Authors
Hendrik Poorter, Yvonne de Jong-Van Berkel
Author Affiliations
Hendrik Poorter IBG-2, Forschungszentrum Jülich
Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University
Overview
Nitrate is easy to extract from dried or fresh plant material. Hot or boiling water will extract all the nitrate. The water extract is immediately ready for use in the colorimetric nitrate determination using salicylic acid according to Cataldo et al. (1975). Nitrate can be determined after coupling it to a phenol in concentrated sulphuric acid. After adding a hydroxide a yellow colour develops. Salicylic acid is used as the phenol compound.
Extraction
Materials/Equipment
Reagents
- Deionized water
- Magnesium carbonate/calcium hydroxide (1:2) powder
Equipment
- Water bath
- Table centrifuge
- Tubes with a plastic screw-cap
Procedure
Extraction
- Weigh about 50 mg of dried, ground plant material or 500 mg of fresh plant material in a tube.
- Add 5 ml hot deionized water (90-95ºC), mix thoroughly and add another 5 ml hot deionized water.
- Put the closed tubes in a waterbath at 80ºC for 30 minutes, shake the solution.
- After cooling down centrifuge the open tubes at high speed (4500 rpm – 2649 g) in a table centrifuge.
- Decant the supernatant and weigh the supernatant to determine the exact volume of your extract.
- If leaf material is used, chlorophyll can be removed by adding 0.5 gram of magnesium carbonate/calcium hydroxide to the supernatant. After that, centrifugate at high speed in a table centrifuge.
- The supernatant contains the nitrate extract.
Notes and troubleshooting tips
- There can be problems adding hot water to dry, ground material. The material sometimes floats on the water surface. To avoid this problem, add some TWEEN 20 to the samples.
- Extracts should be stored in the freezer at -20ºC.
- For some species, supernatant may be colored by phenols. These must be removed with PVPP.
Chemical Determination
Materials/Equipment
Reagents
- Salicylic acid
- Sulphuric acid 96%
- Sodium hydroxide (NaOH)
- Sodium nitrate
Equipment
- Spectrophotometer
- Large tubes (25 ml)
Procedure
Preparation of reagent A
- Dissolve 5 g of salicylic acid in 96% sulphuric acid
- Fill up to 100 ml with 96% sulphuric acid
Preparation of reagent B
- Dissolve 40 g of NaOH in deionized water
- Fill up to 500 ml with deionized water
Preparation of standard solution
- Dissolve 0.0680 g of sodium nitrate in deionized water
- Fill up to 100 ml with deionized water (concentration of sodium nitrate is 8.0 mM)
- Dilute the standard solution with deionized water in a range of: 0; 0.1; 0.2; 0.5; 1.0; 2.0; 4.0 and 6.0 mM nitrate.
Determination
- Add 0.8 ml of reagent 1 to 0.2 ml standard or extract
- Mix well
- Leave for 20 minutes at room temperature
- Add slowly 19 ml of reagent 2
- Mix well
- Cool down to room temperature
- The colour is yellow and is stable for 48 hours
- Measure the absorption at 410 nm with a spectrophotometer
Notes and troubleshooting tips
- Prepare reagent A as fresh as possible. Store in a dark bottle.
- Lately a first attempt is made to perform the nitrate determination with smaller volume of reagents and sample. This method is not fully checked. The procedure of this “micro” determination is:
Equipment- Spectrophotometer
- Centrifuge
- Eppendorf vials (1.5 ml)
Procedure
- Add 40 μl of reagent A to 10 μl standard or extract
- Mix well
- Centrifuge for 10 secs with ‘QUICK START’
- Leave for 20 minutes at room temperature
- Add slowly 1 ml of reagent B
- Mix well
- Cool down to room temperature
- The colour is yellow and is stable for 48 hours
- Measure the absorption at 410 nm with a spectrophotometer
Reference values
5-50 mg g-1 NO3, depending on growing conditions. Generally only low values in woody plants.
Literature references
Cataldo, D.A., Haroon, M., Schrader, L.E., Youngs, V.L., 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant. An. 6: 71-80.