The document describes the Kjeldahl method for determining total nitrogen in a sample. It involves three main steps: digestion, distillation, and titration. In digestion, the sample is broken down in sulfuric acid to convert nitrogen into ammonium ions. Distillation transforms ammonium ions into ammonia gas, which is captured in an absorbing solution. Finally, titration uses sulfuric acid to determine the amount of ammonia in the solution, allowing calculation of the nitrogen content in the original sample. The Kjeldahl method is commonly used to indirectly estimate protein content through nitrogen levels and provides important information about water quality and soil fertility.

1. Institute of Environmental Engineering and Research
Environmental Lab Techniques
Lab Manual
Submitted to: Ma’am Rohab Asad
Submitted By:
Maryam Gull (2019-EN-31)
Maryam Abid (2019-EN-32)
Atiqah-Bint-e Fayyaz (2019-EN-33)

2. Experiment No: 05
Estimation of pH Curves
Objective:
To determine the equivalence points of two titrations from plots of pH versus mL of titrant
added.
Apparatus:
 Beaker
 0.02N of H2SO4
 Indicator
 Pipette
 Burette
 pH meter
 0.1N of HCl
 NaOH
RelatedTheory:
Introduction:
A titration curve is a graphical representation of the pH of a solution during a titration. In a
strong acid-strong base titration, the equivalence point is reached when the moles of acid and
base are equal and the pH is 7. In a weak acid-strong base titration, the pH is greater than 7 at
the equivalence point.

3. PH:
pH =−log [H+]
An acidic solution has a high concentration of hydrogen ions (H+). 0r Compound that donates
electron termed as Acid.
A basic solution has a high hydroxyl ion (OH-) concentration. Or Compound that accepts
electron termed as Base
pH is a measure of how acidic/basic water is. The range goes from 0 to 14, with 7 being neutral.
pHs of less than 7 indicate acidity, whereas a pH of greater than 7 indicates a base.
NeutralizationReaction:
The reaction of an acid with a base is called a neutralization reaction. The products of
this reaction are a salt and water.
pH of reaction solution depends upon formed salt;
 Both Acid and Base (Strong/weak) ----> Neutral Salt
 Strong Acid and weak Base ----> Acidic Salt
 Weak Acid and Strong Base ----> Basic Salt
Indicator
In chemistry, pH is a scale used to specify the acidity or basicity of an aqueous solution. Acidic
solutions (solutions with higher concentrations of H+ ions) are measured to have lower pH
values than basic or alkaline solutions.
Indicator is used to determine end point.
Indicators:
 Phenolphthalein
 Methyl Orange etc.
 In Acids, Phenolphthalein is colorless.

4.  In Bases, phenolphthalein gives pink color.
pH Curves
Equivalence point:
The point at which the titrant neutralizes all the titrate, leaving behind the neutral water is
called as equivalence point.
It is the point at which an equal amount of acid neutralizes an equal amount of base or vice
versa.
End Point:
The point at which titration is ended due to some change in indicator.
Average pH of common solutions
Substance pH range Type
Batteryacid < 1
Acid
Gastric acid 1.0 – 1.5
Vinegar 2.5
Orange juice 3.3 – 4.2
Black coffee 5 – 5.03
Milk 6.5 – 6.8
Pure water 7 Neutral
Sea water 7.5 – 8.4 Base

5. Ammonia 11.0 – 11.5
Bleach 12.5
Lye 13.0 – 13.6
Environmental Significance:
 pH is highly important and is used to monitor for safe water conditions. Many animals
cannot live in a pH level below 5 or above 9.
 Once the normal pH range for a water has been established, a rise or fall in pH can
indicate chemical pollution, or acid rain
 In general, fish reproduction is affected at pH levels below 5.0
 Fish begin to die when pH falls below 4.0
Procedure:
 Take a beaker.
 Add 20ml of 0.1N NaOH in it.
 Place pH electrode in beaker and note its pH.
 Now add 0.5ml of 0.1N HCl in the beaker and again note its pH by dipping the pH
electrode in it.
 After every 0.5ml addition, note pH of the solution.
 Continue these steps till 30mL of HCl is added.
 Prepare a table of it and XY scattered graph from it.
Volume(NaOH) pH
0.5 8.1
1 8.2
1.5 8.4
2 8.3
2.5 8.1
3 7.9

6. For HCL:
 Now take 20ml of HCl in the beaker and note its pH.
 Then add 0.5ml of NaOH in the beaker
 Note the pH of solution.
 Repeat the process till 30mL of NaOH is added.
 Then make the graph of obtained data with pH on vertical axis and volume used on
horizontal axis.
 Where the two curves cross each other, that point will be the equivalent point.
Volume(HCl) pH
0.5 7.2
1 7.3
1.5 7.3
2 7.4
2.5 7.4
7.8
7.9
8
8.1
8.2
8.3
8.4
8.5
0 0.5 1 1.5 2 2.5 3 3.5
pH
Volume
For NaOH
1

7. 3 10
Combined Graph:
Comments:
The pH scale is used to measure the acid and alkaline present in various fluids. The pH scaleranges
from 0 to 14. A pH of 7 is neutral, whereas a pH results below 7 is acidic and above 7 is alkaline.
Urine has the highest range of pH compared to other bodily fluids.The American Association for
ClinicalChemistry says the normal urine pH range is between 4.5 and 8. Any pH higher than 8 is
basic or alkaline,and any under 6 is acidic.
References:
 https://www.fondriest.com/news/whatis-ph.htm
 https://www.google.com/search?q=ph
 https://www.almadartebio.org
6
7
8
9
10
0 1 2 3 4
For HCL
2
7
7.5
8
8.5
9
9.5
10
10.5
0 0.5 1 1.5 2 2.5 3 3.5
1
2

8. Experiment No # 06
ESTIMATION OF SO4
-2
USING TURBIDITY METER
Objective:
To find sulphate ions in the water sample using turbidity meter.
Apparatus:
 Whatman No. 1 filter paper
 Magnetic stirrer
 Volumetric Flasks
 Turbidity Meter
 Funnel
 Sample Cells
Chemicals:
 Standard Sulphate Solution
 Barium Chloride
 Sodium Chloride-hydrochloric acid
 Glycerol-alcohol Solution
RelatedTheory:
Introduction:
Sulfate are found in appreciable quantity in all natural waters, particularly high in arid and
semi-arid regions where natural waters in general have high salt content. Sulfate salts are
mostly soluble in water and impart hardness. Water with high concentrations has a bitter taste.
Sulfate may cause intestinal disorders.

9. Turbidity:
It is the measure of relative clarity of a liquid. It is an optical characteristic of water and is a
measurement of the amount of light that is scattered by material in the water when a light is
shined through the water sample. Turbidity makes water cloudy or opaque.
Unit of Turbidity:
Turbidity is measured in NTU: Nephelometric Turbidity Units.
Measurement of Turbidity:
The instrument used for measuring it is called nephelometer or turbidimeter, which measures
the intensity of light scattered at 90 degrees as a beam of light passes through a water sample.

10. Stock Solution:
A stock solution is a large volume of common reagent, such as hydrochloricacid or sodium
hydroxide, at a standardized concentration. This termis commonly used in analytical chemistry
for procedures such as titrations, where it is important that exact concentrations of solutions
are used.
Preparationof Stock Solution:
A stock solution is prepared by weighing out an appropriate portion of a pure solid or by
measuring out an appropriate volume of a pure liquid, placing it in a suitable flask, and diluting
to a known volume.
Environmental Significance:
Sulfates contribute to acidification of surface water and soil, and contribute to acid rain and
fog that damage ecosystems, forests and plants. High concentrations of sulfate in the water we
drink can have a laxative effect when combined with calcium and magnesium, the two most
common constituents of hardness. Bacteria, which attack and reduce sulfates, form hydrogen
sulfide gas (H2S). That’s why it is essential to determine sulphates in water and its removal.
Procedure:
 Prepare 100ppm sulphate solution in 100mL of water using copper sulphate penta
hydrated compound.
 Take 25.989 mg (0.0259g) of CuSO4.5H2O in 100 ml of distilled water.
 Take 5 100 ml volumetric flasks and label them as 0, 10, 20, 30 and 40 ppm.
 Take 10 ml stock solution in 10 ppm flask.

11.  Take 20 ml stock solution in 20 ppm flask.
 Take 30 ml stock solution in 30 ppm flask.
 Take 40 ml stock solution in 40 ppm flask.
 No stock solution in 0ppm (blank)
 Add 10 ml 2% BaCl2 solution in each of the 5 flasks.
 Now add distilled water in each of the 5 flasks up to 100 ml mark.
Turbidity
 Shake solutions well and place each in cell and measure turbidity.
 Plot calibration curve.
Determination of unknown sample
 Take 10 ml of sample in 100 ml volumetric flask and add 10 ml 2 % BaCl2 in it.
 Add 80 ml of distilled water in it.
 Place in cell and measure turbidity.
 Identify reading in Y-axis and extend to cut calibration curve. Draw perpendicular which
gives unknown concentration.
ObservationandCalculations:
Sample conc. Turbidity
0 0.203
10 0.204
20 0.227
30 0.229
40 0.231

12. Comments:
Sulfate levels above 250 mg/L may make the water taste bitter or like medicine. High sulfate
levels in water may also corrode plumbing, particularly copper piping. In areas with high sulfate
levels, plumbing materials more resistant to corrosion, such as plastic pipe, are commonly used.
It also damages the ecosystem and forests. So, it is important to determine the concentration
of sulphates ions in water.
References:
 https://www.google.com/search?q=what+is+turbidity
 https://web.iitd.ac.in/~arunku/files/
 https://www.jstor.org/stable/44111423
 https://www.google.com/search?q=preparation+of+stock+solution
Experiment No # 07
CALCULATING TOTAL AMOUNT OF NITROGEN
Objective:
We have to find out total nitrogen in a sample.
Apparatus:
 Burette
 Burette stand
 Kjeldhal Flask
 Titration Flask
 Beaker
 Pipette
0.2
0.205
0.21
0.215
0.22
0.225
0.23
0.235
0 5 10 15 20 25 30 35 40 45
Turbidity Curve

13.  Hot Plate
 Glass Beads
Chemicals:
 Sample
 Digestion Reagent
 Alkali Thiosulphate
 Boric Acid
 Phenolphthalein
 0.02N H2SO4
Related Theory:
Introduction:
Using a conversion factor, the kjeldahl technique assessed total nitrogen and protein as
nitrogen content of the sample. Copper sulphates are used as a catalyst to digest the sample in
sulfuric acid. Nitrogen is converted to ammonia, which is then distilled and titrated. This
approach is used to determine nitrogen levels in organic and inorganicmaterials.
Kjeldhal Method:
Protein of organic matter is estimated indirectly by determining their nitrogen content. There
are different methods used for determination of nitrogen content. The most commonly used

14. method for determination of nitrogen content is kjeldahl method. It is assumed that mixture of
protein contain about 16% of nitrogen. Thus protein of a sample is determined by calculating
nitrogen content. A conversion factor is used for this purpose is 6.25. It is applicable in most of
foods because their non-proteinic nitrogen is negligible.
Steps:
There are three steps involves in this method:
 Digestion:
The basic purpose of digestion is to break all nitrogen bonds and converted into
ammonium ions. In this step we add sulfuric acid at temperature which lie in range of
350 to 380 degree. Higher temperature is used for fast digestion. This speed and
efficiency of digestion is increased by using catalyst copper sulphates. Potassium
sulphates is added to increase the boiling point of Sulfuric acid.
Sample + sulfuric acid → carbon dioxide+ water
Protein (N) + H2 SO4→ (NH4)2 SO4 + H2O+ CO2
After completion of digestion, sample is placed to cool down and prepared for distillation
process. Sample formed ammonium sulphate and settle down in flask. Bluish shade is present
in the sample due to copper ammonium sulphate. Before placing our sample at distillation unit,
we have to remove copper from our sample and make pH basic from acidic. It is diluted with
distilled water. We add alkali Thiosulphate, it breaks copper ammonium sulphate complex.
Alkali makes basic solution. Now sample is ready for distillation.
 Distillation:
Ammonium ions are transformed to ammonia in this process. Steam distillation is used to
move ammonia gas into the receiving vessel. Ammonia gas is captured in the receiving vessel by
an absorbing solution. Boric acid was placed in a receiving flask. Continue distilling until the
volume in the receiving flask reaches 250ml. Stop the distillation process after one and a half
hours.
 Titration:
We titrate our receiving flask solution with Sulfuric acid as a titrant by using indicator. Boric
acid form ammonium borate and give green color and its react with sulfuric acid again form
ammonium sulphate. Green color disappear and after titration it changes into violet color. This
is end point of titration.

15. Environmental Significance:
When nitrogen enters water by runoff from agricultural land or surface runoff, it combines
with oxygen to generate nitrogen dioxide, which causes respiratory issues when humans
consume it and also causes an algae bloom. TKN also provides information on the protein
content of food samples. Nitrates induce a deficiency in vitamin A and impair the thyroid
gland's ability to operate. Nitrogen is necessary for plant development, but also upsets marine
life by diminishing oxygen in the water, thus we must treat any water with nitrogen levels
greater than 50 mg/l first. Because nitrogen improves soil fertility, it is also utilised to
manufacture fertilisers.
Procedure:
Sample Preparation:
 Take a kjeldhal flask. Add 140ml of sample in it and if it is solid add 1g of sample in it. Add
140ml of distilled water in it and if solid sample is taken then add 280ml of distilled water.
 Add 20ml digestion reagent in it.
 Place flask on hot-plate at 2500C. Keep on heating till all liquid evaporates.
 Add 100ml of water in flask. Shake it well till we have clear solution.
 Add 20ml of alkali Thiosulphate solution in it.

16.  Add 1ml phenolphthalein in it.
 If pink color doesn’t appears then add 20ml more alkaliThiosulphate. Color becomes pink.
 Add distilled water to make total volume up-to 300ml.
Distillation:
 Take a titration flask. Add 50ml of indicating boric acid solution.
 Place kjeldhal flask at heating end of distillation apparatus and place titration flask at
receiving end.
 Keep on distillation till volume in titration flask reaches up-to 250ml.
Titration:
 Take 100ml from titration flask in a beaker and titrate it against 0.02N H2SO4 till violet
color appears again.
 Note volume of H2SO4. Suppose that volume is ‘A1’.
 Repeat same procedure for blank solution. Suppose volume is ‘B’.
 Titrate remaining 150ml solution in titration flask. Suppose that volume of H2SO4 is ‘A2’.
Observation andCalculations:
N(
mg
l
) =
(A1 + A2 − B) × Nof H2SO4 × eq.wt.of N × 1000
volume of sample
Calculationfor sample:
Initial Final Difference
0 11.5 11.5
11.5 24.2 12.7
Sum = 24.2
Calculationfor blank:
Initial Final Difference
24.2 24.6 0.4
N= (24.2-0.4)*0.02*14*1000/140
= 48mg/L

17. For Inorganic nitrogen:
N (
mg
l
) =
(A1 + A2 − B) × Nof H2SO4 × eq. wt. of N × 1000
volume of sample
N=21.5*0.02*14*1000/140
=43 mg/l
For Organic Nitrogen:
Total Nitrogen= Inorganic Nitrogen + Organic Nitrogen
For Inorganic Nitrogen:
 No Digestion.
 Take 140mL of sample and add 140mL of Dist. Water in it.
 Add 20mL of alkali thio sulphate solution.
 Remaining process is same as distillation and titration as in previous experiment.
Comments:
The TKN value for municipal wastewater is between 35 and 60 mg/l, according to the US EPA's
NPDES (National Pollutant Discharge Elimination System). Our sample comes from our
university's dormitories and has a TKN value of 52.6mg/l, which is in the range of municipal
wastewater.
References:
 https://www.google.com/search?q=total+nitrogen+in+wastewater
 https://www.thewastewaterblog.com/single-post/2017/11/01/total-kjeldahl-nitrogen
 https://www.coleparmer.com/tech-article/kjeldahl-method-for-determining-nitrogen
 https://www.velp.com/en-ww/kjeldahl-method-1.aspx