PRINCIPLE

1. Nitrate is reduced quantitatively to nitrite by cadmium metal in the form of an open tubular coil. The nitrite thus found plus any originally present in the sample is determined as an azo dye at 540 nm, following its diazotization with sulfanilamide and subsequent coupling with N-1-naphthylenediamine dihydrochloride. A dialyzer is added to the system in order to dilute the sample such that analysis values will lie within the linear range of the system.
2. This is a combination flow injection - segmented flow technique. 100 1 of sample extract is collected and injected into a carrier stream. The stream is segmented after injection using helium gas.

EQUIPMENT

1. Perstorp EnviroFlow 3000 AutoAnalyzer, equipped with a NO₃-N cartridge.
2. Random access auto sampler.
3. 24 inch cadmium coil.
4. Dialyzer.
5. Dialysis membranes

REAGENTS

Sulfanilamide:	C6H8N2O2S
N-1-naphthylenediamine dihydrochloride:	C12H14N22H2O
Potassium Chloride:	KCl
Ammonium Chloride:	NH4Cl
Ammonium Hydroxide:	NH4OH
Phosphoric Acid:	H3PO4
Disodium EDTA:	C10H14N2Na2O82H2O
Brigj-35, 30% W/V:

STANDARDS

1. Stock Solutions:
   1. 1000 ppm NO₃-N: Use certified, NIST traceable standard.
   2. 100 ppm NO₃-N: Pipet 50 mL of 1000 ppm NO₃-N standard into a 500 mL volumetric flask. Dilute to volume with deionized water and mix well.
2. Instrument Calibration Standards: Pipet the designated amounts of 100 ppm NO₃-N standard into 100 mL volumetric flasks. Dilute to volume with deionized water and mix well.

   Standard #	Stock Solution mL	Final Concentration ppm	Instrument Readout ppm
   CAL1	50	50	20000
   CAL2	30	30	12000
   CAL3	20	20	8000
   CAL4	10	10	4000
   CAL5	5	5	2000

AUTOANALYZER SOLUTIONS

1. Ammonium chloride - EDTA buffer, pH 8.5:
   1. Weigh 85 g ammonium chloride into a 1 liter beaker containing 900 mL deionized water. Stir until dissolved.
   2. Weigh 0.1 g disodium EDTA into the beaker and stir until dissolved.
   3. Adjust the pH to 8.50 using ammonium hydroxide.
   4. Transfer to a 1 liter volumetric flask and dilute to volume with deionized water.
   5. Add 2 mL Bridj-35, 30% W/V and mix well.
2. Color Reagent:
   1. Add 50 mL phosphoric acid to a 500 mL volumetric flask containing 400 mL deionized water.
   2. Weigh 1 g N-1-naphthylenediamine dihydrochloride into the flask. Swirl to dissolve.
   3. Dilute to volume with deionized water and mix well.
   4. Filter using Whatman #1 paper and store in an amber bottle. This solution is stable for several months.

EXTRACTING SOLUTION

1. 0.1 N Potassium Chloride:
   1. Weigh 7.456 g potassium chloride into a 1 liter volumetric flask containing 800 mL deionized water.
   2. Dilute to volume with deionized water and mix well.

EXTRACTION

1. Weigh 0.100 g cotton petiole into a 25x200 mm test tube.
2. Reweigh each eighth sample and place at the end of the set.
3. Add 40 mL of 0.1 N potassium chloride solution (1/400 extraction ratio.)
4. Shake on a reciprocal shaker at high speed (250 oscillations per minute) for 15 minutes.
5. Filter using Whatman #1 paper.
6. After the tenth week of the cotton petiole sampling season, change the extraction ratio to 1/200 by adding 20 mL of 0.1 N KCl solution in step 3 above. Make the corresponding changes in the Instrument Readout Values for the Instrument Calibration Standards.

PROCEDURE

1. Place reagent lines in 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 sample, and deionized water blank in 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 NO3PET from the sample menu table menu.
6. Construct a sample table consisting of standards and samples in the following manner:

   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 sample it may be necessary to refill cup three during the analysis of the set.
9. Run a baseline check after each CCV 1.
10. Select NO3PET method from the data collect menu.
11. At the OPERATOR prompt, enter the range of sample numbers in the set, and begin the analysis.
12. When the analysis is complete, 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. Results are reported as ppm NO₃N. Round off the value to the nearest 100 ppm NO₃N.

QUALITY CONTROL

1. Values for the CCV (continuing calibration verification) must agree within 10% of the known value.
2. Values obtained on the quality control sample must agree within the limits established by the originating organization.
3. Values on the duplicate samples must be within 20% of the average of the two samples.

NOTES

1. The quality control sample and standard stock solution should be from different sources.
2. The peak numbers on the sample graph will not correspond to the cup positions listed on the report.

REFERENCES

1. Dorich, R.A. and D.W. Nelson. 1984. Evaluation of Manual Cadmium Reduction Methods for Determination of Nitrate in Potassium Chloride Extracts. Soil Sci. Soc. Am. J. 48:72-75.
2. Huffman, S.A. and K.A. Barbarick. 1981. Soil Nitrate Analysis by Cadmium Reduction. Comm. Soil Sci. Plant Anal. 12(1):79-89.
3. Methods for Chemical Analysis of Water and Wastes. March 1984. EPA- 600/4-79-020. Nitrogen, Nitrate-Nitrite, Method 353.2 (Colorimetric Automated, Cadmium Reduction) Storet No. Total 00630.