Soil Ammonium Nitrogen Content Assay Kit (IPB, Micro Method)

Cat. No.: S1506761

GRADE & PURITY BioReagent

Overview

  Soil ammonium nitrogen (NH₄⁺-N) is a crucial component of soil available nitrogen. As an essential fundamental nutrient for plant growth and development, nitrogen plays a vital role in plant morphogenesis and development. Ammonium nitrogen can be directly absorbed and utilized by plants. Its content significantly impacts agricultural production and serves as an important indicator for assessing soil fertility and nitrogen nutrition levels.

Detection Principle

  Ammonium nitrogen in the soil reacts with hypochlorite and phenol in a strongly alkaline medium to form the water-soluble dye indophenol blue, which has a characteristic absorption peak at 625 nm. The absorbance value is proportional to the ammonium nitrogen content.

Applicable Sample: Soil samples

S1506761	Component	96T	Storage
S1506761A	Extraction Buffer	70 mL×2	2-8℃
S1506761B	Reagent Ⅰ	2 EA	2-8℃. Store in the dark.
S1506761C	Reagent Ⅱ	11.2 mL	2-8℃. Store in the dark.
S1506761D	Reagent Ⅲ	2.8 mL	2-8℃
S1506761E	Standard	1 mL	2-8℃

Note: Before formal testing, it is recommended to perform a preliminary assay using 2-3 samples expected to have significant differences.

Required Instruments and Reagents

1. Microplate reader or visible spectrophotometer (capable of measuring absorbance at 625 nm)

2. 96-well plate or micro glass cuvettes, adjustable pipettes, and tips

3. Constant temperature shaker, benchtop centrifuge

4. Deionized water

Experimental Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Working Reagent Ⅰ	Prepare before use. Dissolve each vial in 5.6 mL deionized water according to the required amount.	Unused reagent can be stored at 4°C protected from light for up to 2 weeks. Reagent I is toxic and has an irritating odor. Perform subsequent steps in a fume hood.
Reagent Ⅱ	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Reagent Ⅲ	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Standard	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.

Standard Preparation: Using the 500 μmol/mL standard, prepare a dilution series as shown in the table below:

Tube	Standard Volume Added (to previous tube)	Extraction Buffer Volume (µL)	Final Concentration (μmol/mL)
Std.1	5 µL of 500 μmol/mL Std.	995	2.5
Std.2	100µL of Std.1 (2.5 μmol/mL)	100	1.25
Std.3	100µL of Std.2 (1.25 μmol/mL)	100	0.625
Std.4	100µL of Std.3 (0.625 μmol/mL)	100	0.313
Std.5	100µL of Std.4 (0.313 μmol/mL)	100	0.156
Std.	100µL of Std.5 (0.156 μmol/mL)	100	0.078
Std.	100µL of Std.6 (0.078 μmol/mL)	100	0.039
Blank	0	100	0

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

2. Sample Preparation

Note: Air-drying or oven-drying soil can easily alter ammonium nitrogen content. Therefore, it is recommended to use fresh soil or samples processed identically. If not used immediately, samples can be stored at -80°C for several weeks. Control thawing temperature and time during assay. If thawing at room temperature, complete sample thawing within 4 hours.

Add Extraction Buffer to the soil sample at a ratio of 1:5-10 (soil mass (g): Extraction Buffer volume (mL)) (recommended: weigh approximately 0.1 g soil, add 1 mL Extraction Buffer). Shake at 37°C for 1 hour for extraction. Then centrifuge at 25°C, 10,000 × g for 10 minutes. Collect the supernatant for assay.

3. Assay Steps

3.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 625 nm. Zero the visible spectrophotometer with deionized water.

3.2 Operation Table (perform the following steps in a 96-well plate or micro glass cuvette):

Reagent (μL)	Blank Well	Standard Well	Test Well
Sample Supernatant	0	0	20
Extraction Buffer	20	0	0
Standards	0	20	0
Working Reagent Ⅰ	80	80	80
Reagent Ⅱ	80	80	80

Mix thoroughly and incubate at 25°C for 1 hour.

Reagent Ⅲ

20

20

20

3.3 Mix thoroughly and immediately measure the absorbance at 625 nm. Record the values as A_blank, A_standard, and 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. Preliminary tests with 2-3 samples expected to have significant differences are recommended before the formal experiment. If ΔA_test is less than 0.02, consider increasing the sample amount appropriately. If ΔA_test is greater than the ΔA_standard of the 2.5 μmol/mL standard, the sample supernatant can be further diluted with Extraction Buffer (multiply the result by the dilution factor) or the sample amount used for extraction can be reduced.

4. Calculation of Results

Note: We provide two formulas, including the detailed derivation formula and the simplified formula. They are exactly equivalent. The simplified formula (in bold) is recommended as the final calculation formula.

4.1 Plotting the Standard Curve

Plot the standard solution concentration (x-axis) against ΔA_standard (y-axis) to draw the standard curve. Obtain the standard equation y = kx + b. Substitute ΔA_test into the equation to calculate x (μmol/mL). 

4.2 Calculation of Ammonium Nitrogen Content NH₄⁺-N content (μg/g soil) = x × V_{sample total} × 18 ÷ W = 180 × x 

Parameter Description: 

V_{sample total}: Volume of extraction buffer added, 1 mL;

W: Sample mass, 0.1 g;

18: Molar mass of NH₄⁺, μg/μmol.

Precautions

1. When comparing ammonium nitrogen content between different samples, ensure the same sampling depth. Calculate based on dry weight after oven-drying the soil samples before comparison.

2. Take appropriate protective measures during operation.

3. This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats 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.