/vol)HCl
HCl Ulrex brand                                                                   20 ml
Milli-Q water                                                          2000 ml
Working Stock Standard Solution
Phosphorous Primary Stock Solution (50,000 µg P/L)                   1 ml
Nitrate Primary Stock Solution (44,000 µg NO3-N/L)                               6 ml
1. Place poly bottle on balance. Tare. Add stock P, record weight.
2. Add stock N, record weight.
3. Dilute to 100 g with 1% HCl(Ultrex), and record weight.
4. Calculate the ∑N and∑P concentrations as shown below.
     g P stock * conc P stock ÷ total solution weight = µg/l (ppb) P
     g NO3 stock - g P stock = g NO3 stock
     g NO3 * conc NO3-N stock ÷ total solution weight = µg/L (ppb) N
Working Standards
          Record weights after each addition to poly bottle.
Standard 1         20 ml working stock. Dilute to 100 ml with 1% HCl.
Standard 2         15 ml working stock. Dilute to 100 ml with 1% HCl.
Standard 3         10 ml working stock. Dilute to 100 ml with 1% HCl
Standard 4           5 ml working stock. Dilute to 100 ml with 1% HCl.
Standard 5           1 ml working stock. Dilute to 100 ml with 1% HCl.
Standard 10       20 ml working stock. Dilute to   40 ml with 1% HCl.
Blank                   0 ml working stock.   Dilute to 100 ml with 1% HCl.
Calculate working standard concentrations as follows:
P conc.=wt working stock* conc. P in working stock ÷ solution wt.
N conc.=wt working stock* conc. N in working stock ÷ solution wt.
Analytical Procedure:
1. Switch to the ∑P module. Connect the tubing(make sure to             switch the sample tube from nitrate to ∑N), plug in the
          heating element, and change the filters.
2. Turn on the Technicon(see start up in Daily Operations.)
          --must have one liter of ammonium chloride to run a full day.
3. Place the NO2 Color Reagent, the ammonium chloride, and the
          newly MIXED ∑P color reagent lines into proper bottles.
4. Run pump on high speed to get reagents through the lines. After
          the ammonium chloride is in lines, place cadmium column in
5. Allow baseline to stabalize for about an hour after the cadmium
          column is in place. 
1. ∑N&∑P vials must be removed from specifically dedicated acid        bath shortly before use. They should be rinsed with MQ six          times and wet-capped.  
2. Allow time for outsides of vials to dry and then use tape to label      the glass sample vials.
3. Aliquot 5 ml sample (well mixed) or standard into glass culture         tube.
4. Deliver 2.5 ml of Digestion reagent to each tube with a repipet         dedicated for that use.
5. Cap tube and vortex, then vent.
6. Autoclave samples and standards for 60 minutes at 18-20 psi. 
7. Remove samples and standards from autoclave after temperature    dial indicates less than 70 °C.
Samples are ready to be run for total N and total P.
Instrument conditions:
-Ortho-phosphate module: Analytical cartridge has a heating bath that runs at 37 °C. Filter on colorimeter is 880 nm. The standard calibration setting is typically around 0.0. Settings 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 the sample volume introduced into the reagent stream by reducing the sample pump tubes from .32 ml/min. to .1 ml/min. Otherwise, set up the autoanalyzer system as for dissolved nitrate plus nitrite analysis.
The filter on colorimeter is 550 nm. The standard calibration setting is typically around 10.7. The Perkin-Elmer recorder settings are as follows:
                   chart speed: 5mm/min.
                   recorder range: 10 mV
          The autosampler tray holds 40 cups. Conical-bottomed cups must be rinsed first with 1 N HCl then rinsed twice with milli-Q water . All residual water should be thoroughly aspirated from the cups to avoid any dilution or contamination effect.
          When preparing the tray for analysis, sequence the samples and standards as follows: positions 1-5 are occupied by standards 1-5, position 6 by a standard blank, and positions 7, 22 or 24, and 40 by milli-Q. Position 7 is occupied by an ICV. TLBL’s must also be run at the beginning of the first run. Run a standard midway through each tray to monitor the cadmium columns efficiency. All trays should contain 10% sample blanks and 10 % sample replicates.

D’Elia, C., P. Steudler, and N. Conwin. 1977 Determination of total
     nitrogen in aqueous samples using persulfate digestion. Limnol.
     Oceanogr. 22: 760-764.
Kalff, J., and E. Bentzen. 1984. A method for the analysis of total
     nitrogen in natural waters. Can. J. Fish. Aquat. Sci. 41: 815-819.
Langner, C.L., and P.F. Hendrix. 1982 Evaluation of a persulfate
     digestion method for particulate nitrogen and phosphorus. Water
     Res. 16: 1451-1454.
Nydahl, F. 1978. On the Peroxodisulphate oxidation of total nitrogen
     in waters to nitrate. Water Res. 12: 1123-1130.
Solorano, L., and J.H. Sharp. 1980. Determination of total dissolved
    nitrogen in natural waters. Limnol. Oceanogr. 25: 751-754.
Last revised 8/24/97 by James Thoyre
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