BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
氮素是作物生长需求量和增产贡献率最大的营养元素，土壤中硝态氮指硝酸盐中所含有的氮元素， 作为高等植物吸收氮的主要形式之一，其含量直接关系到作物的产量与品质，并且能够反映土壤短期氮素供应状况，对土壤中氮素营养的控制具有重要意义。
检测原理：NO₃⁻在浓酸条件下能够与水杨酸反应生成硝基水杨酸，在碱性条件下呈黄色，产物在410nm处具有特征吸收峰，通过吸光值变化即可定量检测土壤硝态氮的含量。
| S1505722 | 组分 | 48T | 96T | 储存条件 |
| S1505722A | 试剂 I | 1EA | 2EA | 2-8℃. 避光 |
| S1505722B | 试剂 II | 28.5 mL | 57 mL | 2-8℃. |
| S1505722C | 标准品 | 1EA | 1EA | 2-8℃. |
注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
自备仪器和试剂
1、酶标仪或可见分光光度计（能测410nm处的吸光度）
2、96孔板或微量玻璃比色皿、可调节式移液枪及枪头
3、分析天平、恒温水浴锅、恒温摇床、台式离心机
4、去离子水、浓硫酸
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
试剂 I | 临用前配制，根据用量每支加入1.2mL 硫酸充分溶解后使用。 | 4℃避光保存1周；有强腐蚀性，建议在使用过程中做好个人防护。 |
试剂 II | 即用型；使用前平衡到室温 | 4℃保存；有强腐蚀性，建议在使用过程中做好个人防护。 |
标准品 | 临用前配制，加入1mL去离子水配成10 mg/mL的NO₃⁻标准液， 取100μL上述溶液于1.5mL EP管中，加入900μL去离子水，即为1mg/mL的NO₃⁻标准液。 | 用不完的10 mg/mL的NO₃⁻标准液4℃保存2周。 |
2、样本制备
按照土壤质量（g）：去离子水体积（mL）为1：5-10的比例（建议称取约0.1g，加入1mL去离子水）加入去离子水，37℃振荡提取1h，然后25℃，10,000g离心10min，取上清待测。
注意：土壤经风干或者烘干很容易引起硝态氮含量的变化，所以建议采用新鲜土壤或者同样处理的样品进行测定。如果不立即进行实验，样本可在-80℃保存数周。测定时，应控制解冻的温度和时间。室温环境下解冻时，需在4h内完成样品解冻。
3、实验步骤
3.1 酶标仪或可见分光光度计预热30min，调节波长到410nm。可见分光光度计用去离子水调零。
3.2 操作表（下述操作在1.5mL 离心管中操作）：
| 试剂 | 空白管 (μL) | 标准管 (μL) | 测定管 (μL) |
| 样本上清 | 0 | 0 | 10 |
| 去离子水 | 10 | 0 | 0 |
标准品 | 0 | 10 | 0 |
试剂 I | 20 | 20 | 20 |
充分混匀，25℃静置30min；
| 试剂 II | 475 | 475 | 475 |
3.3 混匀，涡旋振荡，使出现的沉淀充分溶解，取200μL至微量玻璃比色皿或96孔板于410nm测定吸光度，记为A空白、A标准、A测定，计算ΔA测定=A测定-A空白、ΔA标准=A标准-A空白。 注意：
(1) 空白孔和标准孔只需测定1次。实验之前建议选择2-3个预期差异大的样本做预实验。
(2) 如果ΔA测定小于0.002可适当加大样本量。如果ΔA测定大于1.5，样本可用去离子水进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
(3) 土壤胶体不吸附硝酸根离子，且其易溶于水，在土壤内部移动，所以测定多个样本或者重复样本时注意保持相同的采样深度，建议在土壤表面以下十公分左右处取样，不同样本的含量跟区域有关。
4、结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。 NO₃⁻含量 (μg/g 样本)=ΔA测定÷(ΔA标准÷CStandard)×V总样÷W=1,000×ΔA测定÷ΔA标准÷W
参数说明：
W：样本质量，g；
CStandard：标准溶液浓度，1,000μg/mL；
V总样：提取液体积，1mL。
5、结果展示 取0.1g土壤加入1mL去离子水震荡提取1h，离心取上清之后按照测定步骤操作，使用96孔板测得并计算ΔA测定=A测定-A空白=0.062-0.047=0.015，ΔA标准=A标准-A空白=1.515-0.047=1.468，按土壤质量计算NO₃⁻含量得：
NO₃⁻含量 (μg/g 土样)=1,000×ΔA测定÷ΔA标准÷W=1,000×0.015÷1.468÷0.1=102.18 (μg/g 土样)。
注意事项
1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2、本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
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 ABlank, AStandard, and ATest. Calculate ΔATest = ATest - ABlank and ΔAStandard = AStandard - ABlank. 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 ΔATest is less than 0.002, consider increasing the sample amount. If ΔATest 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 ) × Vtotal extract / W=1000 × ΔATest / ΔAStandard / W
Parameter Definitions:
W: Sample mass (g)
CStandard: Standard solution concentration (1,000 μg/mL)
Vtotal extract: Total extraction volume (1 mL)
5. Results Example
Soil: 0.1 g extracted with 1 mL deionized water for 1h, centrifuged.
Measured: ATest=0.062, ABlank=0.047, AStandard=1.515
ΔATest = 0.062 - 0.047 = 0.015
ΔAStandard = 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.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737529 |
分析证书 | S1505722 |
| ZJ26F0737530 |
分析证书 | S1505722 |
| ZJ25F1129770 |
分析证书 | S1505722 |
危险品化学品经营许可证（带存储）