ENVE 208

ENVIRONMENTAL CHEMISTRY LABORATORY

DETERMINATION OF NITROGEN COMPOUNDS

Nitrogen
o Nitrogen – essential in atmosphere (N2 gas) - is very important to
environmental engineering because it is a very crucial element in
the life processes of plants, animals, and humans.

o Chemistry of nitrogen is complex because of there are several

oxidation states of nitrogen.

o The oxidation state of nitrogen can be changed depending on the

environmental conditions: aerobic or anaerobic

o The oxidation state of nitrogen can be changed by the actions of

different microorganisms.
What are different oxidation states of Nitrogen?
From the viewpoint of inorganic chemists, nitrogen can exist in seven
oxidation states, and compounds in all are of interest to them:
-III 0 I II III IV V
NH3 N2 N2O NO N2O3 NO2 N2O5

However, in aquatic systems only a few of the oxidation states dominate.

These are: NH3, N2 , N2O3 and N2O5 but they actually form the most
important four forms of nitrogen we are concerned with!

Note: N2O3 is the acid anhydride of nitrous acid and N2O5 is the acid
anhydride of nitric acid

The most important 4 forms of nitrogen in an environmental sample:

NH3 + H2O NH4+ + OH-

N2O3 + H2O 2H+ + 2NO2-
N2O5 + H2O 2H+ + 2NO3-

o Organic nitrogen – (Original form of N-compounds, it is in the form of

proteins)
o Ammonia nitrogen (NH3 – NH4+) (ox. state: -3)
o Nitrite nitrogen (NO2-) (ox. state: +3)
o Nitrate nitrogen (NO3-) (ox. state: + 5)
Nitrogen Cycle
The relationship that exist
between the various forms of
nitrogen compounds and the
changes that can occur in nature
are best illustrated by a diagram
known as the nitrogen cycle.

Atmosphere serves as a reservoir from which

nitrogen is constantly removed
Atmosphere Reservoir
Electrical storms
N2 Nitric acid (rain)

Nitrogen fixing bacteria

N2 organic – N (protein)

Direct oxidation of N2 (or NH3)

N2 NO3-(nitrate)

occur during production of

commercial fertilizers
Nitrogen is crucial for life… (protein!)
**Humans and animals are dependent on plants for protein:
The nitrates serve to fertilize plant life and are converted to proteins:

• NO3- + CO2 + green plants + sunlight protein (Organic – N)

Atmospheric nitrogen is also converted to proteins by “nitrogen-fixing” bacteria:

• N2 + special bacteria protein (Organic – N)

Ammonia and ammonium compounds applied to soils to supply plants with ammonia for
further production of proteins:
• NH3 + CO2 + green plants + sunlight protein (Organic – N)

*Note that although humans and animals need organic – N (protein), not all is used. Therefore, waste
(feces) contains un-used organic-N.

Unassimilated protein is then converted…

The proteins remaining in the bodies of dead animals and plants are converted
to ammonia by heterotrophic bacteria under aerobic and anaerobic conditions:
Biological
oOrganic-N (protein) + aerobic/anaerobic bacteria NH3 decomposition of dead
(urea etc. amino acids) (hydrolysis) plants and animal
tissues, fecal matter
If the ammonia is released in excess of plant requirements, the excess is
oxidized by nitrifying bacteria (under aerobic conditions):
(nitrosomonas)
2NH3 + O2 2NO2- + 2H+ + 2H2O
(ammonia) (nitrite)
NITRIFICATION
2NO2- + O2 2NO3-
(nitrobacter) (nitrate)
(fertilizer for plants)
Nitrate conversion
NO3- * However, if nitrates are in excess of the needs of PROBLEM

(nitrate) plant life, then carried away in water to groundwater

* Methemoglobinemia in babies (Blue baby syndrome!)
NO3- NO2- (interferes with hemoglobin)
NO2 can bind to iron on hemoglobin, reducing transfer of O2 to cell tissues
-

Under anaerobic conditions (denitrification):

6NO3- + 2CH3OH 6NO2- + 2CO2 + 2H2O

6NO2- + 3CH3OH 2N2 + 3CO2 + 3H2O + 6OH-

* So, if anaerobic conditions prevail Nutrient loss from soil. Thus knowing the
N-content of soil is important.

Environmental Significance of N-data:

Indicator!
Source of pollution?
Time of pollution

Org-N Recent pollution

NH3 (great danger potential)

NO2- Pollution long time ago

NO3- (little threat)

Changes occurring in forms of nitrogen present in polluted

water under aerobic conditions
Environmental Significance of N-data (cont.):

2. In order to provide sufficient nutrients for microorganisms in biological

treatment
3. Conversion of NH3 requires O2, O2 depletion in rivers
4. N-control is becoming more and more important in biological
wastewater treatment plants
NH4+ NH3 + H+ pKa = 9.3 When pH < 8: no problem
(not toxic) with toxicity (for fish mostly)
(toxic)

Methods of N – Analysis:
Two parameters will be determined:
1. Organic nitrogen
2. NH3 – N
 Methods of N – Analysis: organic-N
o Most organic nitrogen is present in organic compounds as in the from of
amino acids, amines, amides, imides, nitro-derivatives.
o For our concern most significant ones are proteins and their degradation
products (polypeptides and amino acids). Therefore the methods
employed in water analysis have been designed to ensure measurement of
these forms without particular regard to other organic forms.
The method used for organic- N determination is called Kjeldahl Method
and it gives Total Kjeldahl Nitrogen (TKN) measuring both organic and
ammonia nitrogen.
Since we do not have a method to measure organic nitrogen directly we use
a method that measures the sum of organic-N and ammonia-N.

Methods of N – Analysis:
o TKN determination is a two-step process because it is composed of two
things (Organic – N and Ammonia – N):
• Digestion step
• Distillation step
o NH3 – N determination has only one step
• Distillation step
oIn this experiment, we determine total nitrogen by Kjeldahl – N digestion
method, which includes both Organic – N and Ammonia – N.
 Ammonia-N Measurement: distillation ( first step)
Procedure for Ammonia - N
I. DISTILLATION UNIT

The distillate is
collected in an acid
solution.
When pH goes down,
nitrogen is in NH4+
form.

NH4 NH3(g) + H+ NH3 + H3BO3 NH4+ + H2BO3-

Borate buffer (pH = 9.5)
(Higher pH values might be
When pH > 8, and under heat, the
dangerous) Excess boric acid
reaction shifts to the right and
NH3(g)

Ammonia-N Measurement: titration ( second step)

Procedure for Ammonia - N v Ammonium in the distillate
II. Concentrated is then determined by
H2SO4 titration of borate ion with
standard sulfuric acid
solution

v Acid measures the amount

Boric acid
of borate ion in solution
+
Absorbed NH3
H2BO3- + H+ H3BO3 v When the pH of the boric
acid solution is decreased to
its original values = ends
(TITRATION)
The Kjeldahl Method
o The Kjeldahl Nitrogen is defined as the nitrogen present in ammonia +
nitrogen present in organic compounds.
oIn this case FIRST all the organic-N forms must be converted to ammonia.
How to do that?
By DIGESTION in a concentrated sulfuric acid solution.
Then the same procedure: distillation + titration used for Ammonia-N will
be followed.

The Kjeldahl Method (Step 1: digestion)

This includes the N in organic
compound (proteins), also the
I. Digestion Step already existing N in NH3/NH4+)

CH3CHNH2COOH + 7H2SO4 3CO2 + 6SO2 + 8H2O + NH4HSO4

(alanine) catalyst

So, TKN cannot differentiate these

o Organics oxidized to CO2 and H2O
two. Do a distillation separately.
o N in organics is catalytically reduced to NH4HSO4
o Sulfate reduced to sulfur dioxide
o Amino group is released as ammonia, ammonia under aerobic conditions cannot
vaporize and held as ammonium salt (NH4+ )
The Kjeldahl Method (Step 1: digestion)
I. Digestion Step (cont’d)

o Temperature is above the boiling temperature of H2SO4 (340oC)

(360 – 370 oC is kept by the use of K2SO4) to increase the rate of oxidation.

o To speed up the reaction CuSO4 is added as catalyst

o H2SO4: concentrated acid solution

The Kjeldahl Method (Digestion Step)

Procedure for Total – N (Org – N + NH3 – N)
I. Digestion Step Additional step!

Sample Digestion reagent

-
conc. H2SO4
K2SO4
Heat CuSO4
-
½ hour
digestion

COMPLETE DIGESTION VERY IMPORTANT

The Kjeldahl Method (cont.)
The Kjeldahl Nitrogen Method Procedure
During digestion the N-contained in organic compounds is released using a
concentrated sulfuric acid solution for the oxidation of the organics. Nitrogen of
organic compounds is catalytically reduced to ammonia.
Then the rest follows the same procedure as in ammonia-N.
A. The following reactions took place during distillation step under basic
conditions:

heat Note: NH4 + NH3: referred to as

NH4 NH3(g) + H+ pKa = 9.3 ammonia nitrogen

o When pH > 8, and under heat, the reaction shifts to the right and NH3(g)

The Kjeldahl Method (cont.)

o The more reaction shifts to the right: pH
o To prevent drop of pH a borate buffer is used.
o Since we want to keep pH ~ 9.5 because higher pHs might be dangerous
o Experience has shown that essentially all the free ammonia will be expelled
from solutions whose pH is maintained at 9.5 by the time 200 mL of water has
been distilled when samples of 500 – 1000 mL used.
o Released NH3(g) is absorbed by the boric acid solution in another flask placed as
given by (when pH drops):
NH3 + H3BO3 NH4+ + H2BO3- NH4+: can’t volatilize

o
Calculations: %N in soil
!.#
% N in soil = (T – B) x N x $ T: Volume of standard acid used for
the sample titration, mL.
, ./ x !.#
mL x 10*+ -, x , 0 B: Volume of standard acid used for
!# 0/-BC.
Equivalent weight of N = the blank titration, mL.
! .//-BC.
0 N: Normality of standard acid.
14 x 10*+ 0 x 100 (%)
S: Sample weight, g.
= 14 x 10*! %

= 1.4 g/g

Calculations (cont.)
Calculation of Org – N using TKN and Ammonia-N data:
o Total – N (Kjeldahl) = Organic – N + NH3 – N
o So, this does not mean that
TKN ≠ NH3 – N + NO2 – N + NO3 – N

o Remember in TKN method all the organic forms of N were converted to

ammonia.

o To find out how much organic – N is present in the sample, we have to analyze
NH3 – N and then do:
(Total – N (Kjeldahl)) – (NH3 – N) = Organic – N content of soil
The apparatus used for TKN
Kjeldahl Method System

Distillation unit

Heaters

Digestion unit

You analyze a solid sample in this experiment

o In environmental engineering, solid samples are not good for chemical
analysis without pretreatment. This is because in solids there is crystal
lattice structure, that needs to be broken before or during the analysis.
o Often we would like to analyze solid samples which can be a sample from
waste silo, a compost sample, a solid waste sample from a sanitary landfill,
or some kind of sludge sample. These samples are generally heterogeneous
mixtures of organic and inorganic substances. They may contain some
metals, which need to be sorted out.
oOnce the sample is sorted, it is called the laboratory sample.
Pretreatment of the solid sample:
Solid sample = Crude sample
(at the sampling point)
Sorting (remove metals etc.)

Laboratory sample
Dry and grind to pass through 0.2 mm sieve

Store the sample under proper conditions

(refrigeration required or not)

Analyze it