Environmental Engineering Laboratory Technique

University Of Engineering and Technology, Taxila

Lab Manual

Submitted to:
“Engr. Nayab Zahra”
Submitted by:

Urwah Azam
Registration No:
18-ENV-23
Date of Submission:
30th March,2022
1
“ENVIRONMENTAL ENGINEERING LAB

TECHNIQUES”

(LAB MANUAL)

2
 Table of Contents
Layout of Laboratory:................................................................................................................5

“List of Apparatus”....................................................................................................................6

“Description of Apparatus”........................................................................................................7

“Determination Of Solids (TS, TDS, TSS, TFSS, TVSS) in Water and Wastewater Samples”

..................................................................................................................................................17
“Measurement of Dissolved Oxygen by Winkler Method”.....................................................31

“Biological Oxygen Demand in Wastewater Sample”............................................................36

“Determination Of COD by Using Close Reflux Titrimetric Method”...................................40

“To Find Maximum Wavelength of Given Sample by using Spectrometer”……………………

“Estimation of copper ion concentration in a given solution by UV-spectrophotometer”......48

“Estimation of Nickel ion Concentration in a Given Solution by UV-Spectrophotometer”…….

“Determination of chlorine by iodometric method”................................................................53

“Determination of chlorine in waster water sample”...............................................................57

“Determination of nitrogen by Kjeldhal Method”...................................................................62

3
 “List of Experiments”

Sr. No Experiment

Determination of solids (TS, TDS, TSS) in water and wastewater samples.

1

Measurement of Dissolved Oxygen by Winkler Method

2

Measurement of BOD in wastewater sample

3

4 Determination Of COD by Using Titrimetric Method.

To Find Maximum Length by Spectrometer

5
6 Estimation of copper ion concentration in a given solution by UV-
Spectrophotometer

7 Estimation of Nickel ion concentration in a given solution by UV-

Spectrophotometer

8 Determination of chlorine by iodometric method

9 Determination of Nitrate-Nitrogen in water

10 Determination of Nitrogen by Kjeld Hal Method

4
 Layout of Laboratory:

1 2 3 4 5 6 7

8
OFFICE

17
9

10

16 15 14 13 12 11

2 3 4 5 6

1
7

9
12 11 10
OFFICE

Figure 1: Layout of environmental engineering techniques laboratory

5
 “List of Apparatus”
LAB contains the following equipment:

1. Furnace.

2. Hot plate/magnetic stirrers.

3. PH meter.

4. Refrigerator.

5. Turbidity meter.

6. Digital weight balance.

7. Oven.

8. Distillation apparatus.

9. Flame photometer.

10. COD digester.

11. Vacuum PUMP.

12. Filtration assembly.

13. Water bath.

14. Electrical conductivity meter.

15. Desiccator.

16. TSS portable.

17. Titration assembly.

6
 “Description of Apparatus”
1. Furnace:

Figure 2: Furnace

A furnace is equipment used to provide heat for a process or can serve as reactor which

provides heats of reaction. The highest temperature that it provides is around 1100 oC.

Furnace designs vary as to its function, heating duty, type of fuel and method of introducing

combustion air.

2. Hot Plate / Magnetic Stirrer:

Figure 3: Hot plate

7
Hot plates contain a magnetic stirrer, provided with knobs for controlling heating

temperature and stirring speeds thus allowing the liquid to be heated and then stirred or

mixed automatically. They are used to stir and heat the substance at uniform temperature.

3. pH Meter:

Figure 4:pH Meter

A pH meter is a digital device used for potentiometrically measuring the pH, which is either

the concentration or the activity of hydrogen ions, of an aqueous solution. Usually, a pH

meter is provided with two probes, one is used for measuring temperature in degree

centigrade and other is used for measuring pH.

4. Refrigerator:

Refrigerator is used for a variety of reasons where temperature control is desired. It may be

used to preserve certain chemical reagents or can be used to grow certain types of bacteria. It

is also used to preserve cultures of bacteria, yeast, and other microbes. Optimum

temperature for preservation is 4oC.

8
 Figure 5:Refrigerator

5. Turbidity Meter:

Figure 6: Turbidity meter

Turbidity meter is used to find turbidity of sample in NTU i.e., Nephelometric turbidity unit.

It has a detector set up to the side of the light beam. More light reaches the detector if there

are lots of small particles scattering the source beam than if there are few.

9
6. Digital Weight Balance:

Figure 7: Digital weight balance

Digital weight balance, as the name indicates, is a digital device used to measure weight or

calculate mass. The unit of weight is mostly Newton and that of mass is grams or kilograms.

The limit till which it can measure accurately is 120 g.

7. Oven:

Figure 8: Oven

10
Oven in laboratory is used for many purposes such as dry heat sterilization and controlling

of temperature, humidity, and other conditions such as CO 2 and O2 content of atmosphere in

which microbiological culture is being grown. It can also be used for annealing or for drying

of samples etc.

8. Distillation Apparatus:

Figure 9: Distillation Apparatus

Distillation apparatus is used for separation of component substances from liquid mixture by

selective evaporation and condensation. Distillation may result in essentially complete

separation (nearly pure components), or it may be a partial separation that increases the

concentration of selected components of the mixture.

11
9. Flame Photometer:

Figure 10: Flame photometer

Flame photometer is a device used in inorganic chemical analysis to determine the

concentration of certain metal ions; among them are sodium, potassium, lithium, and

calcium and Barium.

10.COD Digestor:

Figure 11: COD digester

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COD Digestion Apparatus is commonly used to indirectly measure organic contents in

wastewater influent and effluent, industrial process water, and more. This instrument is used

by Pollution Control Labs, Chemical’s labs, QC & Research labs etc.

11. Vacuum Pump:

Figure 12: Vacuum Pump

A vacuum pump is a device that removes gas molecules from a sealed volume in order to leave

behind a partial vacuum.

12.Filtration Assembly:

Figure 13: Filtration Assembly

13
Glass Vacuum Filtration Assembly (47mm) is meant for water microbiology or liquids for

particulate contaminant removal, HPLC solvent purification or analysis of particulate

contaminants retained on a membrane filter membrane disc filters and similar applications.

13.Water Bath:

Figure 14: Water Bath

A water bath is laboratory equipment made from a container filled with heated water. It is

used to incubate samples in water at a constant temperature over a long period of time. All

water baths have a digital or an analogue interface to allow users to set a desired

temperature.

14.EC Meter:

Figure 15: Electrical Conductivity meter

14
An electrical conductivity meter (EC meter) measures the electrical conductivity in a

solution. It is commonly used in hydroponics, aquaculture and freshwater systems to

monitor the amount of nutrients, salts or impurities in the water.

15.Desiccator:

Figure 16: Desiccator

Desiccators are sealable enclosures containing desiccants used for preserving moisture-sensitive

items such as cobalt chloride paper for another use. A common use for desiccators is to protect

chemicals which are hygroscopic or which react with water from humidity.

16.TSS Potable:

Figure 17: TSS Potable

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TSS portable Hand-held instrument is ideal for remote monitoring in municipal and

industrial wastewater, drinking water and river monitoring. In wastewater, the TSS Portable

in the suspended solids mode provides an easy way to check the sludge blanket level for

both depth and concentration.

17.Titration Assembly:

Figure 18: Titration Assembly

Titration assembly is basically used for titration. The technique of titration is used to find out

accurately how much of a chemical substance is dissolved in a given volume of a solution,

that is, the concentration of the solution.

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Experiment no 1:

“Determination Of Solids (TS, TDS, TSS, TFSS, TVSS) in Water

and Wastewater Samples”

 Theoretical Background:

“Solids refer to matter suspended or dissolved in water or wastewater”. Waters with high

dissolved solids generally are of inferior palatability and may induce an unfavorable

physiological reaction in the transient consumer. For these reasons, a limit of 500 mg

dissolved solids/L is desirable for drinking waters. Solids analyses are important in the

control of biological and physical wastewater treatment processes and for assessing

compliance with regulatory agency wastewater effluent limitations.

 Total Solids (TS):

 Total solid: is the term used for the material left in the vessel after evaporation of a

sample and its subsequent drying in an oven at a definite temperature. “Total solids are a

measure of the suspended and dissolved solids in water”. less than 500

 Total Suspended solids(TSS): are those that can be retained on a filter paper of pore size

2.0 µm or smaller during the filtration of sample water and remained after subsequent

drying at 1030C-1050C. The limit for TSS according to NEQs is 150 mg/L.

 Total Dissolved solids (TDS): are those that pass through a filter paper of pore size 2µm

or smaller during the filtration of sample water and remained after subsequent drying at

1030C-1050C. For drinking water, the maximum concentration level set by NEQs is 20 to

17
 1000mg/L.

World Health Organization (WHO) has also prescribed an acceptable limit of 500 mg/L.

 Total Fixed Suspended Solids (TFSS): The residue of TSS left on ignition for a

specified time at a specified temperature is known as total fixed suspended solids

 Total Volatile Suspended Solids (TVSS): The weight loss of TSS on ignition is known

as total volatile suspended solids.

 Environmental Significance of Total Solids:

Some of the significances are:

 Total solids will be higher in highly mineralized waters, which result in unsuitability

for many industrial applications.

 It indicates effectiveness of sedimentation process, and it affects effectiveness of

disinfection process in killing microorganisms.

 It is used to assess the suitability of potential supply of water for various uses. In the

case of water softening, number of total solids determines the type of softening

procedure.

 Corrosion control is frequently accomplished by the production of stabilized waters

through pH adjustment. The pH stabilization depends to some extent upon the total

solids present as well as alkalinity and temperature.

 Limits for Total Solids:

The limit for total solid is = 500 mg/l according to WHO standard.

 Procedure:

“PREPARTION OF EVAPORATION DISHES FOR TS, TSS, TDS &VS”

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For TS determination, heat clean China dishes to a temperature of 103-105 ˚C and then cool

them in a desiccator till required. If TDS are to be measured, heat the dishes at 180±2 ˚C for

one hour and transfer them directly into a desiccator and store until usage. If volatile solids

are to be measured, then ignite the China dish at 550 ˚C for 1 hour in a muffle furnace.

Partially cool in air until most of the heat has been dissipated then transfer to desiccator for

final cooling. Always weigh the dishes immediately before use.

“SAMPLING AND STORAGE”

Collect original samples in plastic or glass bottles. Fill completely and cap tightly. Avoid

excessive agitation and prolonged exposure to air. Sample should be analyzed as soon as

possible after collection but can be stored up to 7 days by cooling to 4°C or below.

“MATERIALS AND METHODS”

China dishes, aluminum dish, Fiberglass filter, Suction apparatus, oven, steam bath

apparatus, 50-mL measuring flask, Analytical balance, Distilled water.

 PROCEDURE FOR TOTAL SOLDS:

1. Mix the sample well in a beaker with help of a stirrer. Transfer its 50ml to a pre-

weighed (heat treated) China dish with the help of a pipette.

2. Evaporate the sample in an oven at 103-1050C or on a water bath. The oven should

be preheated to ensure adequate drying.

3. After completion of drying, take the dish out of the oven and cool to room

temperature in a desiccator.

4. Weigh out the dish to the nearest 0.1 mg using an2 analytical balance. Repeat drying

for approximately 15 min at 103-1050C until the result does not differ by more than

0.4 mg.

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 FORMULA FOR TOTAL SOLIDS:

Then calculate the TS as under.

( – 𝐁)
Total Solids (TS) as mg/l =
𝐬𝐚𝐦𝐩𝐥𝐞 𝐕𝐨𝐥𝐮𝐦𝐞 𝐢𝐧 𝐦𝐋
× 𝟏𝟎^𝟔

Where: A = Weight of dried residue + weight of dish (mg)

B = weight of dish only

 PROCEDURE OF TOTAL DISSOLVED SOLIDS:

The procedure is as follows:

1. Connect a clean and dry filter flask to a filter holder (parts of filtration assembly).

2. Place a 47 mm filter disc (pre-treated) in the filter holder with the wrinkled surface

facing upwards. Assemble the other parts of assembly.

3. Mix the sample well and take 100 ml (or more if solids content is low) in a clean

measuring cylinder.

4. Filter the above 100 ml (or more) of the sample through the filter under vacuum applied

to the flask.

5. Apply vacuum for 2-3 minutes after the sample has passed through the filter, then

disconnect the vacuum.

6. Using tongs, transfer the evaporating/China dish (pre-treated from the desiccator to the

balance. Weigh to nearest 0.1 mg and record the weight.

7. Place the evaporating dish on preheated steam bath.

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8. Pour 100 ml filtrate sample from the filter flask into the evaporating dish and evaporate

to dryness.

9. Using tongs transfer the evaporating dish residue to a drying oven and dry at 1800C for

one hour. Transfer to desiccator and cool.

10. Weigh the evaporating dish to nearest 0.1 mg on an analytical balance and record the

weight.

11. Repeat step 9 and 10 until a constant weight is obtained.

FORMULA FOR TOTAL DISSOLVED SOLIDS:

Then calculate the TDS as under

(𝐀−𝐁)×𝟏𝟎^𝟔
Total dissolved solids (TDS) in mg/L =
𝐒𝐚𝐦𝐩𝐥𝐞 𝐕𝐨𝐥𝐮𝐦𝐞 𝐢𝐧 𝐦𝐋

Where: A = Weight of dried residue + dish (mg).

B = Weight of the dish (mg)

 PROCEDURE OF TOTAL SUSPENDED SOLIDS:

The procedure consists of following main steps to follow:

1. Carefully remove the filter paper from the filtration assembly and transfer it to an

aluminum dish or a watch glass.

2. Place it in a drying oven at 1030C for one hour. Preheat the oven to ensure adequate

drying of the filter disc.

3. Remove the filter disc (with aluminum dish or watch glass) from the oven and place it in

desiccators. Cool to room temperature.

4. Remove the disc from the desiccator and weigh to the nearest 0.1 mg on the balance.

21
5. Return the disc to the watch glass if the Volatile Suspended Solids (VSS) are to be

determined. If not, discard the disc.

FORMULA FOR TOTAL SUSPENDED SOLIDS:

(𝐀−𝐁)×𝟏𝟎^𝟔
Total suspended solids (TSS) in mg/L =
𝐒𝐚𝐦𝐩𝐥𝐞 𝐕𝐨𝐥𝐮𝐦𝐞 𝐢𝐧 𝐦𝐋

Where: A = Weight of dried residue + filter paper

(mg) B = Weight of the filter paper (mg)

 PROCEDURE OF TVSS and TFS:

The procedure is as follows:

1. Take the watch glass and filter disc out of the desiccator (from the total suspended

solids section, weight of the disc residue has already been determined). Place them in a

muffle furnace and ignite at 5500C for 15 min. The muffle furnace may be partially

preheated before inserting the watch glass. However, placing the watch glass in a 550 0C

furnace could cause it to shatter. The temperature should be brought to 550 0C after

placing the filter and watch glass in the oven and held at that temperature for about 15

min. Use tongs to transfer the watch glass and filter disc into the furnace as a unit.

2. Remove the watch glass and filter from the furnace and carefully transfer into the

desiccator. Allow to cool at room temperature. Use metal tongs to transfer the watch

glass from the furnace directly into the desiccator. Cover immediately. Allow the vessel

to cool before sealing the desiccator as pressure from the heated air inside the desiccator

can force the cover off.

3. Carefully remove the filter disc from the desiccator and weigh it to the nearest 0.1 mg

using the analytical balance. Take extreme care when removing the lid of the

desiccator
22
 as to not disturb the dried suspended matter on the disc. Use plastic tweezers to transfer

the disc to and from the weighing pan of the balance.

4. Calculate the total volatile suspended solids and fixed solids by using the formula for it.

FORMULA FOR TVSS AND TFSS:

(𝐀−𝐁) × 𝟏𝟎^𝟔
Total volatile Solids (TVSS) in mg/l =
𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐬𝐚𝐦𝐩𝐥𝐞 𝐢𝐧 𝐦𝐋

Where: A= Weight of residue + weight of filter disc before ignition

(mg) B = Weight of residue + weight of filter disc after ignition (mg)

(𝐁−𝐂) × 𝟏𝟎^𝟔
Total Fixed Solids (TFS) in mg/l =
𝐯𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐬𝐚𝐦𝐩𝐥𝐞 𝐢𝐧 𝐦𝐋

Where: B = Weight of residue + weight of filter disc after ignition

(mg) C = Weight of filter disk only (mg)

 Observations and Calculations:

Volume of sample= 50 mL

A. TOTAL SOLIDS (TS)

Where: A = 54.953g

B
=54.901g
( 𝟒.𝟗𝟓𝟑 – × 𝟏𝟎^𝟔
Total Solids (TS) in mg/L =
𝟓𝟒.𝟗𝟎𝟏)

𝟓𝟎

TS = 1040 mg/L

B. TOTAL DISSOLVED SOLIDS (TDS)

23
Where: A = 56.389g

24
 B = 56.355g

Total dissolved solids (TDS) in mg/L = (𝟓𝟔.𝟑𝟖𝟗 – 𝟓𝟔.𝟑𝟓𝟓) × 𝟏𝟎𝟎𝟎 × 𝟏𝟎𝟎𝟎

𝟓𝟎

TDS = 680mg/L

C. TOTAL SUSPENDED SOLIDS (TSS)

Where: A =

0.191g B =

0.182g
(𝟎.𝟏𝟗𝟏−𝟎.𝟏𝟖𝟐)
Total suspended solids (TSS) in mg/L = × 𝟏𝟎𝟎𝟎 × 𝟏𝟎𝟎𝟎
𝟓𝟎

TSS =180mg/L

D. TOTAL VOLATILE SOLIDS (TVSS):

Where: A= 0.009g

B = 0.004g

(𝟎.𝟎𝟎𝟗−𝟎.𝟎𝟎𝟒) × 𝟏𝟎^𝟔
Total volatile Solids (TVSS) in mg/l =
𝟓𝟎

TVSS=100mg/L

E. TOTAL FIXED SOLIDS:

Where: B =

0.187g C =

0.184g

(𝟎.𝟏𝟖𝟕−𝟎.𝟏𝟖𝟒) × 𝟏𝟎^𝟔
Total Fixed Solids (TFS) in mg/l =
𝟓𝟎

TFSS =60mg/L

25
 Results:

26
Total Solids (TS)= 1040mg/L

Total dissolved solids (TDS) = 680mg/L

Total suspended solids (TSS) =

180mg/L

Total volatile Solids (TVSS) in mg/L

=100mg/L Total Fixed Solids (TFS) in mg/

L=60mg/L

 Conclusion:

From the above calculation we can concluded that the total solid in wastewater is1040 mg/l

by comparing it with the standards for drinking water it shows that it is not safe for drinking

purpose.

27
Experiment no 2:

“Measurement of Dissolved Oxygen by Winkler Method”

28
29
Experiment no 2:

“Measurement of Dissolved Oxygen by Winkler Method”

 Theoretical Background:

“Dissolved oxygen (DO) is the amount of oxygen that is present in the water”. It is

measured in milligrams per liter (mg/L), or the number of milligrams of oxygen dissolved in

a liter of water. The dissolved oxygen test is one of the most important analyses in

determining the quality of natural waters.

 Factors Effecting DO:

The amount of DO that can be held by water depends on 3 factors.

1. WATER TEMPERATURE:

Amount of DO increases with decreasing temperature (cold water holds more oxygen).

2. SALINITY: Amount of DO increases with decreasing Salinity (fresh water holds more

oxygen than salt water does).

3. ATMOSPHERIC PRESSURE: Amount of DO decreases with decreasing atmospheric

pressure (Amount of DO absorbed in water decreases as altitude increases)

 DO Measurement “ (Winkler Method- Azide Modification)” :

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The Winkler method with Azide modification is used to determine level of DO in the water

samples. Samples are treated with manganese sulfate and alkaline iodide azide reagent (NaOH

+ KI + NaN3) to form orange- brown flocks or

precipitates. Reaction involve in winkler process are:

a.
Mn2+ + 2OH- → MnO2 + H2O

b.
Mn(OH)2+ 1/2O2 → MnO2 + H2O

This MnO2 is appearing as a brown flock. Upon acidification of the samplze, the flocks react

with iodine (from KI) to produce free iodine (I2) in proportion to oxygen concentration.

c. MnO2+ 2 I- + 4H+ → Mn2+ + I2(aq) + 2H2O

 Interference:

The nitrite ion causes the most frequent interference in the determination of dissolved

oxygen. It occurs pricincipally in effluent from waste water treatment plants that employ

biological process, in river and in BOD samples. NO2- oxidizes I- to I2 under acidic

conditions making DO measurements in accurate. This nitrite interference is eliminated by

the azide in reagent.

2NO2- + 2I- + 4H+→ N2O2 +I2 +

2H2O N2O2+ ½ O2 + H2O

→2NO2-+ 2H+

When sulphuric acid is added following reaction occur:

NaN3 + NO2-→ HN3 +Na+

HN3 + NO2- + H+ →N2 + N2O +H2O

 Sampling and Storage:

Collect samples very carefully. Collect water samples in narrow-mouth glass stoppered

31
BOD bottles of 300-mL capacity. Avoid entraining or dissolving atmospheric oxygen. Let

bottle overflow two or three times its volume and replace stopper so that no air bubbles are

entrained.

32
Analyze the sample as soon as possible if storage is necessary run the steps 1,2,3 of the

procedures and store in the dark at 100C to 200C. Sample stored in this manner are can be

held for 4 to 8 hrs.

A single dissolved oxygen is rarely a representative of overall condition of the body of

water. Therefore, several samples taken at different location are recommended for reliable

results. Protect stored samples from strong sunlight and titrate as soon as possible.

 Apparatus:

 Pipette Burette

 Conical Flask

 Volumetric Flask

 Beaker

 BOD bottles

 Chemicals:

 Maganese sulphate

 Alkaline potassium iodide sodium azide solution

 Sulphuric acid

 Starch used as indicator

 Sodium thiosulphate as titrant

 Procedure:

1. Take BOD bottle of 300ml.Add water sample in it.

33
2. Add 1ml of Manganese sulphate (MnSO4) and mix uniformly with the help of stirrer.

3. Add 1ml of Alkali Iodide Azide reagent in BOD bottle. (If white precipitate is formed

then there is no DO present in water sample. Formation of reddish brown precipitate

indicates the presence of DO).

4. If Reddish brown precipitate is formed, stopper the bottle and shake the bottle upside

down for 20 times and allow the precipitate to settle down for about 2 inches.

5. Add 1 ml Conc.H2SO4 and Shake it again for about 8 times. Allow the precipitate to

settle down.

6. Now take 200 mL of this sample from BOD bottle in to titration flask.

7. Now titrate the sample with standard 0.025 N Na2S2O4 till the light yellow colour appears.

8. Add 1ml starch solution in titration flask. Colour will change from yellow to blue on this

addition.

9. Now again titrate it with Na2S2O4 till blue colour will disappear.

10. Note down mL of titrant used.

 Observation and Calculations:

S/N Initial Burette Reading Final Burette Reading Difference (mL)

(mL) (mL)

1 14.7 20.5 5.8

Reading after color disappear

3 20.5 24.1 3.6

DO (mg/l) = {(B-A)× N×8×1000}/ml of sample

34
 DO=3.6 mL

 Conclusion:

As we can see that DO in our sample is high enough for the microbes to perform their activities

i.e. decomposition of organic matter. It is also enough for other processes i.e. water or

wastewater treatment.

35
Experiment no 3:

“Biological Oxygen Demand in Wastewater Sample”

 Theoretical Background:
Biological oxygen demand is the amount of dissolved oxygen needed by aerobic biological

organisms to break down organic material present in a given water sample at certain

temperature over a specific time period.

 Units of Measurements:

The bod value is most commonly expressed in milligrams of oxygen consumed per liter of

sample during 5 days of incubation at 20 oc and is usually used as a surrogate of the degree of

the organic pollution of water.

 Importance of BOD test:

BOD test is important because:

i. It determines the pollution strength of domestic and industrial wastes.

ii. It is one of most important in stream-pollutional-control activities

iii. It is used to evaluate the purification capacity of receiving bodies of water.

 TYPES OF BOD TEST:

36
1. Seeded: In which a diverse group of organisms are added to sample for proper

results.e.g., industrial water is seeded.

2. Unseeded: In which such organisms are not required for the test results. E.g., domestic

waste or sewage.

 Conditions for Test:

Following are the conditions applied for this test:

 Freedom from toxic materials

 Favourable pH and osmotic conditions

 Presence of available accessory nutrients

 Standard temperature

 Presence of significant population of mixed organisms of soil origin.

 Methods for the Measurement of BOD:

There are two methods for measuring BOD which we have studied:

1. Direct Method:

This method is valid for organic matter whose concentration is less than 7 mg/L. We

cannot maintain conditions in this process so this method cannot be used.

2. Dilution Method:

In this bioassay procedure, we can maintain conditions that are written above so we use

this method for measuring BOD.

 Dilution Media:

37
 Wide variety of water is used in this test. Natural surface water appear ideal but has

variable BOD , variable microorganisms population and variable mineral content.

Tap water also has some limitations due to possibilities of toxicity from chlorine residuals.

So it has developed that a synthetic dilution water prepared from distilled water is best

for BOD test because most of the variable are kept under control.

 Preparation of Dilution Media:

Proper conditions are given to prepare this dilution media. Take 1L distil water and add

all chemicals to remove toxic substances and maintain ph. Attach to aerator to shake and

dissolve DO. Introduce microorganisms if we use industrial water but seeding is not

required for domestic water.

 Materials and Method:

MATERIALS:
 Incubator

 Burette

 Pipette

 BOD bottle.

CHEMICALS:

 Maganese sulphate

 Alkaline potassium sodium azide solution

 H2SO4

 Starch

38
 Sodium thiosulphate

 Procedure:

1. Take 9 BOD Bottles of 300ml. Mark each bottle and make three sets.

2. Fill each bottle half with dilution media and ensure filling without any bubbles.

3. Then take 1 bottle from each set and add 2ml sample in each bottle.

4. Then take remaining bottle from each set and add 2ml in first set remaining bottles

,5mlin second set and 10ml sample in 3rd set remaining bottle.

5. Then completely fill them with dilution media and waste extra so no air bubbles are

entrapped.

6. Determine DO of the 3 bottles and put other in incubator.

7. Place the samples at 20C in incubator for 5 days.

8. After 5 days determine the DO of the sample bottles.

 FORMULA:

BOD = DO0 – DO5 / P

where

P = Vol of sample÷ total volume of bottle

 Observation and Calculations:

Sample Initial DO Final DO BOD5

Blank 8.2 8.0 Exceed from Standard

39
 2 8.1 5.6 Exceed from Standard

5 8.0 1.7 Exceed from Standard

10 8.1 0.0 Exceed from Standard

 Result:

The BOD of our Sample is more than 80mg/L.

40
Experiment no 4:

Determination Of COD by Using Close Reflux Titrimetric Method.

 COD:

The chemical oxygen demand is an indicative measure of the amount of oxygen that can be

consumed by reactions in a measured solution. It is commonly expressed in mg/L.

 Significance:

The Chemical Oxygen Demand (COD) method determines the quantity of oxygen required

to oxidize the organic matter in a waste sample, under specific conditions of oxidizing agent,

temperature, and time. In biological wastewater treatment process the part of chemical

oxygen demands (CODs) removed is converted into bio solids which makes up sewage

sludge. Sewage sludge usually represents 1–2% of the treated wastewater volume the

existing wastewater treatment plants in the United States, for instance, generate over 6.5

million tons of dry solids annually; it is estimated to be around 3.0 and 2.0 Mt per year

produced in different countries.

 NEQS Guidelines:

According to NEQS standards COD in the wastewater should be lies in range of 150 mg/L.

41
 Principle:

In this procedure, the sample is heated for 2 hours with strong oxidizing agent such as

potassium dichromate. It is hexavalent salt. Oxidizible organic compounds react with

potassium dichromate, reducing the dichromate from hexavalent to trivalent green chromic

ion.

 Sampling:

Collect samples in glass bottles. Use plastic containers only if you are sure that there is no

organic matter present in container. Biologically active sample should be analyzed as soon

as possible. Sample containers containing settleable matter should be completely mixed.

Sample should be preserved with sulphuric acid with pH less than 2 and maintained at 4oC.

 Interferences:

Traces of the organic material either from the glassware or the atmosphere may cause a

gross positive error. Volatile materials may be lost when the sample temperature rises during

sulphuric acid addition step. To minimize this loss the flask should be cool during the

addition of sulphuric acid. Chlorides are quantitively oxidized by the dichromate. Mercuric

sulphate is added to complex the chloride interference. Nitrite’s interference is removed by

sulfemic acid.

 Apparatus:

 COD digestor

 Glass vials (Standard 10ml size)

 Titration flask

 Titration stand

 Burette

 Pippette

 Volumetric flask
42
 Beakers

43
 Reagents:

 Standard Potassium Dichromate Solution (0.01667M)

Add to about 500ml distilled water, 4.903g potassium dichromate (Primary standard grade

previously dried at 150oC for 2 hours) then add 167ml Conc sulphuric acid and 33.3g

mercuric sulphate (Secondary digestion catalyst used to oxidize straight chain

hydrocarbons). Dissolve, cool to room temperature and dilute to 1000ml.

 Sulphuric Acid Reagent:

Add silver sulphate to Conc sulphuric acid at the rate 5.5g silver sulphate per kg sulphuric

acid. Let the mixture stand for 1-2 days to dissolve.

 Sulfemic acid

 Standard Ferrous Ammonium Sulphate. Fe(NH4)2(SO4)26H2O

Dissolve 98g FAS in distilled water and add 20ml Conc Sulphuric acid. Cool and dilute

2000ml.

 Procedure:

1. Put 100ml sample in a blender. Blend for 30 seconds or until homogenized for
samples for large amounts of solids.

2. Set the digestion reactor power to on and pre heat at 150oC.

3. Prepare the sample. Remove the cap from a viale and hold the viale at an angle of
45oC.

4. Use a clean pipette to add 2.5ml sample , 1.5ml digestion solution and 3.5ml
sulphuric acid solution.

5. Replace the viale cap tightly. Rinse the outside of COD viale with distilled water
and wipe it with clean tissue. Invert the vials gently several times to mix the

contents.

44
6. Place the vials in pre heated digestor.

45
 7. Prepare a blank by repeating all the steps.

8. Heat the vials for 2 hours.

9. Turn digestor off. Wait about 20 minutes for vials to cool.

10. After 20 minutes, invert each viale several times and place in rack.

11. Wait until the vials are cool at room temperature.

12. Transfer the contents in a viale to 25ml titration flask. Add 1-2 drops of Freon
indicator. Fill burette with standard FAS. Now titrate the sample in a titration flask

against FAS until colour changes from blue green to reddish brown.

 Observations and Calculations:

COD as mg of O2/L = (A-B) * Molarity of FAS * 8000 / ml of

sample A=12 ml

B=7.7 ml

Molarity of FAS= 0.25

Volume of sample=100

ml

(12-7.7)*0.25*8000/100 = 86 mg/L

 Comments:

According to NEQS standards COD in the waste water should be lies in range of 150 mg/L,

As we found 86 mg/L.

46
 Experiment no 5:

“TO FIND OUT MAXIMUM WAVELENGTH OF GIVEN SAMPLE USING

SPECTROMETER”

THEORETICAL BACKGROUND

SPECTROPHOTOMETER
A spectrophotometer is an instrument that measures the amount of light
absorbed by a sample. Spectrophotometer techniques are mostly used to
measure the concentration of solutes in solution by measuring the amount
of the light that is absorbed by the solution in a cuvette placed in the
spectrophotometer.

PARTS OF SPECTROPHOTOMETER
A spectrophotometer consists of four basic components: a light source, a sample holder,
a monochromator, and a detector. The monochromator comprises a fixed entrance slit, a
dispersing element such as a prism or a diffraction grating, and a moving exit slit.

BEER-LAMBERT LAW
The Beer-Lambert law is a linear relationship between the absorbance and the
concentration, molar absorption coefficient and optical coefficient of a solution:

47
 The molar absorption coefficient is a sample dependent property and is a measure of how
strong an absorber the sample is at a particular wavelength of light. The concentration is
simply the moles L-1 (M) of the sample dissolved in the solution, and the length is the length
of the cuvette used for the absorbance measurement and is typically 1 cm.
The transmittance, T, of the solution is defined as the ratio of the transmitted
intensity, I, over the incident intensity, I0 and takes values between 0 and 1

MATERIALS AND METHOD

MATERIAL
Spectrophotometer, volumetric flasks, pipette
REAGENTS
0.1% KMnO4, distilled water.
PROCEDURE
 Take 0.1 g of KMnO4 and dissolve in 100 ml distilled water
 Check this solution for various wavelengths from 200 to 800 nm
 Plot the graph for maximum wavelength

48
OBSERVATIONS AND CALCULATIONS

WAVELENGTH ABSORBANCE
200 3.48
400 2.49
600 3.75
800 0.21

Maximum Absorbance
4
3.5
3
2.5
Absorbance

2
1.5
1
0.5
0
100 200 300 400 500 600 700 800 900

Wavelength (nm)

For accurate value we have measure absorbance form 550-650 nm.

WAVELENGTH ABSORBANCE
550 35.55
560 23.1
570 23.15
580 21.6
590 11.65
600 18.75
610 1.8
620 1.64
630 3.85
640 3.55
650 3.2

49
 Maximum Absorbance
40
35
30
25
Absorbance

20
15
10
5
0
540 560 580 600 620 640 660
Wavelength (nm)

RESULT
The highest value of absorbance is 35.55 which is at wavelength 550 nm.

Experiment no 6:

50
 “Estimation of copper ion concentration in a given solution by
UV- spectrophotometer”
 Theoretical background:

COPPER:
Copper is a chemical element with symbol Cu (from Latin: cuprum) and atomic number 29.

It is a soft, malleable and ductile metal with very high thermal and electrical conductivity. A

freshly exposed surface of pure copper has a reddish-orange color. It is used as a conductor

of heat and electricity, as a building material, and as a constituent of various metal alloys. It

is a transition element and form cuprous (Cu+1) and cupric ions (Cu+2).

COPPER ATOM

It is a chromophore (Those compounds having specific color and visible in UV spectrometer

are called chromophores.) It is found everywhere in water, soils, rocks, atmosphere etc.

 Environmental Significance:

 Used in manufacturing of electric wires, pipes, coins etc.

 Used in food additives

 Used in insecticides and fungicides

 Copper aides in formation of hemoglobin

 Copper helps iron in blood absorption

 Traditionally it has been one of the metals used to make coins, along with silver and gold.

 Copper sulphate is used widely as an agricultural poison and as an algaecide in water

purificatio

51
Excess of copper concentration in drinking water can cause vomiting, nausea, diarrhea and

abdominal pain. In drinking water color of dissolved copper is pale blue. On proclaim (Kaolin)

sink, blue green color is due to copper.

Color of anhydrous CuSO4 is pale blue green. It is also called blue stone or blue vitriol and

hydrated CuSO4.5H2O is dark blue. To measure copper concentration in sample anhydrous

copper is used this is because it gives color.

 Guidelines for Copper Concentration:

 NEQs/WHO guidelines for drinking water is 2mg/l

 NEQs for wastewater is 1mg/l

 Materials and Methods:

MATERIALS:

 Volumetric flask

 Beaker

 Spectrophotometer

 Sample cell

REAGENTS:

Copper sulphate (CuSO4.5H2O)

Ammonium hydroxide

(NH4OH)

 Procedure:

1. Prepare stock solution of copper (1000ppm) by adding 0.392g of CuSO4.5H2O in 100ml

of water.

2. Prepare intermediate solution of copper (100ppm) by adding 10ml of stock solution into

52
a volumetric flask and diluting up to 100ml

53
3. Prepare calibrated solutions of copper of the following concentrations:

4. (Add 5ml of NH4OH in each sample before adding water to enhance color)

 20ppm: By adding 20ml of intermediate solution and adding up to 100ml of water.

 40ppm: By adding 40ml of intermediate solution and adding up to 100ml of water.

 60ppm: By adding 60ml of intermediate solution and adding up to 100ml of water.

 80ppm: By adding 80ml of intermediate solution and adding up to 100ml of water.

5. Find absorbance of each calibrated sample at 620nm (wavelength at which maximum

absorbance of copper takes place).

6. Plot calibration curve between absorbance and concentration.

7. Find absorbance for copper sample of unknown concentration.

 Observation and Calculation:

Concentration (ppm) Absorbance (Abs)

20 0.027

40 0.051

60 0.065

80 0.076

Unknown 0.039

54
 GRAPH:

 COMMENTS:

Using spectrophotometer is most convenient method to measure the unknown concentration

of copper but for this it is first necessary to establish relationship between concentration and

absorbance for copper solution. Then we can find unknown concentration from the

calibration curve.

In this experiment, absorbance for sample of unknown concentration is 0.039 and from the

graph it is deduces that respective concentration is 30ppm or 30mg/L.

55
56
57
8. Prepare calibrated solutions of copper of the following concentrations:

9. (Add 5ml of NH4OH in each sample before adding water to enhance color)

 20ppm: By adding 20ml of intermediate solution and adding up to 100ml of water.

 40ppm: By adding 40ml of intermediate solution and adding up to 100ml of water.

 60ppm: By adding 60ml of intermediate solution and adding up to 100ml of water.

 80ppm: By adding 80ml of intermediate solution and adding up to 100ml of water.

10. Find absorbance of each calibrated sample at 620nm (wavelength at which maximum

absorbance of copper takes place).

11. Plot calibration curve between absorbance and concentration.

12. Find absorbance for copper sample of unknown concentration.

 Observation and Calculation:

Concentration (ppm) Absorbance (Abs)

20 0.027

40 0.051

60 0.065

80 0.076

Unknown 0.039

58
 GRAPH:

 COMMENTS:

Using spectrophotometer is most convenient method to measure the unknown concentration

of copper but for this it is first necessary to establish relationship between concentration and

absorbance for copper solution. Then we can find unknown concentration from the

calibration curve.

In this experiment, absorbance for sample of unknown concentration is 0.039 and from the

graph it is deduces that respective concentration is 30ppm or 30mg/L.

59
 Experiment no:7

“ESTIMATION OF NICKEL BY SPECTROMETER”

THEORETICAL BACKGROUND
Nickel is a chemical element with the symbol Ni and atomic number 28.
It is a silvery-white lustrous metal with a slight golden tinge. Nickel belongs to the transition
metals and is hard and ductile.
ENVIRONMENTAL SIGNIFICANCE OF NICKEL
Nickel is the 24th most abundant element in the earth's crust and can be found in the air, soil,
sediments and water. Nickel and nickel-containing materials play an important role in the
technologies and applications that are helping achieve key environmental policy objectives,
such as reducing carbon emissions.
GUIDELINES FOR NICKEL CONCENTRATION
Reference values for nickel concentrations in serum and urine from healthy persons without
occupational exposure to nickel compounds have recently been compiled. Values for
serum/plasma are in the range 0.14-0.65 µg/liter; values of around 0.2 µg/liter seem to be
the most reliable.

MATERIALS AND METHOD

MATERIALS
 Volumetric flask
 Beaker
 Spectrophotometer
 Sample cell

REAGENTS
 Nickel Nitrate
 Ammonium hydroxide (NH4OH)

PROCEDURE
 Prepare a solution of Ni by adding 2 grams of Nickel Nitrate

 We have found that 646.3mg of Ni was prepared.

60
  Prepare intermediate solution of (100ppm) by adding 10ml of stock solution into a
volumetric flask and diluting up to 100ml

 Prepare calibrated solutions of nickel of the following concentrations:

 (Add 5ml of NH4OH in each sample before adding water to enhance color)
 32.2ppm: By adding 32.2ml of intermediate solution and adding up to 100ml of water.

 64.4ppm: By adding 64.4ml of intermediate solution and adding up to 100ml of water.

 96.7ppm: By adding 96.7ml of intermediate solution and adding up to 100ml of water.

 128.9ppm: By adding 128.9ml of intermediate solution and adding up to 100ml of water.

 Find absorbance of each calibrated sample at 570-610nm (wavelength at which maximum

absorbance of nickel takes place).

 Plot calibration curve between absorbance and concentration.

 Find absorbance for copper sample of unknown concentration.

OBSERVATIONS AND CALCULATIONS

CONCENTRATION (ppm) ABSORBANCE

32.2 0.027

64.5 0.027

96.7 0.028

128.9 0.029

GRAPH
61
 Concentration vs Absorbance
0.03

0.029

0.028
Absorbance

0.027

0.026

0.025

0.024
20 40 60 80 100 120 140
Concentration(ppm)

RESULT
Using spectrophotometer is most convenient method to measure the unknown concentration
of nickel but for this it is first necessary to establish relationship between concentration and
absorbance for nickel solution. Then we can find unknown concentration from the calibration
curve. In this experiment, absorbance for sample of unknown concentration from the graph is
deduced.

62
Experiment no 8:

“Determination of chlorine by iodometric method”

 Theoretical background:

Iodometry also known as iodometric titration is a method of volumetric chemical analysis, a

redox titration where the appearance or disappearance of elementary iodine indicates the end

point. Chlorine is used to protect the public health by killing the microorganisms found in

water which cause diseases. Chlorine has the ability to take electrons from both bromide

ions and iodide ions. Bromine and iodine can't get those electrons back from the chloride

ions formed. That means that chlorine is a more powerful oxidizing agent than either

bromine or iodine.

Consider this, chlorine is toxic enough to be a chemical weapon and categorized as a

“choking agent”. Inhalation of chlorine gas can cause difficulty breathing, chest pains,

cough, eye irritation, increased heartbeat, rapid breathing, and death. Exposure would be a

very traumatic experience.

 Working Principle:

Chlorine liberates free iodide from KI at a pH of 8 or less. The liberated iodine is then

titrated with standard sodium thiosulphate with starch as indicator. Titration is done at a pH

of 3-4 because the reaction is not stoichiometric at neutral pH due to partial oxidation of

thiosulphate to sulphate

I3− + 2e− → 3I−

S4O 62− + 2e− → 2S O2 2−

3

I3− + 2 S2O32− → S O 42−6 + 3 I−

 Sampling and Storage:

63
Plastic and glass containers can be used for collecting sample.

Chlorine in a water sample is not stable as a result its concentration rapidly decreases

exposure to sunlight or other strong light air or agitation will further reduce its quality in the

water solution. Therefore sample to be analyzed for chlorine cannot be stored or preserved.

Test must be started immediately after sampling.

 Interferences:

Oxidize form of manganese, other oxidizing agents and reducing agents such as organic

sulphite may cause interference. Although the neutral titration minimizes the interference

effects of nitrite and ferric ion interference but acid titration is preferred because some forms

of combined available elements doesn’t react with pH-7. Only acidic acid should be used for

titration. Sulphuric acid will increase interference and HCL should never be used.

 Materials and Method:

Materials:

 Burette with stand

 Volumetric flask

 Pipette

 50ml cylinder

Reagents:

1. Conc. Acetic acid (pH 3-4)

2. Potassium iodide crystals

3. Standard sodium thiosulphate solution(Na2S2O3) 0.01N:

0.01N of Na2S2O3 in 1 litre

Molecular weight of Na2S2O3.5H2O = 248g

64
 Wt. of solute = Normality x Equivalent Wt. x Volume /1000

Wt. of solute = 2.5g

Dissolve 2.5g of Na2S2O3 in freshly boiled and cooled distilled water and dilute into 1-liter

water. Standardize it against primary standard of K2Cr2O7 solution.

4. Potassium dichromate solution (K2Cr2O7) 0.1N:

Dissolve 0.49g of anhydrous potassium dichromate in distilled water and dilute for 1

litre and store in a glass stopper bottle.

5. Starch solution:

Make a paste from 1g of laboratory grade soluble starch with little water. Add the paste

with constant stirring to 100ml of boiling water and boil for 1 minute allow to cool and

add 2-3g of KI. Keep in stopper bottle.

6. Standard Iodine Solution 0.0282N:

Dissolve 2.5g of KI in 25ml of distilled water add 0.37g of re-sublimed iodine and stir

until dissolved. Transfer it into 100ml volume flask and dilute up to the mark. Keep in

stopper bottle.

 Procedure:

1. Volume of the sample:

Select an appropriate sample volume. For chlorine range of 1-10mg/l. Use 500ml of

sample.

For a chlorine range of >10mg/l sample: Take 100ml of sample.

2. Preparation for titration:

Place 5ml of acetic acid or enough to reduce pH 3-4 in the flask. Add about 1g of

potassium iodide. Pour sample acid mix with stirring rod.

3. Titration:

65
 Titrate above sample away from sunlight. Add standardized Na 2S2O3 from burette until

yellow color of liberated iodine disappears. Add 1ml of standard solution acid titrate

until blue color disappears. Note volume of Na2S2O3 used.

4. Blank Titration:

Take a volume of distilled water corres to sample volume. Add 5ml of acetic acid, 1g of

KI, 1ml starch solution. Perform blank titration as below whichever implies:

If blue color appears titrate with standard Na2S2O3 till disappearance of blue color

and record result. Note Na2S2O3 used. B is noted as negative volume.


If no blue color appears titrate with 0.0282N iodine solution until blue color appears.

Back titrate with Na2S2O3 and record result. B is positive volume in this case.

 Formula:

Chlorine as (mg/l) = (A±B) x Normality x 35450 / Sample volume

 Observation and Calculations:

S/N Initial Burette Reading Final Burette Reading Difference

(mL) (mL) (mL)

1 Blank 11.8 12.0 0.2

2 Sample 2.2 11.4 9.2

Chlorine as (mg/l) = (9.2+0.2) x 0.0094x 35450 / 100

=31.32 mg/L

66
 Experiment no 9:

“DETERMINATION OF CHLORIDE IN WASTEWATER SAMPLE”

THEORETICAL BACKGROUND
The chloride ion is the anion Cl⁻. It is formed when the element chlorine gains an electron or
when a compound such as hydrogen chloride is dissolved in water or other polar solvents.
Chloride salts such as sodium chloride are often very soluble in water.
Chloride in water is a tough problem to solve. It takes only a small amount — 1 teaspoon
per 5 gallons of water — to pollute water permanently. At high concentrations, chloride
can harm fish and plant life. But there's no easy and affordable way to remove chloride in
wastewater.
The presence of chloride in municipal wastes and sewage effluent ultimately increases the
chloride content in fresh and wastewater. It comes from activities carried out in
agricultural areas, as well as from industrial activities and chloride stores. The high
content of chloride is mostly because of human activities.

SOURCES IN WASTEWATER
Chlorides may get into surface water from several sources including:
 Rocks containing chlorides.
 Agricultural runoff.
 Wastewater from industries.
 Oil well wastes.
 Effluent wastewater from wastewater treatment plants.
 Road salting.

SIGNIFICANCE
 Its high concentration reacts with metals and causes corrosion.

 Its high concentration has bad effects on agriculture.

 Its high conc. causes yellow color leaf , Cl water intake by roots then salt blocks capillary
tubes and water cannot moves upward.

67
EFFECTS
 Its guidelines are based on taste, not health effects.

 EPA suggests its level not higher than 250mg/l.

 High levels cause dehydration(kidney disease).

PRINCIPLE
Mercuric ions in the titrant react with chloride in the water sample to form mercuric chloride.
Afterall of the chloride, the mercuric ions react with diphenylcarbazone indicator to show
purple color which is end point of titration.

MATERIALS & METHOD

REAGENTS
1. Mercuric Nitrate (Titrant)
2. Mixed Indicator
a. Phenyl Carbazone.
b. Xylene Syanol.
c. Nitric Acid.

MATERIALS
 Measuring cylinder

 Pippete

 Burette

 Titration flask

 Titatration stand

PROCEDURE
1. Take 25ml of sample in measuring cylinder.
2. Add sample in titration flask.
3. Add mixed indicator, 2drops and will give light green color.

 Now if pH of sample is 2.3, then it will give green color.

 If pH is between 2.3-3.8, then it will give blueish green color.

 If pH is greater than 3.0, then it will give blue.

68
 4. Add titrant Mercuric Nitrate in a burette.
5. Now titrate your sample against the titrant and note down the readings at end point(purple
color).

FORMULA
( A−B )∗N∗35.5∗1000
Cl2 (mg/l)=
ml of sample used
N=0.0141N

OBSERVATION & CALCULATIONS

Volume of Sample = 25 ml

Initial Burette Reading Difference

Sr. No Final Burette Reading (ml)
(ml) (ml)

1 (Blank) 0.0 2.8 2.8

2 (Sample) 2.8 3.5 0.7

(2.8−0.7)× 0.141 ×35.5 ×1000

Total chloride as (mg/l) =
25
= 420.4mg/l

69
Experiment no 10:

“Determination of nitrogen by Kjeldhal Method”

 Theoretical background:

The Kjeldhal method is used to determine the nitrogen content in organic and inorganic

samples. For longer than 100 years the Kjeldhal method has been used for the determination

of nitrogen in a wide range of samples. The determination of Kjeldhal nitrogen is made in

foods and drinks, meat, feeds, cereals and forages for the calculation of the protein content.

Also, the Kjeldhal method is used for the nitrogen determination in wastewaters, soils and

other samples. It is an official method and it is described in different normative such as

AOAC, USEPA, ISO, DIN, Pharmacopeias and different European Directives.

 Steps:

The Kjeldhal method consists of three steps:

 Digestion

 Distillation

 Titration

Digestion:

Digestion of the sample Digestion is 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. The

general equation for the digestion of an organic sample is shown below: Protein + H2SO4 →

(NH4)2SO4(aq) + CO2(g) + SO2(g) + H2O(g) (1) Sulfuric acid has been used alone for the

digestion of organic samples. The amount of acid required is influenced by sample size and

relative amount of carbon and hydrogen in the sample, as well as amount of nitrogen. A

very

70
fatty sample consumes more acid. Also, heat input and digestion length influence the

amount of acid loss due to vaporization during the digestion process.

Distillation:

Distillation is 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. This is

accomplished by; 1) raising the pH of the mixture using sodium hydroxide (NaOH solution).

This has the effect of changing the ammonium (NH4 + ) ions (which are dissolved in the

liquid) to ammonia (NH3), which is a gas.

(NH4)2SO4(aq) + 2NaOH → Na2SO4(aq) + 2H2O(l) + 2NH3(g)

separating the nitrogen away from the digestion mixture by distilling the ammonia

(converting it to a volatile gas, by raising the temperature to boiling point) and then trapping

the distilled vapors in a special trapping solution of boric acid (H3BO3). The ammonia is

bound to the boric acid in the form of ammonium borate complex.

H3BO3+ NH3 → NH4 + + H2BO3-

(3) The majority of the NH3 is distilled and trapped in the receiving acid solution within the

first 5 or 10 minutes of boiling. But depending on the volume of the digestion mixture and

the method being followed, 15 to 150 ml of condensate should be collected in the receiving

flask to ensure complete recovery of nitrogen. Further extension of the distillation times and

volumes collected simply results in more water being carried over to the receiving solution.

Titration:

There are two types of titration: back titration, and direct titration. Both methods indicate the

ammonia present in the distillate with a color change and allow for calculation of unknown

concentrations.

 Materials and

Methods: Materials:

71
 Erlenmeyer flasks

 Burette

 Digestion tubes

Reagents:

 Potassium sulphate, K2SO4+Se tablets (2 for each digestion tube)

 Concentrated H2SO4 (in the hood)

 35 % (w/v) NaOH for each digestion tube (already prepared).

 4.0 % (w/v) Boric acid, H3BO3 (already prepared)

 0.1 M HCl (already prepared, exact concentration will be given)

 Methyl orange (in droppers)

 Procedure:

1. After sterilization of equipment’s at 1210C with 15p pressure about 15-30 minutes.

2. Place 1g of dry sample in 100ml kjheldhal flask dilute to 50ml.

3. Then add few glass beads (for control of boiling chips) carefully.

4. Add 10ml digestion reagent.

5. After mixing, heat the flask under a wood of a digestion rack at 3600C-4000C, until the

solution clear/pale green/transparent.

6. Continue heating for additional 30 minutes.

7. Allow to cool & dilute up to 30ml.

8. Total volume of distilled apparatus should not exceed 30ml.

9. Add 10ml NaOH-Na2S2O3 reagent.

10. Place the distillation flask on the distillation stand and connect to the condenser, start the

cooling of water flowing the condenser.

11. Distilled and collect 30-40ml distillate below the surface of 10ml, indicating boric acid

contain in 125ml flask (Titration Flask).

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12. As the ammonia is absorbed in the indicating boric acid it changes into green colour.

13. Titrate the distillate against the 0.02N sulphuric acid to countify the ammonia absorbed

by the boric acid.

14. Solution it at the end point.

15. The indicating boric acid regains its reddish pink colour.

16. Prepare 50ml of the blank with distilled water + all the reagent.

17. Perform all the procedure steps for balance and titrate the distilled water with 0.02N H2SO4.

Formula:

Total nitrogen as (mg/l) = (A-B) x280

Gram weight of sample

A= Volume of H2SO4 titrated for sample.

B= Volume of H2SO4 titrated for blank.

 Observation and Calculation

Volume of Sample = 50 ml

S/N Initial Burette Reading Final Burette Reading Difference (mL)

(mL) (mL)

1 Blank 0.0 0.2 0.2

2 Sample 3 47.5 44.5

Total nitrogen as (mg/l) = (44.5-0.2) x280

50

= 248.08mg/L

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 Result:

So, the nitrogen in the sample is 248.08 mg/L. Nitrogen determination by kjeldhal method is

very effective and accurate method. Also, the Kjeldhal method is used for the nitrogen

determination in wastewaters, soils and other samples. It is an official method and it is

described in different normative and used excessively.

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