Method 76 Determination of nitrate in plant material by autoanalysis

See the discussion with references in Chapter 4 'Nitrate and water soluble carbohydrate'. Soil extracts low in colour may also be analysed by this procedure by taking a 10-ml scoop of fresh or thawed soil. The official Bran+Luebbe AutoAnalyzer method for nitrate and nitrite in soil, plant and fertilizer extracts is reproduced with permission in Appendix 5.


• Ammonium chloride buffer, pH 7.5 - dissolve 400 g ammonium chloride (NH4Cl), 40 g EDTA disodium salt, 40 g sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O) and 0.08 g copper sulphate pentahydrate (CuSO4.5H2O) in 1400 ml previously heated water (5 min in a domestic microwave) contained in a 3-l beaker. Adjust the pH to 7.5 ±0.1 with 10% w/v NaOH and make up to 2 l.

• Benzoic acid solution, saturated - see Chapter 4 'Nitrate and water soluble carbohydrate', for details.

• Cadmium filings - using a wood rasp, file cadmium rod held in a vice, carefully collecting the filings in a surrounding sheet of polythene. Sieve the filings, retaining those between 16 and 25 mesh, and save in a stoppered sample tube. Note: cadmium is toxic, so wear gloves and clean the work area carefully afterwards. The filings are used to fill a 380 x 2 mm ID glass tube, plugged at each end for approximately 15 mm with a bunch of fibreglass fibres.

• Orange reagent - warm 1640 ml water containing 200 ml acetic acid, glacial, to approximately 50°C on a magnetic stirrer-hotplate. Add 1.0 g sul-phanilamide and stir until dissolved. Add 1.0 g 1-naphthylamine-7-sulphonic acid (Cleve's acid) previously finely ground in a pestle and mortar. Stir to dissolve, using a thick glass rod to crush any remaining particles. Add 280 ml 10% m/v NaOH, cool and adjust the pH to 4.00 ±0.05 using acetic acid or 10% NaOH added dropwise from a disposable polythene Pasteur pipette. Make up to 2 l and store in a dark glass reagent bottle.

• Sodium acetate buffer - dissolve 20 g NaOH in water and add to a solution of 200 ml acetic acid, glacial, in 1400 ml water. Make up to 2 l and adjust the pH to 4.00 ±0.05.

• Stock standard nitrate solution, 100 pg ml-1 of NO3-N - dissolve 0.3034 g sodium nitrate (NaNO3, previously dried ) in saturated benzoic acid solution and make up to 500 ml and mix.

• Working standard nitrate solutions, 2-15 pg ml-1 of NO3-N - pipette 2, 4, 6, 8, 10 and 15 ml of the stock standard nitrate solution into a series of 100-ml volumetric flasks and make up to 100 ml with saturated benzoic acid solution and mix.

Procedure. Weigh 0.1 g dried herbage into a 250-ml wide-mouth high-density polyethylene screw-cap bottle. The square type bottles fit best the square box of the reciprocating shaker. Add 50 ml saturated benzoic acid solution and shake for 30 min. Filter through a Whatman No. 4 filter paper, rejecting the first few millilitres and save in polythene capped sample tubes. The flow diagram is given in Fig. 7.2.

Switch on the modules for autoanalysis and commence pumping reagents. The wash solution is saturated benzoic acid. Load the standards into a sample tray and analyse at a rate of 20 samples per hour, adjusting the sensitivity of the detection-readout to bring the baseline and top standards on scale. If the samples are all low, the sensitivity should be increased and appropriate lower standards used. After a baseline, analyse a tray of samples plus standards, and follow with a baseline before analysing the second tray of samples and standards. Conclude by aspirating the wash solution to obtain a final baseline.

Cadmium column

Colorimeter/ Spectrophotometer 470 nm

To chart recorder

Double mixing coil

Cadmium column

Double mixing coil

Colorimeter/ Spectrophotometer 470 nm

Sampler 20 h-1 ratio 2:1 sample:wash

Pump sample amm. chloride

sample amm. chloride sodium acetate orange reagent satd benzoic flowcell waste

Fig. 7.2. Flow diagram for determination of nitrate in plant materials.

Calculation. Draw a baseline on the chart under all the sample peaks by connecting the baseline from aspirating wash at the start, between trays and at the end. Read the concentration of the sample solutions by comparing the peak heights of the samples with the standards using a chart reader (see Chapter 1, 'Chart reader'). Divide the concentration of the sample solution in pg ml-1 of NO3-N by 20 to obtain the percentage NO3-N in the sample. If a 10 ml soil sample was extracted into 50 ml saturated benzoic acid solution, the concentration of NO3-N in the extract should be multiplied by 5 to give the concentration of NO3-N in pg ml-1 of NO3-N in the fresh soil. Otherwise, divide by 2000 to obtain the percentage NO3-N in the fresh soil.

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