Primary Method Feature: Kjeldahl (Part 1: Kjeldahl Foundations)

Why are we talking about Kjeldahl on the NIR blog?

This blog has made many mentions to the fact that near-infrared (NIR) spectroscopy is a secondary method. That means that NIR doesn’t measure things like moisture, fat, protein, ash, %-polymerization or anything else directly. Instead, we use an acceptable primary method, like Kjeldahl, to produce reference data (i.e. %-protein) that gets assigned to a sample that we have NIR spectra of. Then, we use a set of samples that have both an NIR spectrum and reference measurement data to train the NIR to make those measurements for us in the future via a calibration. Once the calibration is developed, we can take a routine sample (e.g. ground meat) and measure the protein content in a matter of seconds. Another benefit of NIR is the ability to measure all calibrated properties simultaneously, as illustrated in the flow chart below. More details on calibration can be found in this earlier blog post.

Flow of NIR method development. The spectrum of a sample is matched with a corresponding reference (primary method) measurement. Sets of these paired data are then to create an NIR calibration model for each property. Once validated, the NIR method can replace the primary method for routine sample measurements that fall within the calibration range and range of spectral variation captured by the calibration model.

The Kjeldahl method allows for the calculation of protein content in various samples based on the determined nitrogen which is a general constituent of all proteins. Today, the scope of Kjeldahl nitrogen determination also includes applications in the fields of environmental analysis, R&D, pharmaceutical, chemical, and cosmetics industries.

Johan Kjeldahl published his method for nitrogen determination in 1883. At that time in history, the electric lamp had just been patented. While the lighting industry has definitely made some progress since that time, the Kjeldahl method has remained relatively unchanged.

The Kjeldahl method consists of a procedure of catalytically supported mineralization of organic material in a boiling mixture of sulfuric acid and sulfate salts at boiling temperatures between 340 and 370 degrees C. In the digestion process, the organically bonded nitrogen is converted into ammonium sulfate. Alkalizing the solution liberates ammonia which is quantitatively steam-distilled and determined by titration.

BUCHI introduced its first Kjeldahl system in 1961 with a simple, but first automatic nitrogen determination apparatus with integrated steam generator. Digestion and scrubbing products were later added to the portfolio, and offerings became increasingly more sophisticated over time. The latest innovation offered the first nitrogen distillation instrument with integrated titration for potentiometric and colorimetric titration and sample changer for a fully automated process.

This post serves as part 1 of a 2-part series.

  • Part 1 will focus on some of the fundamentals of the Kjeldahl method, starting with some basic theoretical background and experimental considerations.
  • Part 2 will directly compare Kjeldahl and NIR methods for protein determination, providing insight into which is the better solution for a variety of scenarios

The importance of bringing this content into the BUCHI NIR blog? It all goes back to the argument “you only get out what you put in.” That is to say, the quality of the Kjeldahl measurements will profoundly impact NIR method accuracy when Kjeldahl serves as the primary source of sample reference data.

For a deeper dive into the theory, or for those who prefer listening over reading, please refer to the 2017 BUCHI webinar: Kjeldahl Chemistry, embedded below. Or, if you’re good on the Kjeldahl side of things and want to skip ahead to how this relates back to NIR, scroll down to the final section of this blog: “Optimizing NIR-Kjeldahl Synergy.”

Check out this 2017 webinar to learn: ✔️ fundamental Kjeldahl chemistry ✔️ why we digest ✔️ why we use distillation ✔️ develop what & how to best fit the uniqueness of your laboratory needs ✔️ and view applications notes to demonstrate how to optimize your process.

The Kjeldahl Process

At its most basic, Kjeldahl is a 5-step process: sample preparation, digestion, distillation, titration and calculation of results.

Sample Preparation

Whether food, beverage, feed, chemical, pharmaceutical or environmental, samples must be homogeneous. The weight of sample prepared will depend on the nitrogen content of the sample, as well as the degree of heterogeneity of the sample. In general, the higher the sample’s nitrogen content and the more homogeneous the sample, the lower the sample weight can be. As a general rule of thumb, the relative standard deviation (rsd) expressed in % of the mean value is a useful indicator of homogeneity. For protein contents of 6 to 30%, homogeneous samples will have rsd values < 1%. For rsd values greater than 1%, samples require additional homogenization steps (like grinding) to maintain as small a sample size as possible.

Sample drying is required for any results based on dry matter. Alternatively, the water content of a sample may be determined by classical water analysis like Karl-Fischer titration or gravimetric methods.

There are various tubes that can be used for sample preparation. Optimally, the sample should have a nitrogen content within the range of 1 to 200 mg per sample tube. Macro-, standard-, and micro- Kjeldahl reflect the amount of nitrogen per sample tube, which also dictates the appropriate sample tube size. For example, macro Kjeldahl refers to a sample with 10 – 30 mg N and requires a 300 or 500-mL tube size.

Sample preparation can be aided using weighing tables. Different weighing tables exist for solid and liquid samples. Start by selecting the expected nitrogen content of the sample [N%], then select titrant concentration [Normality, N], then choose sample weight (in grams) that will correspond to a 3-17 mL titrant consumption. Worked examples, including tables for calculating the necessary volume of sulfuric acid, are provided in the BUCHI BUCHI Kjeldahl Practice Guide booklet. BUCHI also offers the Kjeldahl Optimizer app app to help optimize process parameters for digestion, distillation and titration.


During the digestion step of the Kjeldahl method, organic matter in the sample is destroyed by boiling in concentrated sulfuric acid. In this process, organically-bonded nitrogen is converted to ammonium ions (i.e. mineralized), while organic carbon and hydrogen form carbon dioxide and water. Water will be observed as it condenses at cool parts of the glassware. The formation of carbon dioxide can be visualized by foam in the samples, as shown in the image below. Under good reflux conditions, the foam will decompose over the course of the digestion. Once the digestion has produced a clear liquid, an additional 30 minutes of digestion is typically recommended to ensure complete mineralization.

Sample mid- and post-digestion.

In Johan Kjeldahl’s original procedure, digestion was carried out in boiling sulfuric acid, and oxidation was supported by the addition of potassium permanganate, a strong oxidizing agent. Now, scientists use salts (e.g. potassium sulfate) and catalysts or hydrogen peroxide, which further speed up digestion. Selenium and metal salts, particularly mercury, copper or titanium, are common catalysts today.

It is important to keep foam formation under control to avoid poor reflux conditions. Optimization of digestion parameters can be aided by using the Kjeldahl Optimizer app, which will provide critical parameters including the recommended number of Kjeldahl tablets (or powder weight) and a calculation of the required volume of sulfuric acid.

Types of digesters

There are two types of digestion: infrared (IR) and block. The IR-digestion uses convection and irradiation, whereas a block digester uses direct physical contact of the block’s metal surface with the glass, providing higher heat energy for reflux. Due to faster heat transfer and automation potential, block digesters are characterized by higher throughput.

Functional principle of block digester, BUCHI KjelDigester. The aluminum block (1) generates high temperatures in the samples (2). The sample is digested in constantly boiling sulfuric acid. Hot acid fumes rise into the condensation zone (3), and condense and rinse back down to the sample creating a constant reflux. Residual fumes (4) which escape the condensation zone are highly corrosive and must be withdrawn and efficiently neutralized (e.g. with the Scrubber K-415).

The main advantage of IR digestion (e.g. BUCHI SpeedDigester) is flexibility in both experimental setup and applications. For example, one SpeedDigester can be set up with various sample tube sizes, as well as for reflux digestion by water or air by simply changing the accessory. The water reflux set-up dramatically expands the range of applications possible, including heavy metal determination in soil, electronic waster, water, food, feed, and textiles. The air reflux set-up is used to determine COD (chemical oxygen demand) in water to evaluate water quality.

Principles of IR digestion (BUCHI SpeedDigester).
The infrared heating (1) generates high temperatures in the sample (2). The sample is digested in constantly boiling acid. Hot acid fumes rise into the condensation zone (3), condense and rinse back down to the sample, creating a constant reflux. Residual fumes which escape the condensation zone is highly corrosive and must be withdrawn and efficiently neutralized (i.e. with the Scrubber K-415).

Acids & Catalysts

In Kjeldahl applications, 98% sulfuric acid is used for digestion. Special applications may call for modifications in the concentration of sulfuric acid or mixtures of acids. For example, digestions meant for the analysis of phosphates in wastewater use a mixture of sulfuric and nitric acid.

The amount of acid required for a reaction can be calculated by adding the individual acid consumption per constituent in the sample (e.g. fat, protein, carbohydrates). An excess of sulfuric acid is required to ensure formation of ammonium ions and nitrogen loss by evaporation of ammonia is avoided.

There are a variety of catalysts used in digestion. Usually, Kjeldahl tablets consist mainly of an inert salt (potassium or sodium sulfate) which increases the boiling point of sulfuric acid. Additionally, 1- 3% of one or several metal catalysts is added to speed up the chemical reaction. Some samples require additives like silicone to reduce the formation of foam at the beginning of digestion.

Advantages and disadvantages of common digestion catalysts. For more detailed information and practical guidance on selecting the appropriate catalyst and salt concentrations, consult the Kjeldahl Knowledge Base eBook.

Kjeldahl tablets are introduced to the sample solution to raise the boiling point and catalyze the process. BUCHI has designed a mobile and web Kjeldahl Tablet Configurator app that can help identify the appropriate tablets based on user input regarding: safety and ecological aspects, digestion and time and potential for sample foaming. Possibilities include: titanium, copper, Missouri, specialty anti-foaming and economy versions.


After digestion, the sample is allowed to cool to room temperature. Then the acidic digestion mixture is diluted with distilled water and the sample tube is transferred to a distillation unit. The digestion mixture is next alkalized with sodium hydroxide (NaOH) prior to distillation to free up ammonia. The ammonia is steam distilled into an acidic receiver solution.

The receiving vessel for distillate collection is filled with an acidic absorbing solution in order to capture the dissolved ammonia gas. Common solutions are aqueous boric acid (for boric acid titration) or sulfuric acid (for back titrations). Distillation method parameters will vary depending on the titration method selected. Distillation time will vary depending on instrumentation, but typically falls between 150 to 300 seconds.


After steam distillation is complete, ammonia may be quantified. This is most commonly done by means of titration. The pH in the acidic receiver solution rises upon the addition of ammonia. The nitrogen and protein content is then determined by titration of the borate complex.

Two titration types are commonly used in Kjeldahl methods: boric acid and back titration. Boric acid is most common, as it is the direct detection and allows for automation without additional equipment. In back titration, the receiving solution is a standardized acid of which an accurate volume is dispensed into the receiving vessel. After collecting ammonia the excess acid is titrated with a basic titration solution pH 7.00. Back titration is often used to avoid health concerns with boric acid use but comes at a trade-off of being more expensive, as additional dosing unit and more volumentric solutions are required.

The most sophisticated procedure is the use of an automated Kjeldahl distillation unit with a built-in titrator and having the result calculation done by the software of the instrument or an optional PC software (e.g. KjelLink PC for KjelMaster K-375). In any case, the chemical reaction used for calculation is the tetrahydroxyborate anion with a generalized strong acid.

Potentiometric or colorimetric titration may be employed for pH detection. These options are characterized by unique advantages and disadvantages.

When boric acid titration is employed, potentiometric or colorimetric detection of the titration is possible. Colorimetric detection does not work with back titration.


Calculations can aim at results expressed in absolute nitrogen (mgN) per sample or in terms of concentration (%N, mgN/kg, mgN/L, etc). The calculations will differ based on boric or back titration, as described in the Kjeldahl Knoweldge Base eBook. The KjelMaster K-375 performs the calculation automatically.

To calculate the protein content, nitrogen is multiplied by a sample-specific protein factor. For a “general protein”, there is an assumption of an average nitrogen content o 16%, which leads to a “general protein factor” of 6.25.

Due to improvements in analytical technology that have made more precise protein determinations feasible, adjusted protein factors have been derived for individual protein containing foodstuffs, and are supported by official bodies like AOAC , ISO and DIN.

More Resources

Watch an automated Kjeldahl process in this application video highlighting dog biscuit analysis.

For more detailed information and downloadable content on this topic, please refer to the BUCHI Kjeldahl Practice Guide or Kjeldahl Knoweldge Base eBook. You can also find detailed information for specific applications in our applications database.

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