Nitrogen is a necessary part
of all living cells, proteins,
enzymes , and metabolic
processes involved in the
synthesis and transfer of energy. The
element is one of the most important
mineral nutrients and is known to play
a crucial role in a plant’s growth, reproduction,
and survival. Nitrogen is present
in chlorophyll, a green pigment that
allows plants to capture energy from the
sun and produce food for themselves in
a process called photosynthesis. This
vital nutrient facilitates rapid plant
growth, increasing seed and fruit production
and improving the quality of
leaf and forage crops.
Although nitrogen is the most abundant
element in the atmosphere, plants are
unable to use it unless it is naturally processed
in the soil or added using the commercial
fertilizers that are popular among
farmers and gardeners. However, despite
its necessity in certain concentrations,
deficiency or excess of nitrogen can negatively
impact plant development. Overapplication
of nitrogen-containing fertilizers
can result in rapid, lush growth and a
diminished root system. In extreme cases,
too much nitrogen can cause burning of
the leaf tissue and lead to plant death.
As a result, analysis of nitrogen content
in soils is important both for the
evaluation of organic matter and the calculation
of the ideal fertilizer quantities
required to maximize plant growth.
Nitrogen content analysis is also necessary
in order to determine the quality
of various types of crops for feeding and
processing, as well as for N-cycle and
N-fixation monitoring in agricultural
and environmental research.
For many years, the Kjeldahl method has
been the worldwide standard for nitrogen
analysis in a wide variety of materials.
The method was introduced more than
100 years ago to facilitate the quantitative
determination of nitrogen content in a
variety of organic and inorganic substances,
such as meat, feed, grain, wastewater, and
soil. The method consists of five main steps:
sample digestion in boiling sulfuric acid,
neutralization with sodium hydroxide solution,
distillation of the resulting ammonia
gas into a trapping solution, titration with
an acid solution, and determination of
nitrogen content by calculation.
Despite the Kjeldahl method generating
accurate and reproducible results, the
technique is limited by a number of key
disadvantages, including that it is a very
time-consuming technique and requires
more than 4 hr to complete a single analytical
cycle. The method also demands constant
operator interaction throughout the
entire process, while the use of catalysts as
well as strong, concentrated sulfuric acid
at high temperature poses considerable
health hazards to users of the method and
generates harmful chemical waste.
The Dumas combustion method has been
developed to overcome some of the limitations
of the traditional Kjeldahl method.
It is an easy-to-use, automated instrumental
technique capable of rapidly measuring
nitrogen concentration in soils without
the use of toxic chemicals or catalysts.
However, the technique requires a high
initial investment and, as with the Kjeldahl
method, does not offer measurements of
the true protein content. In addition, the
method uses a small sample size, making it
difficult to obtain a representative sample.
As the demand increases for improved
sample throughput, reduced operational
costs, and a decrease in human error, it has
become very important to have a simple
and automatic technique that enables fast
nitrogen analysis combined with excellent
reproducibility. Adding a stage to the
Dumas combustion method in which samples
undergo dynamic flash combustion
significantly increases the method’s safety,
accuracy, speed, and dependability.
Benefits of dynamic flash
The dynamic flash combustion method uses
an innovative CO2 adsorber regenerating
technology, which is automatically activated
to adsorb the CO2 generated during the combustion.
Contrary to conventional technologies,
this self-cleaning filter does not need to be
changed several times a week, saving time and
money and enhancing the system’s autonomy
while reducing instrument maintenance. The
method also does not require sample digestion
of toxic chemicals, eliminating health hazards.
The dynamic flash combustion method
also uses a Peltier water elimination device
and a safe, sensitive, and reliable thermal conductivity detector (TCD) that covers the quantitative nitrogen/protein determination
from low ppm to high percentage concentrations.
Compared to the Kjeldahl and Dumas
combustion methods, the novel technique
achieves a much higher sample throughput
within 5–7 min, depending on the nature of
the samples. The large sample size capacity of
the method enables maximum accuracy and
reliability of results, while reducing sample
handling time and minimizing matrix effects. An experiment was performed to demonstrate
the analytical capabilities of the
dynamic flash combustion method.
The Thermo Scientific FLASH 4000 elemental
analyzer (Thermo Fisher Scientific,
Cambridge, U.K.) based on the dynamic
combustion of the sample was used to demonstrate
the efficiency of the method for
reliable nitrogen analysis in soils and plants.
The samples were selected on the basis of
their differing nature and nitrogen content,
meaning that the combustion and the
amount of oxygen required were completely
different for each sample. The samples were
weighed in a tin capsule and introduced
into the combustion reactor via the Thermo
Scientific MAS 4000 autosampler,
with the correct amount of oxygen, which
was automatically calculated using the
OxyTune® function of the Thermo Scientific
Eager Xperience software platform on
which the analyzer operates. Determining
the exact amount of oxygen is necessary in
order to ensure the complete combustion of
Following combustion, the produced gases
were passed by a helium flow to a second
reactor that was filled with copper. Water
was trapped through a water condensation
drainage device, while the CO2 was
adsorbed by no-stop twin traps. Nitrogen
was then passed through a GC column
and detected by a thermal conductivity
detector. The temperatures for the
left tube, right tube, and oven were set at
950 °C, 840 °C, and 50 °C, respectively,
and the carrier and reference flow were
both 300 mL/min. A total of 500 mg of the
9.59 %N standard were used.
Analyzer performance evaluation
by WEPAL round-robin tests
The precision of the analyzer was evaluated
through the participation in international
round-robin tests conducted as part
of the Wageningen Evaluating Programs for
Analytical Laboratories (WEPAL) of the
Wageningen University in The Netherlands.
For soil samples, data were compared
with the range accepted by WEPAL statistic
studies, including all methods for nitrogen
determination. For plant samples, the results
were compared with the range accepted for
both Kjeldahl and total nitrogen methods
including the combustion method.
The calibration of the system was performed
with EDTA STD (9.59 %N) using K factor
as the calibration method. The samples
were analyzed as they were received by
WEPAL without treatment, and the data
obtained demonstrated the no-matrix effect in the determination of nitrogen, indicating
complete combustion for all sample types.
Table 1 shows the reproducibility of 10 consecutive
runs of soil WEPAL samples using
a sample weight of about 1000 mg. Nitrogen
data obtained were inside the range of
nitrogen concentration approved by the
WEPAL statistic studies.
Table 2 displays the reproducibility of 10
consecutive runs of plant WEPAL samples
using a sample weight of about
1000 mg. Nitrogen data obtained were
inside the range of nitrogen concentration
approved by WEPAL statistic
studies for both Kjeldahl and total
Table 3 shows the reproducibility of
nitrogen determination in soils in a wide
level of concentration. Samples were
analyzed in triplicate, and the weight of
sample used was 500–1500 mg, depending
on the matrix.
Table 4 shows the reproducibility of
nitrogen determination in plants in a
wide level of concentrations. Samples
were analyzed in triplicate, and the
weight of sample used was 500–1500
mg, depending on the matrix.
The precise determination of nitrogen
content in soils is necessary in order
to get the maximum benefit from fertilization
and facilitate rapid plant
growth with minimal pollution hazard.
Dynamic flash combustion is the
ideal technique for analyzing nitrogen
in soils and plants, with experimental
data demonstrating excellent reproducibility
and no memory effects when
changing the type of sample, indicating
the complete detection of this vital
nutrient. The method is capable of analyzing
nitrogen in a wide range of concentrations,
from low to high, with optimal accuracy
and without matrix effects.
Dr. Krotz is Product Specialist, OEA, and Dr.
Giazzi is Product Manager, OEA, Thermo Fisher
Scientific, Strada Rivoltana, 20090 Rodano,
Milan, Italy; tel.: +39 02 95059336; fax: +39 02
95059276; e-mail: email@example.com.