2.0 Definition
The concentration of reactive nitrate is given in mmol kg -1 in seawater.

3.0 Principle of Analysis
The determination of nitrate is based on the method of Morris and Riley (1963) and modified by Strickland and Parsons (1968). Nitrate is reduced to nitrite using a cadmium-copper column. The nitrite produced reacts with sulfanilamide in an acid solution. The resulting diazonium compound is coupled with N-(1-Naphthyl)-ethylenediamine dihydrochloride to form a colored azo dye, the extinction of which can be measured spectrophotometrically.
The following stoichiometric equations apply.

3.1 Nitrate is reduced using a copper-cadmium column:
        NO₃^- + Me _(s) + 2H⁺ ->NO₂^- + Me²⁺ + H₂O

3.2 NO₃^- can easily be reduced further to NO due to the similar electromotive forces (E o ) of the reactions:
        NO₃^-  + 3H⁺ + 2e^- -> HNO₂ + H₂O (Eo = 0.94 V)
        NO₃^-  + 4H⁺ + 3e^- -> NO + 2H₂O (Eo = 0.97 V)

3.3 To ensure that this does not occur, the reaction takes place in a neutral or slightly
alkaline solution.
        NO₃^-  + H₂O + 2e^- -> NO₂^- + 2OH^- (E o = 0.015 V)

3.4 Ammonium chloride in the sample stream acts as both a complexant and as a buffer.
2NH₄ + <--> 2NH₃⁺ 2H⁺
Cd²⁺ + 2NH₃ ->[Cd(NH₃)₂ ]

4.0 Apparatus
Spectrophotometer

5.0 Reagents
5.1 Concentrated ammonium chloride solution: Dissolve 125 g of reagent grade ammonium chloride (NH₄Cl) in 500 ml of de-ionized water. This solution may be stored in a glass or plastic bottle.

5.2 Dilute ammonium chloride solution: Dilute 50 ml of the concentrated ammonium chloride (NH 4 Cl) solution to 2000 ml with de-ionized water. Store in a glass or plastic bottle.

5.3 Sulfanilamide solution: Dissolve 5 g of sulfanilamide in a mixture of 50 ml of concentrated hydrochloric acid and about 300 ml of de-ionized water. Dilute to 500 ml with de-ionized water. This solution is stable for many months.

5.4 N-(1-Naphthyl) - ethylenediamine dihydrochloride solution: Dissolve 0.50 g of the dihydrochloride in 500 ml of de-ionized water. Store in a dark bottle and renew monthly or if a brown coloration develops.

5.5 Copper sulfate stock solution: 2% w/v in de-ionized water. Dissolve 20 g cupric sulfate pentahydrate, CuSO 4 *5H 2 O in 1 liter de-ionized water. Stable at room temperature.

6.0 Sampling
6.1 Samples are collected on deeper casts after the oxygen, CO₂ and salinity samples have been drawn. Shallow samples (upper 250 m) are collected on the gases cast after oxygen, CO₂ , DOC and salinity samples are drawn. An in-line filter (0.8 mm Nuclepore filter) is connected to the Niskin bottle. The spigot is opened and three sets of samples are collected from the water that passes through the filter by gravity filtration. Each bottle is rinsed three times and then filled just below the shoulder. Care must be taken to avoid overfilling of samples to be frozen. These bottles are transferred to a freezer (- 20°C) and kept frozen until analysed.

6.2 Contamination is a major problem with nutrient samples, especially in the upper ocean where the ambient concentrations are low. All the nutrient bottles are rigorously cleaned before use. The cleaning begins by a wash with a detergent (Aquet) followed by a rinse with 10% HCl, three rinses with deionized water, and a final
rinse with de-ionized water.

6.3 Prolonged storage of samples is not advisable, even if frozen.

7.0 Procedures
7.1 Cadmium - copper column material: Stir 100 g of acid-washed cadmium filings with 500 ml of a 2% (w/v) solution of copper sulphate pentahydrate, CuSO₄ * 5H₂O, until all blue coloring has left the solution and copper particles enter the supernatant. This material is then used to pack the reduction columns, utilizing a small plug of copper “wool” as a plug at the bottom and top of the column. Columns are about 30 cm long and have a flow rate of about 10 ml/minute. The columns are washed with dilute ammonium chloride solution and the column material completely covered by dilute ammonium chloride solution when not in use. Note: About 50 g of cadmium filings are required for a column. In addition, cadmium that has become inefficient at reduction may be regenerated by washing with 5% (v/v) hydrochloric acid (300 ml for the cadmium from four columns) and rinsing with 300 ml portions of deionized water until the pH of the wash is greater than 5. The cadmium is then retreated with the copper sulphate solution before use.

7.2 Sample analysis

7.2.1 Samples should be at a temperature between 15° and 30°.

7.2.2 Add 1.0 ml of concentrated ammonium chloride solution to 100 ± 2 ml of sample in a 125 ml Erlenmeyer flask. Mix the solution.

7.2.3 Pour about 5 ml onto the top of the column and allow it to pass through.

7.2.4 Add the remainder of the sample to the column and collect the effluent back into the Erlenmeyer flask. Use the first 45–50 ml to wash the flask and a 50 ml graduated cylinder. Collect the next 50 ml in the graduated cylinder and pour this back into the flask. Allow the remaining sample to drain out through the column. Some dilute ammonium chloride may then be used to wash the column although this is not necessary unless the columns are not to be reused for periods exceeding one hour.

7.2.5 As soon as possible after reduction, add 1.0 ml of sulfanilamide solution to the sample in the flask and mix. Permit the reagent to react for between 2 and 8 minutes.

7.2.6 Add 1.0 ml of N-(1-Naphthyl)-ethylenediamine dihydrochloride solution and mix immediately.

7.2.7 Measure the extinction of the samples at 543 nm between 10 minutes and 2 hours after the addition of the naphthylethylenediamine reagent. Absorbances of less than 0.1 in a 1 cm cell should be re-read in a 10 cm cell.

7.3 Reagent Blank Determination: A reagent blank is barely significant when working with a 1cm cell but gains considerable importance when a 10cm cell is used. In either case it should be checked throughout each analysis. The reagent blank is determined using deionized water as sample and following the procedure outlined in section 7.2. Add the concentrated ammonium chloride solution to 100 ml of deionized water in a clean Erlenmeyer flask and use a column previously flushed with at least 50 ml dilute ammonium chloride solution just before. The absorbance of the blank should not exceed 0.1 using a 10 cm cell.

7.4 Standardization

7.4.1 Primary nitrate standard:
Dissolve 1.011 g of analytical reagent quality potassium nitrate in 1000 ml of deionized water. 1 ml=10 mmol N

7.4.2 Working nitrate standard:
Dilute 4 ml of primary nitrate standard up to 2000 ml in low nutrient seawater.  The solution should be stored in a dark bottle and prepared fresh immediately  before each use (20 mM).

7.4.3 Run approximately 100 ml of working standard solution as described in section 7.2. This should be performed in triplicate initially for each column. Thereafter, standards are to be run with each batch of samples to check the efficiency of the reduction columns.

8.0 Calculation and expression of results
8.1 A standardization factor F can be calculated as:

                    F = (20mmol/kg ) / ( E_s - E_b)
where:
                    20 mmol/kg = concentration of the standard
                    E s = mean absorbance of the standards
                    E b = mean absorbance of the blanks

8.2 The nitrate concentration is calculated by:
                    mM NO 3 = corrected absorbance ´ F - 0.95C
where:         F = standardization factor
                    C = concentration of nitrite present in the sample
                    corrected absorbance = sample absorbance - reagent blank

With good columns, 5% of the nitrite is reduced leading to a correction of 0.95 times the nitrite concentration of the sample is made.

9.0 Notes
9.1 The cadmium-copper column:
The column deactivates through continual use. The addition of the ammonium chloride should slow this process. A well-packed column should be capable of reducing at least 100 samples. There is no need to wash the columns between the samples, but if the columns are not to be used for over an hour, 50 ml of dilute ammonium chloride should be run through the system. This aids in extending the life of the column.
The columns should be stored completely covered in liquid.

10.0 References
Morris and Riley. (1963). Analytica Chimica Acta, 29, 272–279.
Strickland, J.D.H., and Parsons, T.R. (1968). Determination of reactive nitrate. In: A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada, Bulletin 167, 71–75.
Grasshoff, K, M. Ehrhardt, M. and K. Kremling (1983). Determination of nutrients. In: Methods of Seawater Analysis. p. 143.