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Determination of nitrate in plant and soil


This method is used to know the quantity of Nitrate-N in soil and plant extracts. Nitrate in plants and soil is calculated quantitatively by salicylic method using Spectrometer. Nitrate content present in soil tells about the nutrients required for the growth and good yield in plants. This method is also used to determine the concentration of phosphate present in both natural and wastewater sources.


Spectroscopy is the technique used to calculate the concentration of a chemical element or chemical compound in a solution with the help of a colour reagent. The intensity of the color of a solution and the concentration of an analyte species present in this solution has a linear relationship.


3.1 LCR – Linear Calibration Range


4.1 Health effects of nitrate

Till now no serious health hazards such as carcinogenicity or toxicity is found in adults. But in some rare cases, methemoglobinemia, which is a serious health condition, can occur in babies, which is also known as blue-baby syndrome. This occurs in infants who are less than 6 months of age and is not yet properly able to digest high concentration of nitrates. The baby turns blue due to the decrease in the amount of oxygen carried by blood as the nitrate was not converted to nitrite in the body.

4.2 Health effects of sodium hydroxide

Inhalation can cause severe breathing problems such as irritation if inhaled in large amounts. Severe burns, blistering and pain can occur when in contact with skin. Contact with eyes can lead to blurred vision or even permanent loss of vision. Symptoms such as nausea, stomach cramp, vomiting and diarrhea are used to identify ingestion, which may cause severe burns and lead to death.

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4.3 Health effects of salicylic acid

It causes serious burns both thermally and chemically due to dehydration. It is highly corrosive in nature. Ingestion can lead to internal burns, organ damage, and death while exposure with eyes leads to permanent blindness.



6.1 Samples should be collected in clean containers.

6.2 A chain of custody form should be attached to the samples for analysis.

6.3 Samples of soil and plant must be properly stored as soon as the laboratory receive the samples from the clients.

6.4 Soil and plant samples must be dried at temperature 55 ˚C and 65 ˚C

6.5 1 ml of conc H2SO4 can be added to the 1L of sample solution for the preservation of solution.


7.1 Equipment

7.1.1 Cary 60 UV-Vis Spectrophotometer

7.1.2 Graduate cylinder 50 ml, 100 ml

7.1.3 Beaker, 150 ml

7.1.4 Flask

7.1.5 Funnel tubes

7.1.6 Pipette

7.1.7 Dropper

7.1.8 Test tubes

7.1.9 Quartz cells

7.2 Reagents

7.2.1 Salicylic acid-H2SO4

Dissolve 5g of salicylic acid in 100 ml conc H2SO4

7.2.2 N-NaOH

Dissolve 40.g of NaOH pellets into 100 ml of distilled water. Transfer to a 500 ml volumetric flask and make up to 500 ml with distilled water.

7.2.3. Distilled water


8.1 Calibration and Standardization

8.1.1 All the instruments used must be calibrated at required regular intervals when there calibration is expected to be changed

8.1.2 Every six months, LCR (Linear Calibration Range) must be checked as it is expected to be change

8.1.3 A blank and three standard solutions are used to verify the samples

8.1.4 The resulting graph should have linear curve as the intensity of the color of a solution and the concentration of an analyte species present in this solution has a linear relationship

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8.2 Quality Control

8.2.1 All the bottles used must be rinse with water reagent before and after the use

8.2.2 When the sample is collected, it must be cooled at 4 ˚C

8.2.3 1 ml of conc. H2SO4 can be added to the 1L of sample solution for the preservation of solution


9.1 General Testing Procedure

1. Turn on spectrophotometer

2. Pipette an aliquot (e.g. 0.25 mL) of extract or standard into a 50-mL Erlenmeyer flask

3. Mix thoroughly with 0.8 mL of 5% (w/v) salicylic acid in conc. H2SO4

4. After 20 minutes at room temperature, add 19 mL of 2 N NaOH to raise the pH above 12

5. Cool samples to room temperature

6. Measure absorbance at 410 nm

9.2 Reagent Preparation

Salicylic acid-H2SO4

Dissolve 5g of salicylic acid in 100 mL of conc. H2SO4


In a 250 mL beaker dissolve 40.0 g of NaOH in about 100 mL of DI water. Then transfer it to a 500 mL volumetric flask and make up to 500.0 mL with DI water

Standards Stock solution 0.25 g/L NO3-N (=250mg/L, 250 ug/mL)

Dissolve 1.805 g potassium nitrate in DI water in 1.0 L flask, make sure that all KNO3 is dissolved then make it to the mark with DI water Prepare series of standards containing ~0 to 60ug NO3-N in a 0.25 mL aliquot


Beckmann Acta III Spectrophotometer shows maximum absorption at 410 nm. Absorption should be determined directly. Flask contents should be diluted 1:10 with water before reading the absorbance. Salicylic acid method is suitable for sample containing 500 – 30″,000 ug NO3- -N per gram dry weight. With dilution the upper range can be extended 10 times. Colored development is rapid and is stable for at least 48 hours in light or dark. When more water is present sensitivity and response linearity to NO3- -N is lost.

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Nitration of Salicylic acid (2-Hydroxybenzoic acid) should occur principally at Carbon-5 which is para to hydroxyl group. The hydroxyl group is ortho or para directing while the carboxylic group is primarily mata directing. Both functional groups promote nitration of Salicylic acid at Carbon-5. This suggests that nitration occurs primarily at one site on the salicylic acid molecule. The rate of reaction with nitrate ions is rapid enough for binding of other ions at the reactive signs.


• Isolate the waste materials according to their category.

• Do not mix chemicals in a single container for the ease of handling.

• All the waste materials should be packed with suitable materials (liquids in leakproof containers, solid wastes in plastic bags).

• Label all the container with category of chemicals and chemicals used.

• Finally, the waste materials should be sent to an approved waste disposal facility.


12.1 D.A. Cataldo et al. Rapid Colorimetric determination of Nitrate in plant tissue by nitration of Salicyclic acid




12.5 Samira A. Ben Mussa et al. Determination of Available Nitrate, Phosphate and Sulfate in Soil Samples. International Journal of PharmTech Research 2009, Vol.1, pp 598-604.

12.6 Dominic Anthony Cataldo. Researchgate.

12.7… • PDF file

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