Soil Nitrate Nitrogen Content Assay Kit (SA, Micro Method) - BioReagent

Cat. No.: S1505722

GRADE & PURITY BioReagent

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Application

Cell Metabolism

Overview

  Nitrogen is the nutrient element with the highest demand and contribution to yield increase in crop growth. Nitrate nitrogen in the soil refers to the nitrogen element contained in nitrate salts. As one of the main forms of nitrogen absorbed by higher plants, its content is directly related to crop yield and quality. It can reflect the short-term nitrogen supply status of the soil and is significant for controlling nitrogen nutrition in the soil.

Assay Principle

NO₃⁻ can react with salicylic acid under concentrated acid conditions to generate nitrosalicylic acid, which appears yellow under alkaline conditions. The product has a characteristic absorption peak at 410 nm. The nitrate nitrogen content can be quantitatively detected by the change in absorbance.

S1505722	Component	48T	96T	Storage
S1505722A	Reagent Ⅰ	1EA	2EA	2-8℃. Store in the dark.
S1505722B	Reagent Ⅱ	28.5 mL	57 mL	2-8℃
S1505722C	Standard	1EA	1EA	2-8℃

Note: It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

Required Materials Not Provided

1. Microplate reader or visible spectrophotometer (capable of measuring absorbance at 410 nm)

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

3. Analytical balance, constant temperature water bath, incubator shaker, benchtop centrifuge

4. Deionized water, Concentrated Sulfuric Acid

Experimental Procedure

1. Reagent Preparation

Reagent Name	Preparation Steps	Notes & Storage
Reagent Ⅰ	Reconstitute before use: Add 1.2 mL of concentrated sulfuric acid per vial. Dissolve thoroughly.	Stable at 4°C, protected from light, for 1 week. Highly corrosive – use appropriate personal protective equipment (PPE).
Reagent Ⅱ	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C. Highly corrosive – use appropriate PPE.
Standard	Reconstitute before use: Add 1 mL deionized water to the standard vial to prepare a 10 mg/mL NO₃⁻ stock solution. Take 100 μL of this stock and add 900 μL deionized water to make a 1 mg/mL NO₃⁻ working standard solution.	Unused 10 mg/mL stock can be stored at 4°C for 2 weeks.

2. Sample Preparation

• Add deionized water to the soil sample at a ratio of 1:5-10 (soil weight (g) : water volume (mL)). (Adding 1 mL deionized water to approx. 0.1 g soil is recommended).

• Extract by shaking at 37°C for 1 hour.

• Centrifuge at 10,000 g for 10 minutes at 25°C.

• Collect the supernatant for assay.

• Note: Air-drying or oven-drying soil can easily alter nitrate nitrogen content. Using fresh soil or samples processed identically is recommended.

• 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 within 4 hours.

3. Assay Procedure

3.1 Instrument Preparation

• Preheat the microplate reader or visible spectrophotometer for 30 minutes. Set wavelength to 410 nm.

• Zero the visible spectrophotometer with deionized water.

3.2 Assay Setup (Perform in 1.5 mL centrifuge tubes)

Reagent (μL)	Blank Tube	Standard Tube	Test Tube
Sample Supernatant	0	0	10
Deionized Water	10	0	0
Standard (1 mg/mL)	0	10	0
Reagent Ⅰ	20	20	20

Mix thoroughly and let stand at 25°C for 30 minutes.

Reagent Ⅱ

475

475

475

3.3 Measurement 

Mix well and vortex to ensure any precipitate formed is fully dissolved. 

Transfer 200 μL to a micro glass cuvette or 96-well plate. 

Measure the absorbance at 410 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. Notes: 

Notes: 

(1) The Blank and Standard tubes only need to be measured once. A pilot experiment with 2-3 variable samples is recommended. 

(2) If ΔA_Test is less than 0.002, consider increasing the sample amount. If ΔA_Test is greater than 1.5, dilute the sample supernatant further with deionized water and multiply the result by the dilution factor, or reduce the soil amount used for extraction. 

(3) Soil colloids do not adsorb nitrate ions, which are easily soluble in water and mobile within the soil. When measuring multiple samples or replicates, ensure consistent sampling depth (recommended: approx. 10 cm below the surface). Note that content varies by region. 

4. Calculation of Results 

Note: The derived and simplified formulas provided are equivalent. The simplified formula (in bold) is recommended. 

NO₃⁻ Content (μg/g sample) = ΔA _test / (ΔA _standard / C _standard ) × V_{total extract} / W=1000 × ΔA_Test / ΔA_Standard / W 

Parameter Definitions: 

W: Sample mass (g) 

C_Standard: Standard solution concentration (1,000 μg/mL) 

V_{total extract}: Total extraction volume (1 mL) 

5. Results Example 

Soil: 0.1 g extracted with 1 mL deionized water for 1h, centrifuged. 

Measured: A_Test=0.062, A_Blank=0.047, A_Standard=1.515 

ΔA_Test = 0.062 - 0.047 = 0.015 

ΔA_Standard = 1.515 - 0.047 = 1.468 

NO₃⁻ Content (μg/g soil) = 1,000 × 0.015 / 1.468 / 0.1 = 102.18 μg/g soil

Precautions

1. It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.

2. 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.

Specifications

Specifications & Purity

BioReagent

Stability And Storage

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

Storage

Store at 2-8°C, Protected from light

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive