Version: V1.0

Date of Establishment:July 2025
Technical SupportTaizhou Depu Analytical Instrument Technology Co., Ltd. Analysis and Testing Technology Center

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Part One: Introduction

1.1 Background and Significance

Nitrogen, as one of the core components of living organisms, has extremely important indicator significance in the fields of agriculture, food, environment, and chemical engineering. The accurate determination of effective nitrogen content in fertilizers, protein conversion factors in food, and total nitrogen pollution load in water/soil is directly related to the effectiveness of agricultural production efficiency, food safety supervision, and ecological environment protection decisions.

Standard substance for nitrogen content determination(hereinafter referred to as "nitrogen standard substance") is a reference substance with one or more sufficiently uniform and well determined characteristic values (nitrogen content). It serves as a "value transfer carrier" for chemical measurements, and is a key basis for calibrating analytical instruments, validating measurement methods, evaluating laboratory capabilities, and assigning actual sample values. Establishing an accurate, reliable, and traceable method for determining the value of nitrogen reference materials is the cornerstone of ensuring that nitrogen related measurement results are comparable and usable in time and space.

1.2 Purpose and Scope

This white paper aims to systematically explain the complete technical path for nitrogen standard substance determination based on current effective national and international standards. The content covers method selection, experimental equipment requirements, detailed operating procedures, data processing models, and uncertainty assessment systems.

The method described in this article is mainly applicable to the calibration of nitrogen reference materials for the following substrate types:

• Agriculture and fertilizers:Urea, ammonium sulfate, potassium nitrate, compound fertilizer, etc.

• Food and Feed Category:Grains, dairy products, meat products, plant protein, etc.

• Environment and Soil Category:Soil, sediment, and some water quality (requiring corresponding pre-treatment).

• Pure substances and chemical reagents:Nitrogen containing organic compounds of known purity.

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Part 2: Selection and Principle of Fixed Value Method

2.1 Basis for Method Selection

The determination of nitrogen reference materials should follow the principle of "accuracy first, clear traceability". The classic Kjeldahl method is widely used as a benchmark method for determining constant and trace nitrogen standards due to its clear reaction mechanism, mature technology, and high international recognition. For specific matrices (such as samples containing nitrate and nitrite), improved Kjeldahl method or elemental analyzer method (Dumas method) should be used for comparison and verification. This white paper is based onFully automatic Kjeldahl nitrogen determination methodElaborate on the core technology path.

2.2 Determination principle (Kjeldahl nitrogen determination method)

The basic principle of Kjeldahl nitrogen determination method is divided into three chemical stages:

1. Dissolve:In the presence of catalysts (copper sulfate, potassium sulfate), organic nitrogen in the sample is co heated with concentrated sulfuric acid, oxidized and decomposed, and converted into ammonium sulfate.

   $$\\Text {organic nitrogen}+H2SO4 \ \ xrightarrow {\ \ text {heating, catalyst}} (NH4) 2SO4+CO2+H2O$$

2. Distillation:After cooling the digestion solution, excess sodium hydroxide is added to alkalize it, causing the ammonium salt to decompose and release ammonia gas. The ammonia is then carried out through steam distillation and absorbed by the boric acid solution.

   $$(NH_4)_2SO_4 + 2NaOH \\rightarrow Na_2SO_4 + 2NH_3 \\uparrow + 2H_2O$$$$2NH_3 + 4H_3BO_3 \\rightarrow (NH_4)_2B_4O_7 + 5H_2O$$

3. Titration:Titrate ammonium tetraborate in the absorption solution with a calibrated hydrochloric acid standard solution, and calculate the nitrogen content based on the acid consumption.

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Part Three: Instruments, Equipment, Reagents and Consumables

To ensure the metrological traceability of the calibration process, all instruments used must undergo regular calibration, and reagents and consumables must meet specific purity requirements.

3.1 Core Instruments and Equipment

• Fully automatic Kjeldahl nitrogen analyzer:Recommended for useTaizhou Depu Analytical Instrument Technology Co., LtdThe K1160 series fully automatic Kjeldahl nitrogen analyzer produced. This instrument integrates digestion control, automatic distillation, automatic titration, and data processing functions, and has the following key technical advantages:

  • High precision titration system:The titration resolution reaches 0.1 μ L, ensuring the accuracy of trace nitrogen determination.

  • Intelligent endpoint determination:Using high-precision color sensors to accurately capture the color change points of methyl red bromocresol green indicator, eliminating human errors.

  • Full process traceability:The software records distillation temperature, alkali dosage, and titration curve in real-time, in compliance with GLP standards.

• Analytical balance:Sensitivity of 0.01mg or 0.1mg (one in 100000 or one in 10000) requires a valid calibration certificate.

• Dissolving furnace:The aluminum block digestion furnace matched with the Kjeldahl nitrogen analyzer has a temperature control accuracy of ± 1 ° C.

• Auxiliary equipment:Ultra pure water machine (providing ammonia free water), fume hood, dryer, oven, muffle furnace.

3.2 List of reagents and consumables

Special Note:The experimental water must be ammonia free distilled water or deionized water of equivalent purity, with a conductivity of ≤ 0.1 mS/m.

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Part Four: Detailed Process of Fixed Value Measurement

4.1 Calibration of hydrochloric acid standard solution

1. Weighing:Weigh three portions of anhydrous sodium carbonate that have been burned to constant weight at 270-300 ° C (approximately 0.15g per portion, accurate to 0.0001g).

2. dissolution:Place in a conical flask, dissolve in 50mL of water, and add 10 drops of mixed indicator.

3. Titration:Titrate with the hydrochloric acid solution to be calibrated until the solution changes from green to dark red, boil for 2 minutes to remove carbon dioxide, and continue titration until dark red after cooling.

4. calculation:

   $$c(HCl) = \\frac{m}{V \\times 0.05299}$$

   among which$V$For titration volume (mL),$m$The mass of sodium carbonate (g) is 0.05299, which is equivalent to the mass of Na ₂ CO3 (g/mmol) of 1.00 mmol HCl.

4.2 Sample digestion

1. Weighing:According to the estimated nitrogen content, weigh an appropriate amount of uniform standard substance sample (ensuring an absolute nitrogen content within the range of 5-30 mg) into a dry digestion tube, accurate to 0.0001g.

2. Add reagents:Add 6g of potassium sulfate and 0.4g of copper sulfate in sequence, slowly add 12mL of concentrated sulfuric acid along the tube wall, and gently shake well.

3. Dissolve:Place the digestion tube on the digestion furnace and set the temperature program:

   • Stage 1: 180 ° C, 30 minutes (preheating, removing moisture)

   • Stage 2: 420 ° C, 90-120 minutes (Dissolve until the solution is clear and transparent, blue-green in color)

4. Cooling:Remove the digestion tube and place it on a test tube rack to cool naturally to room temperature.

4.3 Distillation and titration

1. Blank preparation:Simultaneously prepare at least two reagent blanks (without adding samples, all other steps are identical) for deducting system blanks.

2. Instrument preparation:StartTaizhou Depu K1160 Fully Automatic Kjeldahl Nitrogen AnalyzerCheck if the alkaline solution, boric acid, and distilled water are sufficient.

3. Machine testing:

   • Place the digestion tube containing the cooled digestion solution on the instrument rack.

   • Enter parameters such as sample quality, dilution factor, and standard acid concentration in the software.

   • Start the 'Automatic Measurement' program, and the instrument will automatically complete:

     • Neutralization with alkali and steam distillation (about 5-8 minutes).

     • Boric acid absorption.

     • Automatically titrate and record the endpoint volume.

     • Clean the pipeline.

4. Result record:The instrument automatically calculates and displays the sample consumption volume$V_1$Record the blank consumption volume$V_0$.

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Part 5: Data Processing and Uncertainty Assessment

5.1 Nitrogen Content Calculation

Mass fraction of nitrogen in the sample$w(N)$Calculate according to the following formula:

$$w(N) = \\frac{(V_1 - V_0) \\times c \\times 0.01401}{m} \\times 100\\%$$

among which

• $V_1$Volume of hydrochloric acid consumed for sample titration (mL)

• $V_0$Blank titration consumes hydrochloric acid volume (mL)

• $c$Concentration of hydrochloric acid standard solution (mol/L)

• 0.01401: millimolar mass of nitrogen (g/mmol)

• $m$Sample quality (g)

5.2 Uncertainty assessment of fixed value results

The value of a standard substance must provide an uncertainty that includes the confidence level. According to JJF 1343 guidelines, the main sources of uncertainty in the determination of nitrogen reference materials are as follows:

1. type A uncertainty$u_A$：Introduced by the repeatability of multiple independent measurement results, the experimental standard deviation is calculated using the Bessel formula$s$, then$u_A = s / \\sqrt{n}$.

2. B-class uncertainty$u_B$：Evaluated by non statistical methods, including:

   • $u(m)$Balance calibration and weighing repeatability.

   • $u(V)$Titration volume error (instrument calibration, temperature effect).

   • $u(c)$The uncertainty introduced in the hydrochloric acid calibration process (purity of reference material, weighing, calibration repeatability).

   • $u(Rec)$The uncertainty introduced by the method recovery rate (evaluated through spiked recovery or standard substance validation).

Synthesis and expression:
Composite standard uncertainty$u_c = \\sqrt{u_A^2 + \\sum u_B^2}$.
Take the inclusion factor$k=2$(Confidence probability of about 95%), extended uncertainty$U = k \\cdot u_c$.
The final value of the standard substance is expressed as:$(\\bar{x} \\pm U) \\%$

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Part 6: Quality Control and Assurance System

To ensure the reliability of nitrogen standard substance values, the laboratory should establish and operate the following quality control measures:

1. Blank monitoring:Each batch of samples should have at least two reagent blanks. If the blank value increases abnormally, it is necessary to investigate the reagent or environmental factors.

2. Standard sample verification:During each batch of testing, a certified standard substance with a known content that matches the matrix of the test substance is synchronously tested, and the measured value is controlled within the range specified in the certificate.

3. Parallel sample:All samples should be tested in parallel at least twice, and the relative range should be controlled within 0.5%.

4. Instrument verification:Regularly using standard ammonium salt solutions (such as ammonium sulfate)Taizhou Depu fully automatic Kjeldahl nitrogen analyzerVerify the distillation recovery rate to ensure that it is between 99.5% and 100.5%.

5. Personnel comparison:Regularly organize different experimental personnel to conduct comparative tests on the same sample and evaluate the reproducibility of the operation.

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Part Seven: Conclusion

This white paper establishes a standard substance calibration technology system for nitrogen content determination, with the fully automated Kjeldahl nitrogen determination method as the core. This method strictly follows national standards by adoptingTaizhou Depu Analytical Instrument Technology Co., LtdThe high-precision automated analysis platform has achieved standardization and automation of the entire process of digestion, distillation, and titration, significantly reducing human operational errors. By combining a complete uncertainty assessment model, the accuracy, reproducibility, and metrological traceability of the fixed value results are ensured, which can fully meet the strict requirements of nitrogen standard substance development, production, and certification.

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Appendix:(can be added as needed)

• Appendix A: Example of Uncertainty Evaluation Calculation

• Appendix B: Guidelines for troubleshooting common problems (incomplete digestion, low recovery rate, etc.)

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Copyright Notice:This white paper was compiled by the Technical Center of Taizhou Depu Analytical Instrument Technology Co., Ltd. with the aim of promoting industry technology exchange. The content is for reference only. Please refer to the current valid standard text for specific experimental procedures.