Photometric Determination of Nitrite in Soil Using Griess Method

Introduction

Nitrite is a rich source of nitrogen found in soil, for plants. In the intermediate step of nitrification, ammonia (NH₃) is aerobically oxidized first to nitrites¹. Nitrites are found in soil amended with composts matured to be used to improve crop yield. It has been studied that nitrites get accumulated in composts after the exudence of ammonia, when only ammonia-oxidizing bacteria get proliferated, with the natural eradication of nitrite oxidizing bacteria (NOB)². Nitrite accumulation leads to nitrous oxide (N₂O), a greenhouse gas³ emitted from soil. It is important to analyze compost-amended soils for nitrite, to mitigate nitrous oxide emission and the consequential ill-impact on the environment.

Experimental

This application note details the photometric determination of nitrite in soils using the Griess method, after extraction with a calcium chloride solution. 

Method

In acidic solution nitrite ions react with sulfanilic acid to form a diazonium salt, which in turn reacts with N-(1-naphthyl) ethylenediamine dihydrochloride to form a red-violet azo dye. This dye is determined photometrically.

The method is analogous to EPA 354.1, APHA 4500-NO₂- B, DIN EN 26777, and ISO 6777.

Measuring Range

Applicable Sample

Soil samples

Reagents, Instruments and Materials

Nitrite Test Kits & Reagents

For the measurement, one of the following Spectroquant^® test kits is necessary.

• Spectroquant^® Nitrite Cell Test (1.14547)
• Spectroquant^® Nitrite Test (1.14776)

Instrument(s) & Devices

For the measurement, one of the following Spectroquant^® photometers is necessary:

• Spectroquant^® VIS Spectrophotometer Prove 100 Plus (1.73026)
• Spectroquant^® UV/VIS Spectrophotometer Prove 300 Plus (1.73027)
• Spectroquant^® UV/VIS Spectrophotometer Prove 600 Plus (1.73028)
• Spectroquant^® Colorimeter Move 100 (1.73632)

Note: Also, legacy Spectroquant^® instruments are suitable.

Software for Data transfer

• Optional Spectroquant^® Prove Connect to LIMS software package (Y.11086) to transfer your data into an existing LIMS system.

Instrument Accessories

• Rectangular cells 10 mm (1.14946) 
• Rectangular cells 20 mm (1.14947) 
• Rectangular cells 50 mm (1.14944)

Other Reagents and Accessories

• Calcium chloride dihydrate for analysis (1.02382)
• Water for analysis (1.16754)
• Charcoal activated for soil tests
• Analytical balance
• Standard laboratory glassware (e.g., glass beakers) and pipettes
• Spatula
• Stirring plate or shaker
• Drying kiln
• Folded filter

Analytical Procedure

Reagent Preparation

• Calcium chloride solution, 0.025 mol/L: Dissolve 3.68 g of calcium chloride dihydrate for analysis with 1 L of water for analysis.

Sample Preparation

• In a glass bottle mix 50 g of naturally moist sample, free from coarse stones, with 100 mL of a calcium chloride solution 0.025 mol/L.
• Add a spatula-tip full of charcoal activated for soil tests and shake the closed bottle in a shaking machine for 1 hour (alternative stir in a beaker).
• Let the suspension settle and filter it through a folded filter.
• For determination of the water content dry a similar sample to constant weight in the drying kiln at 105 °C.
• A drying of the sample before the determination is not advisable because of the fast change in the nitrogen forms.

Note: The analysis should take place very quickly. Storing the sample in plastic bags at room temperature changes the analysis values after a short time already

Using Cat. No. 1.14547: Procedure and Measurement

For more information on the measurement, see the packaging insert for the test.

Procedure

• Pipette 5.0 mL Pretreated sample into a reaction cell, close the cell tightly, and shake vigorously until the reagent is completely dissolved
• Leave to stand for 10 min (reaction time), then measure the sample in the photometer.

Measurement

• For photometric measurement the cells must be clean. Wipe, if necessary, with a clean dry cloth.
• Measurement of turbid solutions yields false-high readings.
• The pH of the measurement solution must be within the range 2.0 – 2.5.
• The color of the measurement solution remains stable for at least 60 min after the end of the reaction time stated above.

Hints for Measurement

• It is recommended to zero the method each new working day. To do this, open the method, either by manually selecting the method or by inserting a barcoded cell. Tap the <Settings> button and select the <ZERO ADJUSTMENT> menu item. After prompting, insert the 16 mm zero cell through the corresponding opening. The zero adjustment is performed automatically. Confirm the performance of the zero-adjustment procedure by clicking on <OK>.
• After the zero has been performed, insert the barcoded Spectroquant^® round cell through the corresponding opening, ensuring that the white position mark on the cell is aligned with the positioning mark on the spectrophotometer. The measurement starts automatically.
• Read off the result in mg/L from the display.

Hint: The above written measurement description is only valid for the Spectroquant® Prove (plus) series photometer. If a different instrument is used, please consult the corresponding instrument manual for more details on how to perform the measurement.

Using Cat. No. 1.14776: Procedure and Measurement

For more information on the measurement, see the packaging insert for the test.

Procedure

• Pipette 5,0 mL Pretreated sample into a test tube
• Add 1 level blue microspoon (in the cap of the NO₂-1 bottle) Reagent NO₂-1 and shake vigorously for 1 minute until the reagent is almost completely dissolved. The pH must be within the range 2.0-2.5. Check with MQuant^® pH-indicator strips. Adjust the pH, if necessary, with sodium hydroxide solution or sulfuric acid.
• Leave to stand for 10 min (reaction time), then fill the sample into a cell, and measure in the photometer. 

Note: For measurement in the 50-mm cell both the sample volume as well as the quantities of reagents Al-1, Al-2, and Al-3 must be doubled. Alternatively, the semi-microcell Cat. No. 1.73502 can be used. When using the 50-mm cell, perform the measurement against a separately prepared blank (preparation as per measurement sample, but with distilled water instead of sample). Configure the photometer for blank measurement. 

Measurement

• Certain photometers may require a blank (preparation as per measurement sample, but with distilled water instead of sample).
• For photometric measurement the cells must be clean. Wipe, if necessary, with a clean dry cloth.
• A minimum bottom sediment may be formed occasionally due to undissolved reagent. This has no influence on the functionality of the test kit. While transferring the measuring solution into the measuring cell, make sure that it remains in the test tube, in order to avoid influences on the result.
• Measurement of turbid solutions yields false-high readings.
• The pH of the measurement solution must be within the range 2.0 - 2.5.
• The color of the measurement solution remains stable for at least 60 min after the end of the reaction time stated above.

Hints for Measurement

• It is recommended to zero the method for each new working day. To do this, open the method by inserting the barcode, tap the <Settings> button and select the <ZERO ADJUSTMENT> menu item. Fill the same cell which will be used for the sample measurement with distilled water. After prompting, insert the filled rectangular cell into the cell compartment. The zero adjustment is performed automatically. Confirm the performance of the zero-adjustment procedure by clicking on <OK>.
• After the zero adjustment, fill the measurement sample into the same or a matched rectangular cell and insert the cell into the cell compartment. The measurement starts automatically.
• Read off the result in mg/L from the display.

Hint: The above written measurement description is only valid for the Spectroquant® Prove (plus) series photometer. If a different instrument is used, please consult the corresponding instrument manual for more details on how to perform the measurement.

Analytical Quality Assurance

Analytical quality assurance (AQA) is recommended before each measurement series.

To check the photometric measurement system (test reagent, measurement device, handling) and the mode of working, Spectroquant® ammonium standard solutions (see section 5 of the respective test kit instruction) can be used.

Sample-dependent interferences (matrix effects) can be determined by means of standard addition or dilution.

To view additional notes, visit SigmaAldrich.com/qa-test-kits.

Calculation

Nitrite content in mg/kg NO₂ = analysis value in mg/L NO₂ x 2