PRINCIPLE

1. Ammonia reacts with alkaline phenol and hypochlorite to form indophenol blue in an amount that is proportional to the ammonia concentration. The blue color is intensified with sodium nitroferricyanide. The absorbance is measured at 660 nm.
2. This is a flow injection technique. 100 l sample extract is injected into a carrier stream prior to the introduction of any reagents into the stream.

EQUIPMENT

1. Perstorp EnviroFlow 3000 AutoAnalyzer, equipped with a NH₄-N cartridge.
2. Random access auto sampler.

REAGENTS

Phenol:	C6H5OH
Sodium Hydroxide:	NaOH
Sodium Hypochlorite, 5.25%:	NaOCl (Commercial household bleach)
Sodium Nitroferricyanide:	Na2Fe (CN)6NO2H2O
Disodium EDTA:	C10H14N2Na2O82H2O
Potassium Chloride:	KCl
BRIDJ-35, 30% W/V:

STANDARDS

1. Stock Solutions:
   1. 1000 ppm NH₄-N: Use certified, NIST traceable standard.
   2. 100 ppm NH₄-N: Pipet 10 mL of 1000 ppm NH₄-N standard into a 100 mL volumetric flask. Dilute to volume with deionized water and mix well.
2. Instrument Calibration Standards:
   1. Pipet the designated amounts of 100 ppm NH₄-N into 100 mL volumetric flasks. Dilute to volume with deionized water and mix well.

      Standard #	Stock Solution mL	Final Concentration ppm	Instrument Readout ppm
      CAL 1	5	5	100
      CAL 2	4	4	80
      CAL 3	3	3	60
      CAL 4	2	2	40
      CAL 5	1	1	20

AUTOANALYZER SOLUTIONS

1. Alkaline Phenol:
   1. Weigh 32 g sodium hydroxide into a 1 liter volumetric flask containing 700 mL deionized water and dissolve.
   2. Cool solution in an ice bath.
   3. Add slowly 83 g phenol (94 mL of liquid phenol) in small increments, cooling in an ice bath after each addition.
   4. Dilute to volume with deionized water and mix well.
   5. Store in a plastic amber bottle. This solution is stable for approximately 1 month.
2. Sodium Hypochlorite Solution:
   1. Add 50 mL of 5.25% NaOCl bleach to a 100 mL volumetric flask.
   2. Dilute to volume with deionized water and mix well.
   3. Store in an amber bottle. Prepare daily.
3. Sodium Nitroferricyanide Solution:
   1. Weigh 0.500 g sodium nitroferricyanide into a 1 liter volumetric flask.
   2. Dilute to volume with deionized water and mix well.
   3. Store in an amber bottle. This solution is stable for approximately 1 month.
4. Stock Complexing Reagent:
   1. Weigh 50 g disodium EDTA into a 1 liter volumetric flask containing 800 mL deionized water and stir with a magnetic stirrer until dissolved.
   2. Add 6 pellets sodium hydroxide.
   3. Dissolve and dilute to volume with deionized water and mix well.
   4. Filter using Whatman #1 paper.
5. Complexing Reagent:
   1. Add 6 drops of Bridj-35, 30% w/v to 250 mL of the stock complexing reagent and mix well.

EXTRACTING SOLUTION

1. 0.1 N KCl - weigh 7.456 g KCl into a 1 liter volumetric flask. Dissolve and dilute to volume with deionized water and mix well.

EXTRACTION

1. Weigh 1 g of plant tissue into a 40 mL cylindrical bottle.
2. Prepare 1 duplicate sample and 1 extraction blank per each 8 samples.
3. Add 20 mL of 0.1N KCl extracting solution.
4. Shake on a reciprocal shaker at high speed (250 oscillations/minute) for 15 minutes.
5. Filter using Whatman #1 paper.

PROCEDURE

1. Place reagent lines into their designated containers.
2. Engage the pump platens and turn on the pump. Allow the pump to run for 10 minutes before beginning sample analysis.
3. Place the instrument calibration standards, quality control standard, and the deionized water blank on the auto sampler tray in the following order.

   Tray Position	Sample ID
   1	CAL 1
   2	CAL 2
   3	CAL 3
   4	CAL 4
   5	CAL 5
   6	QC SAMPLE
   7	BLANK

4. Beginning with position #8, pour up the sample extracts.
5. Select NH4PLNT from the sample table menu.
6. Construct a sample table as follows:

   Cup Position	Name	Type
   1	SYNC	SYNC
   7	BLANK	BLNK
   1	CAL 1	C
   2	CAL 2	C
   3	CAL 3	C
   4	CAL 4	C
   5	CAL 5	C
   3	CCV 1	CCV
   6	QC SAMPLE	U
   8	SAMPLE ID	U

7. Use the letter U to denote sample extracts in the type column.
8. Analyze a CCV 1 check (cup position #3) after each 10 samples and after the last sample. Depending on the number of samples, it may be necessary to refill cup #3 during the run.
9. Run a baseline check after each CCV 1 standard.
10. Select NH4PLNT from the collect data menu.
11. At the operator prompt, enter the number range for the set and begin the analysis.
12. When the set is completed a full report will be printed.
13. Print out the calibration curve, curve statistics, and sample graph separately.
14. Label the calibration curve and sample graph with the date, compound analyzed, and the number range of the samples analyzed in the set.

CALCULATION

1. Sample readout - blank readout = ppm NH₄-N

   Read the value to the nearest whole number.

QUALITY CONTROL

1. The CCV (continuing calibration verification) values must be within 10% of the known value.
2. Values obtained on the quality control sample must agree within the limits established by the originating organization.

NOTES

1. The extraction blank is necessary because filter paper contains a small amount of NH₄-N.
2. The peak numbers on the sample graph will not correspond to the cup positions listed on the report.

REFERENCES

1. Mehlich, A. 1953. Determination of P, Ca, Mg, K, Na, and NH₄. North Carolina Soil Test Division (Mimeo 1953.)
2. Methods for Chemical Analysis of Water and Wastes. March 1984. EPA- 600/4-79-020. "Nitrogen, Ammonia" Method 350.1 (Colorimetric, Automated Phenate) Storet No. Total 00610, Dissolved 00608.