Water and Soil Nitrite Content Assay Kit (NED, Micro Method)

Cat. No.: N1508208

Overview

Nitrite is widely present in water bodies and soil, serving not only as an important intermediate product of organic nitrogen decomposition but also potentially originating from pollution. Excessive intake by humans can induce digestive system cancers.

Detection Principle

Under acidic conditions, nitrite reacts with sulfanilic acid to form a diazo compound, which then couples with N-(1-naphthyl)ethylenediamine to produce a purple-red azo compound with a characteristic absorption peak at 540 nm.

Applicable Samples: Water samples, Soil samples

Note: Before formal testing, it is recommended to perform a preliminary experiment with 2-3 samples expected to show significant differences.

Reagents, consumables and Equipments not provided

• Microplate reader or visible spectrophotometer (capable of measuring absorbance at 540 nm)

• 96-well plate or micro-volume glass cuvette, Adjustable pipettes and tips

• Ice maker, Centrifuge, 2 mm sieve

• Deionized water

Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Reagent Ⅰ	Ready-to-use. Equilibrate to room temperature before use.	Store at 4°C.
Reagent Ⅱ	Ready-to-use. Equilibrate to room temperature before use.	Store at 4°C protected from light.
Reagent Ⅲ	Ready-to-use. Equilibrate to room temperature before use.	Store at 4°C protected from light.

Note: Reagent Ⅲ and the Standard are somewhat toxic. It is recommended to perform the experiment in a fume hood.

2. Standard Curve Setup

Dilute the 1 mmol/mL Standard to 10 µmol/mL using Reagent Ⅰ. Then, dilute the 10 µmol/mL standard as shown in the table below to obtain 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 nmol/mL standards.

Tube	Standard Volume	Reagent Ⅰ Volume (µL)	Standard Concentration (nmol/mL)
Std.1	10µL of 10 μmol/mL Standard	990	100
Std.2	100µL of Std.1 (100 nmol/mL)	100	50
Std.3	100µL of Std.2 (50 nmol/mL)	100	25
Std.4	100µL of Std.3 (25 nmol/mL)	100	12.5
Std.5	100µL of Std.4 (12.5 nmol/mL)	100	6.25
Std.6	100µL of Std.5 (6.25 nmol/mL)	100	3.125
Std.7	100µL of Std.6 (3.125 nmol/mL)	100	1.5625

Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

3. Sample Preparation

3.1 Soil Sample: Take an appropriate amount of soil sample, remove impurities such as stones and twigs, and sieve through a 2 mm mesh. Precisely weigh 0.5 g, add 1 mL of Reagent Ⅰ, shake at room temperature for 1 hour, centrifuge at 8,000 g, 25°C for 15 minutes. Collect the supernatant for testing.

3.2 Water Sample: Test directly. If turbid, centrifuge first before measurement.

4. Experimental Steps

4.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 540 nm. Zero the visible spectrophotometer with deionized water.

4.2 Operation Table (perform in a 96-well plate or micro-volume glass cuvette):

Reagent (µL)	Blank Well	Standard Well	Test Well
Reagent Ⅰ	70	0	0
Standard	0	70	0
Sample	0	0	70
Reagent Ⅱ	65	65	65
Reagent Ⅲ	65	65	65

4.3 Mix rapidly, incubate at 25°C for 15 minutes. Measure the absorbance at 540 nm, recorded as A_Blank, A_Standard, A_Test. Calculate ΔA_Test = A_Test - A_Blank and ΔA_Standard = A_Standard - A_Blank.

*Note: The blank and standard wells only need to be measured once. It is recommended to perform a preliminary experiment with 2-3 samples showing expected large differences before formal testing. If ΔA_Test is less than 0.001, appropriately increase the sample amount. If ΔA_Test is greater than 2.0, further dilute the sample with Reagent Ⅰ and multiply the result by the dilution factor, or reduce the amount of sample used for extraction.*

5. Calculation of Results

Note: We provide two formula sets: the derived calculation formulas and the simplified formulas. They are completely equivalent. The simplified formulas are recommended as the final calculation formulas.

5.1 Plotting the Standard Curve

Plot the standard curve with the standard concentration as the y-axis and ΔA_Standard as the x-axis. Substitute ΔA_Test into the standard curve equation to obtain y (nmol/mL).

5.2 Calculation of Nitrite Content in Samples

(1) Soil Sample

NO₂⁻ (nmol/g) = y × V_Sample × V_Total ÷ V_Sample ÷ W × n = 2y × n

(2) Water Sample

NO₂⁻ (nmol/mL) = y × n = y × n

Parameter Description:

V_Sample: Sample volume, 0.07 mL;

W: Sample mass, 0.5 g;

V_Total: Volume of Reagent Ⅰ added, 1 mL;

n: Dilution factor of the sample.

6. Example Results                

6.1 Typical Standard Curve

y = 89.365x - 0.918, R² = 0.999

             Figure 1: NO₂⁻ Standard Curve

6.2 Experimental Example

 

Figure 2: NO₂⁻ content in soil determined by the kit

Notes

This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

N1508208	Component	96T	Storage
N1508208A	Reagent Ⅰ	70 mL×2	2-8℃
N1508208B	Reagent Ⅱ	10 mL	2-8℃. Store in the dark.
N1508208C	Reagent Ⅲ	10 mL	2-8℃. Store in the dark.
N1508208D	Standard	500 μL	2-8℃