Kjeldahl nitrogen

Kjeldahl analysis is definitely the most common method for performing nitrogen and protein
determination for diversified applications.
Nitrogen determination has a long history in the area of analytical chemistry.
Johan Kjeldahl first introduced the Kjeldahl method in 1883 at a meeting of the Danish Chemical Society.
Kjeldahl, at that time Carlsberg laboratory manager, was assigned to scientifically observe the processes involved in beer
production.
While studying proteins during malt production, he developed a method of determining nitrogen content that was faster
and more accurate than any method available at the time.

Johan Kjeldahl, working at Carlsberg Laboratory in the 1880s

Kjeldahl analysis is extremely versatile, as it can handle a very wide range of samples, from food & feed (grain, meat, fish,
milk, dairy, fruit, vegetables), beverages, environmental (agriculture, oilseeds, soil, fertilizers, water, wastewater, sludge) to
chemical and pharmaceutical industries (paper, textiles, rubber, plastic, polymer).

Total Kjeldahl nitrogen (TKN) is the sum of organic nitrogen, ammonia (NH3), and ammonium (NH4+) in the chemical
analysis of soil, water and wastewater.
To calculate Total Nitrogen (TN), the concentrations of nitrate-N and nitrite-N are determined and added to the total
Kjeldahl nitrogen.

Today, total Kjeldahl nitrogen is a required parameter for regulatory reporting at many treatment plants.
Total Kjeldahl nitrogen is often used as a surrogate for protein in food samples. The conversion from TKN to protein
depends on the type of protein present in the sample and what fraction of the protein is composed of nitrogenous amino
acids. However, the range of conversion factors is relatively narrow. Example conversion factors, known as N factors, for
foods range from 6.38 for dairy and 6.25 for meat, eggs and maize (corn) to 5.70 for wheat flour, and 5.46 for peanuts.

In most cases the key to a successful Kjeldahl analysis can be the sample preparation step (before digestion phase).
This method might not be the fastest method to use but thanks to the high reliability will always give satisfactory results, if
performed correctly (and following Standards).

The Kjeldahl analysis may be broken down into three main steps:
Digestion - the decomposition of nitrogen in organic samples utilizing a concentrated acid solution. This is accomplished
by boiling a homogeneous sample in concentrated sulfuric acid. The end result is an ammonium sulfate solution.
Distillation - adding excess base to the acid digestion mixture to convert NH4+ to NH3, followed by boiling and
condensation of the NH3 gas in a receiving solution.
Titration - to quantify the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be
calculated from the quantified amount of ammonia ions in the receiving solution.

Today, various scientific associations approve the Kjeldahl method, including the AOAC International (Association of
Official Analytical Chemists), AACC (Association of American Cereal Chemists), AOCS (American Oil Chemists Society), EPA
(Environmental Protection Agency), ISO (International Standards Organization), and many others. All VELP Scientifica
equipment for Kjeldahl nitrogen determination work in accordance with the above-mentioned associations.

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 Kjeldahl digestion
Kjeldahl digestion converts nitrogen compounds (proteins, amines, organic compounds) into ammonia
compounds.
The goal of the digestion is to break down the bonds that hold the polypeptides together and convert them into simpler
molecules (such as water, carbon dioxide and ammonium sulphate).

These reactions can be speeded up by the temperature used during Kjeldahl digestion (the higher the temperature used,
the faster the digestion can be obtained) and by the presence of acid, salt and catalysts (selenium, copper, titanium).
Vapors that escape from the tubes are aspirated through the suction cap by a JP recirculating water vacuum pump and
eliminated in an SMS scrubber. This configuration optimizes the efficiency of the operation. Avoid using Kjeldahl digestion
units without an exhaust system: this will dramatically shorten its life and might cause expensive damage. This is the most
time-consuming step of the analysis.

If the sample is solid, weigh out approximately 1 - 3 g of the sample in a VELP weighing
boat (nitrogen-free) (CM0486000 or CM0486001) and record the weight (the particle
size of the sample should be reduced to < 1 mm, for better results. The sample might
need to be homogenized, before any operation).

If the sample is liquid, measure the volume with a pipette and place it in a beaker and
stir it using one of VELP’s hot plate stirrers. If necessary, remove any CO2 (e.g. fizzy
drinks) before measuring the volume.

Place the sample into a VELP glass test tube (where nitrogen content could be quite
low, larger sample amounts need to be used) along with 12 - 20 ml of concentrated
sulfuric acid, as specified in the method. The total amount of acid needed during a
Kjeldahl digestion can vary from one sample type to another. Another factor to consider
is the loss of acid that occurs due to the evaporation through the exhaust system used.
The VELP exhaust system and heat shield control acid loss (around 1.2 ml acid per
sample).

A problem that might occur during the Kjeldahl digestion is the drying out of the
digested sample, a process called “the salting out effect”, due to the too high flow rate.

Add catalyst tablets (select the correct variety according to the protocol):
Copper catalyst (CM, CT0006650) is satisfactory but very slow to react; environmentally friendly. Selenium catalysts (W,
CT0006613) usually react very fast and are typically used for substances that are resistant to Kjeldahl digestion (e.g. fats
and oils). Other formulas are available (ST, CT0006609; TCT, CT0006621).

Select the program from the menu of the Kjeldahl digestion unit (on DKLs the most
used applications are pre-installed and others are user-programmable). Just by pressing
Menu, Programs, you can choose which Standard Program to select or create a new
Customizable Program.

Lower the samples (automatically on DKL Kjeldahl digestion units) into the aluminum
heating block (maintenance-free and highly durable) and heat the mixture to the
temperature indicated in the Standard Method. (The DKL aluminum heating block
ensures the best possible homogeneity across all tubes and a complete Kjeldahl
digestion in each tube. It can reach 450 °C / 842 °F, ensuring a nitrogen recovery higher
than 99% in the following stages).

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 Heat the mixture for the time indicated in the Standard Method in order to obtain a clear and colorless solution. During
this phase the sulfuric acid reacts with the sample, converting all nitrogen in organic form into inorganic form that is stable
and ready to be analyzed.

H2SO4 + Organic N →(NH4)2SO4 + CO2 + H2O + other compounds

If any problem occurs during the Kjeldahl digestion and/or if the sample preparation was not correct, the customer can
notice the presence of carbon residues (black-brown colored) in the digested mixture and on the walls of the tubes. These
are symptoms of an incomplete mineralization of the sample which cannot be processed further.

Raise the samples (automatically on DKL Kjeldahl digestion units) and cool by natural radiation.
Separate the suction cap (press the up arrow on DKL Kjeldahl digestion units) - a drip tray needs to be introduced below
the suction cap to collect any drops of acid that might fall from the suction cap glass bells.
Now the tube rack can be removed and the samples are ready to be moved to the distillation phase.

Typical process for Kjeldahl digestion

Today, various scientific associations approve the Kjeldahl method, including the AOAC International (Association of
Official Analytical Chemists), AACC (Association of American Cereal Chemists), AOCS (American Oil Chemists Society), EPA
(Environmental Protection Agency), ISO (International Standards Organization), and many others. All VELP Scientifica
equipment for Kjeldahl nitrogen determination work in accordance with the above-mentioned associations.

Automatic Kjeldahl Digestion Units - DKL Series

Fully Automatic Kjeldahl Digestion Units for nitrogen analysis and protein determination that
incorporate the revolutionary TEMS™ technology for unprecedented Savings in terms of Time,
Energy - as much as 35%, Money and Space.

High Technology Outstanding Reliability

~ Microprocessor control of precise block
temperature
~ Real time display of process steps
~ 54 programs (30 pre-installed, 24 customizable)
Automated Operation
~ Calibration automatic for top precision &
repeatability
~ No hands lowering and lifting of test tubes
~ Auto separation of manifold from the rack
~ Aluminum block for absolute temperature
homogeneity
~ Unmatched conductivity for fast response up
to 450 °C
~ Conforms to Good Laboratory Practice
standard
~ Premium quality consumables
Practical Functionality
~ LCD display for immediate understanding
~ All necessary items supplied in one package
~ Very small footprint

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 Kjeldahl distillation
Kjeldahl distillation units offers different levels of automation, from simple distillation, to distillation
with automatic reagents addition and residues aspiration, up to fully automated system with integrated
colorimetric titration.
The ammonium sulphate present in the digested sample are converted into ammonia gas, heated and distilled. The
ammonia gas is led into an acid trapping solution where it dissolves and becomes a trapped ammonium ion once again.
Using the Kjeldahl distillation, nitrites and nitrates are not detected. In order to quantify these elements, a reduction of
the sample is necessary (using Devarda alloy) before the Kjeldahl digestion stage.

Add distilled or deionized water to the test tube

containing the digested sample to dilute it
(automatically on UDK 139 & UDK 149 Kjeldahl
Distillation Units and UDK 159 Automatic
Kjeldahl Analyzer). In this way it’s easier to detect
all the ammonia.

Separate the nitrogen from the digested mixture

by steam distilling (steam output regulation 10-
100% on UDK 139 & UDK 149 Kjeldahl Distillation
Units and UDK 159 Automatic Kjeldahl Analyzer),
in order to extract ammonia from the alkaline
solution.

Raise the pH of the digested mixture using sodium

hydroxide (35%) (automatically on UDK Kjeldahl
Distillation Units) to convert NH4+ (in solid
format) into NH3 (gaseous), that will be detected
Steam distillation on VELP Scientifica UDK 159, Automatic Kjeldahl Analyzer
with titration.
Trap the distilled vapors in a dedicated solution of 25-30 ml of boric acid (automatically on UDK 149 Kjeldahl Distillation
Unit and UDK 159 Automatic Kjeldahl Analyzer) to trap all the nitrogen, eliminating the risk of loss.

(NH4)2SO4 + 2NaOH → 2NH3 + Na2SO4 + 2H2O

NH3 + H3BO3 → NH4 H2BO3 + H3BO3

Drain the test tube with the digested sample (automatically on UDK 139 & UDK 149 Kjeldahl Distillation Units and UDK 159
Automatic Kjeldahl Analyzer).

Typical process for Kjeldahl distillation

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 Now perform the final titration of the ammonia distilled from the sample, considering that if the nitrogen content of the
sample is high, a high-concentrated acid for the titration is needed. Another solution is reducing the quantity of the
sample used for the analysis, but in some cases it may cause errors giving wrong results.
The following step is the titration, to determine the amount of ammonia distilled off from the digested solution and hence
calculate the Kjeldahl nitrogen or protein amount, as %.

2 NH4 H2BO3 + H2SO4 → (NH4)2SO4 + 2H3BO3

With UDK 159 Automatic Kjeldahl Analyzer (distillation and colorimetric titration):
Add two indicators to the boric acid (4%) solution, in order to follow the titration
process by a color change. The color is red in absence of ammonia, turns green in
case of significant amount and grey/pink at the end of the analysis.

Put a standardized solution (titrant) of hydrochloric acid (HCl) or sulfuric acid

(H2SO4) in the burette; this solution will be added (automatically) to the colored
boric acid containing the ammonia distilled from the sample. The acid reacts with
ammonia in order to measure it.

Record the volume of the acid titrant solution that was necessary to reach the
endpoint and perform a final calculation to find the amount of nitrogen, expressed
as % N or % proteins, in the original sample (automatically).

With an external potentiometric titrator with a pH electrode (connectable to the UDK 149 Kjeldahl Distillation Unit):
The titrator burette adds the acid titrant solution automatically to the boric acid solution containing the distilled ammonia,
until reaching the endpoint, corresponding to pH=4.7. In this case we don’t check a color change and we don’t use
indicators, but we follow the corresponding change in the pH of the boric acid solution during the titration process using
an external potentiometric titrator with a pH electrode.

Today, various scientific associations approve the Kjeldahl method, including the AOAC International (Association of
Official Analytical Chemists), AACC (Association of American Cereal Chemists), AOCS (American Oil Chemists Society), EPA
(Environmental Protection Agency), ISO (International Standards Organization), and many others. All VELP Scientifica
equipment for Kjeldahl nitrogen determination work in accordance with the above-mentioned associations.

UDK 159 Automatic Kjeldahl Analyzer

Fully automatic Kjeldahl Analyzer (distillation unit with integrated colorimetric titrator), for your analyses concerning
different applications such as determining ammoniacal nitrogen, protein determination, nitrogen content (Kjeldahl or
direct alkaline distillation), nitric nitrogen (after reduction/Devarda), phenols, volatile acids, cyanides and alcohol content.
Automated Operation High Tech
~ Programmable boric acid, water, sodium ~ 6 inch color touch screen
hydroxide addition ~ Library of 55 programs (31 pre-defined, 24
~ Not necessary to set distillation time when customizable)
titrating ~ Choice of 6 languages – from a wide range
~ Incorporates an AOAC recommended downloadable
colorimetric titrator ~ Balance, mouse & printer connections
~ Selectable steam generation output level 10% – ~ No loss of nitrogen: distillate temperature
100% stays below threshold
~ Auto removal of residues from titrator & sample ~ Accurate dosing of reagents with high
tube precision pumps

Versatile Data Handling Outstanding Reliability

~ Multiple data interfaces: Ethernet, USB and RS ~ Maintenance-free, resistant, patented steam
232C generator
~ On-board archiving of data including from ~ Patented titanium condenser for unmatched ~
balance performance for more
~ Data exporting to pen drive or PC in .xls format ~ Durable technopolymer splash head information
~ Data manipulation software can be user ~ Chemically-resistant technopolymer housing
preferred

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