Plant Nitrate Nitrogen Content Assay Kit (SA, Colorimetric Method)

Cat. No.: N1509264

GRADE & PURITY BioReagent

Overview

Nitrate nitrogen is the primary nitrogen source for plants. The nitrate nitrogen content in plant tissues reflects the supply of nitrate nitrogen in the soil and can serve as an indicator of soil nitrogen fertility. Measuring the nitrate nitrogen content in plants not only reveals the nitrogen nutrition status of the plants but is also significant for assessing the quality of vegetables and processed products derived from plants.

Detection Principle

Under concentrated acidic conditions, NO₃⁻ reacts with salicylic acid to form nitrosalicylic acid, which turns yellow under alkaline conditions. The intensity of the yellow color is proportional to the nitrogen content within a certain range. The absorbance at 410 nm is measured using a spectrophotometer or microplate reader, and the nitrate nitrogen content of the sample is calculated based on a standard curve for NO₃⁻.

Applicable Samples: Plant tissues

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 410 nm)
• Centrifuge, electronic balance
• Medium-speed filter paper, centrifuge tubes or test tubes, cuvettes
• Distilled or deionized water, concentrated sulfuric acid

Operating Steps

1. Sample Preparation

1.1 Plant Samples

Take 1 g of plant tissue, clean thoroughly, dry, and cut into 1–2 mm fragments. Add 10 ml of distilled water and extract in a boiling water bath for 30 minutes. After removal, cool to room temperature with tap water, filter through medium-speed filter paper, and bring the volume to 10 ml with distilled water. This is the nitrate nitrogen extraction solution.

1.2 Plasma, Serum, and Urine Samples

Plasma and serum, prepared by conventional methods, can be directly used for detection with this kit for nitrate nitrogen measurement.

1.3 High-Activity Samples

If the sample contains a high concentration of nitrate nitrogen, appropriate dilution with nitrate nitrogen lysis buffer or distilled water can be performed before measurement.

2. Preparation of Salicylic Acid Working Solution

Dissolve salicylic acid in concentrated sulfuric acid at a ratio of 1 g salicylic acid to 20 ml concentrated sulfuric acid. Store at 4°C protected from light. The solution is stable for one week.

Note: Concentrated sulfuric acid is a strong acid with high corrosivity. Handle with care.

3. Preparation of NO₃⁻ Assay Buffer (1×)

Dilute the NO₃⁻ Assay Buffer (2×) with distilled water to achieve a 1× concentration.

4. Preparation of NO₃⁻ Standard Solutions Series

Take the NO₃⁻ Standard (200 μg/mL) and dilute with distilled water according to the table below:

Tube Number	1	2	3	4	5	6	7
NO₃⁻ Standard (200 μg/mL) (mL)	0.05	0.1	0.2	0.3	0.4	0.5	0.6
Distilled Water (mL)	0.95	0.9	0.8	0.7	0.6	0.5	0.4
NO₃⁻ Concentration (μg/mL)	10	20	40	60	80	100	120

5. NO₃⁻ Sample Addition

Set up blank, standard, and test tubes according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the nitrate nitrogen concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.

Substance Added (mL)	Blank Tube	Standard Tube	Test Tube
Distilled Water	0.02	—	—
NO₃⁻ Standard Series (Tubes 1–7)	—	0.02	—
Sample	—	—	0.02
Salicylic Acid Working Solution	0.08	0.08	0.08

Mix well, let stand at room temperature for 20 min, then slowly add NO₃⁻ Assay Buffer (1×).

NO₃⁻ Assay Buffer (1×)

1.9

1.9

1.9

6. NO₃⁻ Measurement

Cool to room temperature. Zero the spectrophotometer or microplate reader with the blank tube, and measure the absorbance of the standard and test tubes at 410 nm (recorded as A_Standard and A_Test) using a 1 cm light path cuvette.

7. Result Calculation

Using the NO₃⁻ Standard Series (Tubes 1–7) concentrations (μg/mL) as the x-axis and the corresponding absorbance values as the y-axis, plot a standard curve. Calculate the NO₃⁻ content of the sample based on the absorbance of the test tube and the following formulas:

7.1 For plant tissue samples:

Nitrate nitrogen (μg/g) = C × V / m

Parameter Explanation:

C: NO₃⁻ concentration obtained from the standard curve (μg/mL)

V: Total volume of the nitrate nitrogen extraction solution (mL)

m: Weight of the plant sample (g)

7.2 For serum, urine, and other liquid samples:

Nitrate nitrogen (μg/mL) = C × N

Parameter Explanation:

C: NO₃⁻ concentration obtained from the standard curve (μg/mL)

N: Dilution factor

Precautions

1. Experimental materials should be as fresh as possible. If not used immediately after collection, store at 4°C.

2. Concentrated sulfuric acid must be prepared by the user. Concentrated sulfuric acid is a strong acid with high corrosivity and oxidizing properties. Handle with care.

3. If a spectrophotometer is unavailable, a standard microplate reader can also be used for measurement.

4. If the concentration of the measured sample is too high, dilute the sample with distilled water and repeat the measurement.

5. For your safety and health, please wear a lab coat and disposable gloves during operation.

6. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.

Appendix

Reference Standard Curve Range:

The absorbance of NO₃⁻ standards at concentrations of 10, 20, 40, 60, 80, 100, and 120 μg/mL was measured at 405 nm using a microplate reader at room temperature. Based on this, the reference standard curve is as follows:

Note: Due to variations in detection instruments, operational techniques, and other conditions, the reference value range may fluctuate. These values are for reference only.

Storage and Shipping

Storage

Store at 2-8°C,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. N1509264A: Store at 2-8℃ long term (12 months). N1509264B, N1509264C: Store at room temperature long term (12 months).

Contents & Storage

N1509264	Component	50T	100T	Storage
N1509264A	NO₃⁻ Standard (200 μg/mL)	0.5 mL	1 mL	2-8℃.
N1509264B	Salicylic Acid	1.5 g	3 g	RT.
N1509264C	NO₃⁻ Assay Buffer (2×)	50 mL	100 mL	RT.