Photometric Determination of Total Kjeldahl Nitrogen (TKN) According to EN ISO 25663
Abstract
This protocol outlines the photometric determination of total Kjeldahl nitrogen in wastewater, raw water, and drinking water using Spectroquant® test kits. Following sample preparation in accordance with the Kjeldahl method (EN ISO 25663), the procedure utilizes a spectrophotometer to quantify total Kjeldahl nitrogen, ensuring precise and reliable results.
Section Overview
Introduction
The Kjeldahl method is a well-established analytical technique for determining nitrogen content in organic compounds and remains fundamental in environmental and industrial analysis. Developed originally for protein analysis, it has been adapted to measure total Kjeldahl nitrogen (TKN) in water and wastewater samples. The method involves digestion of organic nitrogen into ammonium sulfate, followed by conversion and quantification of ammonium ions, which are then used to calculate total nitrogen content. TKN represents the sum of organic nitrogen and ammonium-nitrogen (NH₄⁺-N) while excluding nitrate and nitrite forms. This provides a comprehensive measure of biologically available nitrogen in a sample, making it essential for assessing nutrient loading and wastewater treatment efficiency.1
The Kjeldahl method has been demonstrated to provide robust and precise results across diverse environmental samples. Due to its reliability and standardization through procedures such as EN ISO 25663, it remains suitable for routine laboratory analysis of raw water, drinking water, and wastewater. By offering a comprehensive assessment of total nitrogen content, the Kjeldahl method supports compliance with environmental regulations and continues to serve as a dependable analytical tool across multiple industries.
Experimental
This application note describes the sample preparation using Kjeldahl tablets (1.10958) according to the Wieninger method for the determination of Total Kjeldahl Nitrogen (TKN) in raw water, drinking water and wastewater. The analysis is performed photometrically using the Spectroquant® Ammonium Test (1.14752).
With the Spectroquant® Ammonium Test, the nitrogen content can be determined quickly and easily. The method is preprogrammed in the corresponding Spectroquant® photometers, eliminating the need for calibration. All reagents required for the measurement are included in the test kit.
Method
According to the digestion method outlined in EN 25663, organic nitrogen compounds are converted into ammonium sulfate by digestion with potassium sulfate-containing sulfuric acid in the presence of a catalyst. The resulting Kjeldahl nitrogen is then subsequently quantified photometrically using the indophenol method.
Measuring range
1 mg/L - 500 mg/L N at optical path lengths of 10 mm and at 690 nm
Applicable sample
- Raw water
- Drinking water
- Wastewater
Influence of foreign substances
Foreign substances in the sample solution can:
- Increase the measured value by enhancing the reaction, or
- Lower the measured value by preventing the reaction.
A quantification of these effects is provided in tabular form in the respective package inserts for the most important foreign ions and substances. The tolerance limits have been determined individually for each ion and substance and must not be evaluated cumulatively.
For samples with complex or incompletely characterized compositions (matrices), it is particularly difficult to estimate the potential influence of the foreign substances on the analysis (matrix effect). The following instructions describe a method by means of which the user can test whether a matrix effect is present or not:
Reagents, instruments and materials
Test/reagents
For sample preparation, the following are necessary:
- Sulfuric acid 98 % for the determination of nitrogen (1.00748)
- Kjeldahl tablets for Wieninger method (1.10958)
- Sodium hydroxide solution 50% for analysis EMSURE® (1.58793)
- 4-Nitrophenol pH indicator (1.06798)
- Boiling chips granules (1.07913)
- Kjeldahl flasks and digestion apparatus
- 200-mL volumetric flask
- Pipettes for pipetting volumes 0.6 mL and 8.0 mL
For measurement, the following Spectroquant® test kit is necessary:
- Spectroquant® Ammonium Test (1.14752)
Instrument(s) & devices
For the measurement one of the following Spectroquant® photometers is necessary:
- Spectroquant® VIS Spectrophotometer Prove 100 plus (1.73026)
- Spectroquant® UV/VIS Spectrophotometer Prove 300 plus (1.73027)
- Spectroquant® UV/VIS Spectrophotometer Prove 600 plus (1.73028)
- Spectroquant® Colorimeter Move 100 (1.73632)
Also, legacy system Spectroquant® Prove 600/300/100, and the Nova series are compatible and preprogrammed with this method.
Software for data transfer
- Optional Spectroquant® Prove Connect to LIMS software package (Y.11086) to transfer your data into an existing LIMS system.
Instrument accessories
Rectangular cells are necessary for Spectroquant® Ammonium Test (1.14752)
- Rectangular cells 10 mm (1.14946)
Analytical procedure
Sample Preparation
- Depending on the expected nitrogen content, pipette the corresponding sample volume into the Kjeldahl flask (see Table 1 for recommended sample volumes and dilution factors).
- Prepare a blank using water for analysis in the same manner as the sample.
- Add 10 mL of sulfuric acid 98% and one Kjeldahl tablet to the sample.
- Perform the digestion according to the procedure described in EN ISO 25663.*
- After digestion, allow the solution to cool to room temperature.
- Neutralize the solution with sodium hydroxide solution for analysis, as specified in EN ISO 25663.
- Quantitatively transfer the solution to a 200 mL volumetric flask and make up to the mark with water for analysis.
*Extracts from EN ISO norms cannot be published due to copyright restrictions and licensing agreements.
Photometric analysis
The prepared sample is analyzed using the Spectroquant® Ammonium Test (1.14752).
- Follow the instructions given in the package insert or instrument manual.
Analytical quality assurance
Analytical quality assurance (AQA) is recommended prior to each measurement series.
To check the performance of photometric measurement system (test reagent, measurement device, handling) and the mode of working, ammonium standard solutions can be used.
For detailed instructions on performing the AQA check, refer to the instrument-specific manuals.
A certificate of analysis (CoA) is provided for each batch and can be downloaded online on the product page of the test. In addition, a certificate of quality (CoQ), covering all batches is available. These certificates include performance characteristics determined in accordance with ISO 8466-2 and DIN 38402 A51.
It is nevertheless recommended to determine the performance characteristics independently, ensuring all specific factors that may impact the performance (test reagents, measurement device, handling) are taken into account.