National Institute of Technology Calicut

Department of Chemical Engineering

CH3091 ENVIRONMENTAL AND POLLUTION

CONTROL LABORATORY

SL. NO.
1

NAME

ROLL NO.

DEPARTMENT OF CHEMICAL ENGINEERING

NATIONAL INSTITUTE OF TECHNOLOGY
CALICUT

CH3091 ENVIRONMENTAL AND POLLUTION

CONTROL LABORATORY

CERTIFICATE

Certified that this is a bonafide record of the work done by Mr/Ms.

XYZ

of FIFTH semester (Reg No: BXXXXXXXXXXCH ) in the ENVIRONMENTAL

AND POLLUTION CONTROL LABORATORY during the year 2015.

FACULTY-IN-CHARGE
PLACE

CALICUT

DATE

xx/xx/2015

CONTENTS
Sl.No

Name of the Experiment

1.

DETERMINATION OF ACIDITY, ALKALINITY

2.

DETERMINATION OF DISSOLVED OXYGEN AND BOD

3.

ESTIMATION OF CHEMICAL OXYGEN DEMAND

4.

DETERMINATION OF ORGANIC CARBON CONTENT,

CARBOHYDRATES (ANTHRONE) & POTASSIUM IN SOILS

5.

DETERMINATION OF SOLIDS & VARIOUS HARDNESS IN

WATER

6.

DETERMINATION OF GROWTH CURVE USING E. coli

7.

SEPARATION OF COMPOUNDS BY PAPER CHROMATOGRAPHY

8.

SEPARATION OF COMPOUNDS BY USING THIN LAYER

CHROMATOGRAPHY

9.

Date

Page

DETERMINATION OF
SOLIDS IN WATER

Exp No:
Date: xx/xx/2015

DETERMINATION OF SOLIDS IN WATER

Aim:
To determine the concentration of suspended solids, dissolved solids and total solids in a given
water sample.
Apparatus required:
Porcelain dish, measuring cylinder, weighing balance, beaker, filter paper, funnel.
Reagents:
Sodium chloride, Calcium chloride
Theory:
It is needless to emphasize the importance of water in our life. We need water for
different purposes such as drinking, for industries, for irrigation etc. thus water has different
purposes has its own requirements for the composition and purity and each body of water has to
be analysed on a regular basis to confirm to suitability.
The total amount of solids present in water includes suspended solids and dissolved
solids. Total solids in water sample can be determined by evaporating the sample and weighing
the residue left behind. The suspended solids can be determined by filtering the sample with
filter paper and measuring the weight of residue left in filter paper after drying. The difference
between total solids and suspended solids will represent the dissolved solid content.
paper and measuring the weight of residue left in filter paper after drying. The difference
between total solids and suspended solids will represent the dissolved solid content.

Observations & Calculations:

Sample: 400 ml of water + 4 g of CaCO3 + 1 g of NaCl
W1 = weight of dish = g
W2 = weight of dish + dry residue = g
W3 = weight of filter paper = g
W4 = weight of filter paper + dry residue = g
Total solids in 20ml sample = (W2-W1) g = g
Concentration of total solids, x =
Suspended solids in 20ml sample

mg/ml
= (W4-W3) gm

Concentration of suspended solids, y =

Concentration of dissolved solids

= (x-y) mg/ml

mg/ml

Formula:
Total solids in 20ml sample = (W2-W1) g
Concentration of total solids, x =

mg/ml

Suspended solids in 20ml sample = (W4-W3) g

Concentration of suspended solids, y =

mg/ml

Concentration of dissolved solids = (x-y) mg/ml

where,
W1 = weight of dish (g)
W2 = weight of dish + dry residue (g)
W3 = weight of filter paper (g)
W4 = weight of filter paper + dry residue (g)
Procedure:
The empty dish was weighed and noted as W1 gm provided clean & dry. 20 ml of given
water sample was taken in the dish and evaporated. The dish was then again weighed with the
residue left & noted as W2 gm.
Now, the given filter paper was weighed as W3 gm. 20 ml of given water sample was
filtered through filter paper. This filter paper containing the residue is then dried and weighed as
W4 gm.
Result:
i)

Concentration of total solids in the sample = mg/ml

ii)

Concentration of suspended solids in the sample =

iii) Concentration of dissolved solids in the sample =

mg/ml
mg/ml

DETERMINATION OF
HARDNESS OF WATER

Exp.No:
Date: xx/xx/2015

DETERMINATION OF HARDNESS OF WATER

Aim:
To determine the hardness of the given sample of water.
Apparatus:
Burette and burette stand, porcelain tile, wash bottle, 250 ml graduated cylinder, conical flask.
Chemicals Required:
HCl,

(N/50)

Na2CO3,

EDTA,

Methyl

orange

indicator,

Phenolphthalein

indicator,

Erichrome Black-T indicator, MgSO4, Buffer solution.

Theory:
The hardness of water is due to the presence of water soluble bicarbonates, sulphates and
chlorides and of calcium and magnesium. Hardness is of two types, namely, temporary and
permanent hardness. The hardness which can be removed on heating is termed temporary.
Temporary hardness is due to the presence of bicarbonates of calcium and magnesium, which get
easily decomposed on heating to form carbonates. Permanent hardness is due to presence of
sulphates and chlorides of calcium and magnesium.
To determine the total hardness of water, the EDTA method is used. The disodium salt
of ethylene diamine tetra acetic acid forms stable metal complexes with calcium and magnesium
ions at pH 10. For the titration, Erichrome Black T is used as an indicator. At the pH range (7
11) it has a blue colour. When the indicator is added to the hard water sample containing both
Ca 2+ and Mg2+, the magnesium ions form a stable Mg-EBT complex with the indicator, which
is wine red in colour.

Observations:
Determination of Temporary Hardness:
Burette Reading (ml)
S.No

Initial

Vol. of water

Final

Volume of HCl

sample(ml)

(ml)
V1

100

100

V2

V1

V2

V1

V2

Determination of Total Hardness:

Vol. of water
S.No

Burette Reading (ml)

sample
(ml)

Volume of EDTA
(ml), V3

Initial

100

100

Final

As EDTA is added to the hard water sample Ca-EDTA complex is first formed. When all
Ca 2+ ions are used up, EDTA snatches the Mg ++ ions from Mg-EBT complex to form MgEDTA complex leaving the indicator free and turning the solution blue. This is the endpoint of

the titration and the volume of EDTA consumed corresponds to the EDTA required for
complexion with the calcium and magnesium ions present in the water sample.
Procedure:
Temporary Hardness:
Take 100 ml of water under test into a clean conical flask after rinsing the flask with the same
water. Add one or two drops of phenolphthalein indicator and titrate against approximately 0.01
N HCl. When the colour just disappears, note the titre value (V 1, ml). Add methyl orange
indicator and titration is continued till golden yellow just turns to orange red (V2, ml).
Total Hardness:
Measure out 100ml water under test into a conical flask. Add 3ml of buffer solution followed by
4 to 5 drops of Erichrome Black-T indicator. Mix thoroughly. The solution will become wine red
in colour. It is then titrated against approximately 0.01 N EDTA. Contents of the flask are kept
stirred throughout the titration. End point is the change of purple to blue colour persistent for
about 15 seconds. Titre value is noted as V3 ml.
Standardization of HCl:
Na2CO3 can be used to standardize HCl. Prepare an approximately equal normal solution of
Na2CO3 and titrate against HCl using methyl orange indicator.

Standardization of HCl:

Burette Reading (ml)

Vol. of

Initial

S.No

Final

Volume of HCl
(ml), V4

sample (ml)
1

10

10

Calculations:
Standardisation of HCl:
Normality of Na2C03 Volume of Na2CO3 = Normality of HCl Volume of HCl
NHCl = N

ppm

Temporary hardness of water sample =

=

ppm

Total hardness of water sample =

Permanent hardness of water sample = Total hardness Temporary hardness

=
=

ppm

Equations:
The equations for calculating hardness are:

ppm

ppm

Temporary hardness of water sample =

Where, N2 = Normality of HCl

V1 = Vol. of HCl required for disappearance of pink color.

V2 = Vol. of HCl required for changing yellow to orange red.

Total hardness of water sample =

ppm

Where, N4 = Normality of EDTA

V3 = Volume of EDTA
Permanent hardness of water sample = Total hardness Temporary hardness
Results:
The temporary hardness of given sample of water is
ppm
The total hardness of sample is
ppm
Therefore the permanent hardness of sample is
ppm
Inference:
The water sample which we use every day has some amount of hardness associated with it. This
is the main cause of scum formation etc. The ions like chlorides and sulphates are its basic
reason. Using EDTA and some indicators we can experimentally determine its hardness.

DETERMINATION OF
RESIDUAL CHLORINE

Exp.No:
Date: xx/xx/2015

DETERMINATION OF RESUDUAL CHLORINE

Aim:
To determine the residual chlorine in the given sample of water.
Apparatus:
Burette and burette stand, porcelain tile, wash bottle, 250 ml graduated cylinder, 500 ml conical
flask.
Chemicals Required:
Potassium Iodide crystals, Sodium thiosulphate, Starch indicator, Acetic acid.
Theory:
When chlorine is added to water, some of the chlorine reacts with organic materials and metals in
the water. This phenomenon is called the chlorine demand of water. A breaking point is attained
when whatever chlorine is added to water appears as residual chlorine.
Only the chlorine that remains after satisfying the chlorine demand is available for disinfection.
Thus, total chlorine in water is classified as:

Combined chlorine: The amount of chlorine that has reacted with the nitrates and is
unavailable for disinfection.

Free chlorine: The chlorine available to inactivate disease causing organisms. It helps in
measuring the portability of water.

Residual chlorine in purified water is tested by the starch iodine test. The test is carried out by
the addition of KI followed by starch. When KI is added to water sample, it produces iodide ions
in solution which will react with chlorine to form iodine, giving a yellow colour to the solution.
+2

+2

Observation:
Burette reading (ml)
Sl No.

Volume of sample

Volume of
titrant

Initial

(ml)

Final

chlorine
(ml)

1.

100

2.

100

Calculation:

Residual Chlorine =

=
=

mg/L

Residual
(mg/L)

In presence of starch, the iodine produces a blue colour, which is taken as evidence of residual
chlorine
+ starch Blue colour

The total amount of chlorine residual present in given water can be quantitatively measured by
titrating the iodine released with standard solution of a reducing agent such as Sodium
thiosulphate. The end point of titration is indicated by disappearance of blue colour.
+2

2NaI +

Preparation of Reagent:
0.01N Sodium thiosulphate solution

Weigh approximately 2.482g of sodium thiosulphate (

Transfer it to a beaker and dissolve it in boiled distilled water.

Transfer it to the standard flask and make it up to 100 ml.

).

Procedure:

Rinse the burette with sodium thiosulphate solution and then fill the burette with the
same.

Fix the burette to the stand.

Take 100 ml of the given sample in a conical flask.

Add 5 ml acetic acid to acidify the sample. It is used to reduce the pH between 3 and 4 .

Add about 1g of potassium iodide (KI) measured using the spatula and dissolve it by
thoroughly mixing it with stirring rod.

Perform the titration quickly, since iodine liberates quickly.

Titrate the solution with standard

solution until the yellow colour of liberated

iodine is almost faded out (pale yellow colour).

Add 1ml of starch solution and continue the titration until the blue colour disappears. In
many cases residual chlorine is very low and starch needs to be added before starting up
the titration.

Note down the burette reading to calculate the volume of sodium thiosulphate added.

Result:
Amount of residual chlorine =

mg/L

Inference:

The sample is obtained by mixing a particular amount of bleaching powder in water.

Residual chlorine is measured to be __________mg/L

Active chlorine should be present at each stage of water treatment and distribution.

The residual chlorine at consumers end should be _______mg/L.

Presence of excessive chlorine gives bad odour and taste and is also harmful. It may lead
to cancer, skin and eye irritation.

To avoid excess chlorination, water sample need to be boiled before domestic use.

DETERMINATION OF
ACIDITY AND ALKALINITY

Exp No:
Date:xx/xx/2015

DETERMINATION OF ACIDITY AND ALKALINITY

Aim:
To determine the acidity and alkalinity of the given sample.
Theory:
Ions are present in the sample as a result of dissociation or hydrolysis of solutes. Hydrogen ions
present in the sample react with the standard alkali added and hydroxyl ions present in the
sample react with standard acid. Thus, acidity and alkalinity depends on the end point pH at
indicator used.
Reagents Required
For Determining Acidity:
1)

Std. NaOH titrant: Prepare 0.1N NaOH solution, standardise the solution by titrating it

with 40ml of potassium hydrogen phthalate using 50ml burette.

2)

CO2 free water: Prepare all stock and standardized solutions and dilution water with

distilled or de-ionised water that has been freshly boiled for 15mins and cooled at room
temperature. Final pH of water should be greater than or equal to 6.0 and its conductivity should
be less than 2 micromoles per cm.
3)

Potassium Hydrogen Phthalate: 150-200g of potassium hydrogen phthalate is crushed to

100 Mesh and dried at 1200C for 2 hours. It is cooled in a desiccators. After cooling weigh
100.5g. Transfer it to a 1L volumetric flask and make it up to 1L.
4)

Standard NaOH (0.02N) : Weigh out 800mg of NaOH pellets using weighing machine and

dissolve it in distilled water and make up to 1L.

5)

Methyl orange indicator solution: Dissolve 50mg of methyl orange in distilled water and

dilute it to 100ml.

Observations:
Mineral Acidity

Volume of sample (ml)

Burette reading (volume of

NaOH)ml

Sample 1

25

Sample 1

25

Total Acidity

Volume of sample (ml)

Burette reading (volume of

NaOH)ml

Sample 1

25

Sample 1

25

Phenolphthalein alkalinity
Volume of sample (ml)

Burette reading (ml; Volume of

H2SO4)

Sample 2

25

Sample 2

25

Total alkalinity

Volume of sample (ml)

Burette reading (ml; Volume of

H2SO4)

Sample 2

25

Sample 2

25

6)

Phenolphthalein solution: 500mg of phenolphthalein is dissolved in 50ml of ethyl or

isopropyl alcohol. To this, add 50ml distilled water. Add NaOH solution dropwise until a faint
pink colour appears.

For Determining Alkalinity:

1) Na2CO3 (1N)
Weigh accurately 13.25 grams of anhydrous Na2CO3 (previously dried at 140 0C for 2
hours). Dissolve it in little distilled water and make up to 250 ml in a volumetric flask.
2) H2SO4 (1N)
Add 2.8 ml of H2SO4 in a measuring jar and make up to 100ml with CO2 free distilled
water. Standardize it against 1N Na2CO3 solution using methyl orange as indicator.
3) Phenolphthalein indicator solution
500 mg of phenolphthalein is dissolved in 50ml of ethyl or isopropyl alcohol. To this, add
50ml distilled water. Add NaOH solution (0.02N) drop wise until a faint pink colour
appears.
4) Mixed indicator solution
Dissolve 20 mg of methyl red and 100 mg bromo cresol green in 100 ml of 95% ethyl
alcohol or isopropyl alcohol (if Na salt is used, prepare the solution with distilled water
instead of alcohol)
Procedure:
Determination of Acidity:
1) Mineral Acidity: 25 ml of sample is measured in a measuring jar and taken in a conical flask
(250ml). The sample is dechlorinised using 0.1N Sodium Thiosulphate of very little amount. Add
4-5 drops of methyl orange titrate it with 0.1N NaOH. The end point is the appearance of faint
orange colour. The value is noted for mineral acidity.
2) Total Acidity: 25ml of sample is taken in a conical flask (250ml). Add 4 drops of
Phenolphthalein and titrate against 0.1N NaOH solution. The end point is the appearance of a
faint pink colour.

Calculations:

Acidity in mg/L expressed as CaCO3=

Alkalinity in mg/L =

Acidity :
Mineral acidity of sample
Phenolphthalein acidity of sample

=
=

=
=

mg/L
mg/L

Alkalinity:
Phenolphthalein alkalinity of sample

mg/L

Total alkalinity of sample

mg/L

Determination of Alkalinity:
1) Phenolphthalein Alkalinity: 25ml of the sample is taken in a conical flask (250ml). Add 6
drops of Phenolphthalein. If pink colour is not appearing, the sample has no Phenolphthalein
alkalinity. If appears, titrate it with 1N H2SO4 solution until the pink colour disappears.
2) Total Alkalinity: 25ml of sample is taken in a conical flask and 4 drops of mixed indicator is
added. Then titrate it against 1N H2SO4solution. The end point is the colour change from emerald
green to pink.
Inferences:
Food Sample showed no alkaline character because of no appearance of pink colour on drop
wise addition of phenolphthalein.
Results:
Acidity:
Mineral acidity

mg/L

Total acidity

mg/L

Alkalinity:
Phenolphthalein alkalinity

mg/L

Total alkalinity

mg/L

DETERMINATION OF
DISSOLVED OXYGEN AND
BIOCHEMICAL OXYGEN DEMAND

Exp No:
Date: xx/xx/2015

Determination of Dissolved Oxygen and Biochemical Oxygen Demand

Aim:
To determine the amount of dissolved oxygen and biochemical oxygen demand of a given water
sample.
Apparatus Required:

300ml BOD bottle

conical flask
BOD incubator
Burette
Measuring cylinder
Pipettes

Reagents Required:
1.
2.
3.
4.
5.
6.
7.
8.

Std. Manganese sulphate solution (2M)

N/40 Sodium thiosulphate solution
Starch indicator (1g in 100 ml)
Conc. Sulphuric acid
Calcium Chloride
Magnesium Sulphate
Alkaline Potassium Iodide solution
Potassium Permanganate solution

Theory:
Dissolved Oxygen:
Dissolved oxygen is used to describe the amount of oxygen dissolved in a unit volume of
water. Dissolved oxygen is essential for the maintenance of healthy lakes and rivers .It is a
measure of water to sustain aquatic life. The dissolved oxygen content of water is influenced by
the source, raw water temperature, treatment and chemical or biological processes taking place in
the distribution system. The presence of oxygen in water is a good sign. Depletion of dissolved
Observations and tables:
For DISSOLVED OXYGEN,

Vol. of sample
Sl. No
1

(ml)
102

102

Burette Reading (ml)

Initial
Final

Volume of Na2S2O3
(ml)

0
0

Vol. of blank
(ml)
1

102

2.

102

For BIOCHEMICAL OXYGEN DEMAND,

Sl.

Day

Volume of

Burette

Burette

Volume of Na2S2O3

No

water

reading

reading

initial(ml)
0

Final(ml)

sample(ml)
102

102

Blank

102

Sample

102

Blank

(ml)

Type of
solution
Sample

Calculations:
For DISSOLVED OXYGEN,
Volume of water sample used = 102 ml
Normality of Na2S2O3used =

Dissolved oxygen(D0) =
oxygen in water supplies can encourage the microbial reduction of nitrate to nitrite and sulphate
to sulphide. It can also cause an increase in the concentration of ferrous iron in solution, with
subsequent discolouration at the tap when the water is aerated. In a healthy body of water such as

a lake, or stream, the dissolved oxygen is about 8 ppm. The minimum DO level of 4 to 5 mg/l or
ppm is desirable for survival of aquatic life
Drinking water should be rich in dissolved oxygen for good taste.DO test is used to
evaluate the pollution strength of industrial and domestic waste. Higher values of DO may cause
corrosion of iron and steel. Algae in water may release oxygen during its photosynthesis and DO
may even shoot up to 30mg/l. Oxygen is poorly soluble in water. Its solubility is about 14.6 mg/l
for pure water at 00C under normal atmospheric pressure and drops to 7 mg/l at 35oC
Biochemical Oxygen Demand:
The biochemical oxygen demand determination is a chemical procedure for determiningthe
amount of dissolved oxygen needed by aerobic organisms in a water body to breakthe organic
materials present in the given water sample at o certain temperature over aspecific period of
time.BOD of water or polluted water is the amount of oxygen required for the biological
decomposition of dissolved organic matter to occur under standard condition at astandardized
time and temperature. Usually, the time is taken as 5days and the temperature is 20 0C.The test
measures the molecular oxygen utilizedduring a specified incubation period for the biochemical
degradationof organicmaterial(carbonaeceous demand) and the oxygen used to oxidise inorganic
materials like sulfide and ferrous ions. It may also measure the amount of oxygen needed
tooxidise reduced forms of nitrogen(nitrogenous demand).
Ordinary domestic sewage may have a BOD of 200mg/l. Any effluent to be ischargedinto
natural bodies of water should have BOD lesstham 30 mg/l.this is importantparameter to assess
the pollution of surface waters and ground waters where contamination occur due to disposal of
domestic and industrial effluents.Drinking waterusually has a BOD less than 1 mg/l .But , when
it reaches 5mg/l, the water is doubtfull ofpurity.the determination ofBOD is used in studies to
measure the self purificationcapacity of streams and serves regulatory authorities as a means of
checking on thequality effluents discharged to stream waters.The determination of BOD of
wastes inusefull indesigning treatment facilities.
For BIOCHEMICAL OXYGEN DEMAND,

Normality of Na2S2O3used =

Initial Dissolved oxygen,D0 =

D0 = dissolved oxygen of sample at day 0 =

mg/l

D5 = dissolved oxygen of sample at day 5 =

mg/l

C0 = dissolved oxygen of blank at day 0 =

mg/l

C5= dissolved oxygen of blank at day 5

mg/l

BC, Blank Correction = C0 - C5 =

mg/l

Volume of sample = 10 ml
Volume of dilute sample = 300 ml

mg/l

Principle Of Titration:
Divalent Manganese salt in solution is precipitated by strong alkali to divalent manganese
hydroxide.addition of pottassium iodide or potassium hydroxide is added to create a pinkish
brown precipitate.In the alkaline solution, dissolved oxygen present in the sample rapidly
oxidized to form trivalent or higher valency hydroxide.MnO(OH)2 appears as a precipitate.Iodide

ions are added and acidified(acid facilitates conversion of brown), which reduces tetravalent
hydroxides back to their stable divalent state thereby liberating equivalent amount of
iodine.Thiosulphate solution is used, with starch indicator, to liberate the iodine. This iodine is
equivalent to dissolve oxygen present in the sample.
MnSO4 + 2 KOH

Mn(OH)2 + K2SO4

2Mn(OH)2 + O2

2MnO(OH)2

MnO(OH)2 + 2 KI + H2O
I2 + 2 S2O32-

Mn(OH)2 + I2 + 2 KOH

S4O62- + 2I-

Procedure:
Experimental Procedure:

Take four 300ml glass stoppered BOD bottles(two for sample and two for blank)
Add 10mlof the sample to each of the two BOD bottles and fill the remaining quantity

with dilution water.

The remaining two BOD bottles are for blank, add dilution water alone to these bottles
After the addition immediately place the glass stopper over the BOD bottles and note

down the numbers for identification.

Now preserve one blank and one sample in a BOD incubator at 200C for five days.
Add 0.9 ml of conc. Sulphuric acid and 0.2 ml potassium permanganate. Invert the
contents of the bottle keeping the stopper closed.

Now add 2ml of manganese sulphate and 3 ml of alkaline potassium iodide.

Allow the oxygen to completely react by keeping the bottles for 10-15 minutes.
Add 0.5 ml of potassium permanganate and shake the contents well. The solution turns

yellow. If not add few drops of NaOH/KOH.

Check the pH of the solution .It should be 6.9-7.1.If not add NaOH solution carefully.
Add 1 ml of starch indicator to both the bottles. The solution turns brownish violet.
Rinse the burette with standard thiosulphate solution and fill it upto mark
Pipette out 102 ml of both the solutions from the bottles into 2 separate conical flasks for

titration. Titration must be immediately after the transfer.

Titrate separately both the flasks till colourless and also repeat till concordant values are

obtained.
Note the amount of thiosulfate consumed.
After five days take out the bottles from BOD incubator and analyse the sample and the
blank for DO following the above procedure.

Preparation of Dilution Water:

High quality organic free water must be used for dilution purpose .Usually distilled water is
used .For 5 l of distilled water the following nutrients must be added

Add 5 ml of calcium chloride solution (27.5 g of anhydrous calcium chloride powder in

100 ml of water in a standard flask)

Add 5 ml of magnesium sulphate solution (22.5 g of magnesium sulphate powder in 100
ml of water in a standard flask)

The pH must be in the range 7.0 7.3.else add NaOH solution.

Result:
i)
ii)
iii)

The dissolved oxygen in the sample is

The biochemical oxygen demand of sample is
The dissolved oxygen in the blank solution is

mg/l
mg/l
mg/l

Inference:
On the basis of BOD values, the characteristics of the water and the biological activity of the
incubated micro flora can be determined.

ESTIMATION OF
CHEMICAL OXYGEN DEMAND

Exp No:
Date: xx/xx/2015
ESTIMATION OF CHEMICAL OXYGEN DEMAND
Aim:
To determine the COD in a given water sample.
Theory:
Water is a universal solvent and it dissolves almost anything that it passes by, organic
compounds being no exception. The large amount of organic compounds present in water bodies
are contributed by aquatic plants, animals, industrial effluents etc. Organic compounds oxidize
readily in presence of oxygen to give carbon dioxide, water and ammonia. Hence an excess of
organic compounds in water could deplete oxygen jeopardizing aquatic life.
Chemical Oxygen Demand is used as an indirect measure of the amount of organic
matters. It is actually the amount of oxygen needed to completely oxidize the entire organic
compounds present in water. It is measured as milligrams of oxygen needed to oxidize 1 litre of
water. Potassium dichromate is used as the oxidizing agent owing to its easy availability,
cheapness and strong oxidizing ability. Sulphuric acid provides the acidic environment for
potassium dichromate to carry out its oxidization. Inorganic interference can be a serious concern
especially by chlorine. Chlorine is dealt by using mercuric sulphate which gets converted to
mercuric chloride. Silver sulphate plays the role of catalyst. Mohrs salt oxidizes the excess
dichromate while ferroin acts as the redox indicator.
The organic matter present in sample gets oxidized completely by potassium dichromate
in the presence of sulphuric acid, silver sulphate, and mercury sulphate to produce carbon
dioxide and water. The sample is refluxed with a known amount of potassium dichromate in the
sulphuric acid medium and the excess potassium dichromate is determined by titration against
ferrous ammonium sulphate, using ferroin as an indicator. The dichromate consumed by the
sample is equivalent to the amount of oxygen required to oxidize the organic matter.

Observation:
Sl

Volume (ml)

Burette Reading

No.

Initial

SAMPLE
1

25

BLANK
1

25

Volume of 0.25N
FAS (ml)

Final

Calculation:
Volume of 0.25N FAS used for sample titration (V1) =

ml

Volume of 0.25N FAS used for blank titration (V2) =

ml

Volume of sample used = 25 ml

COD =

g/ml

M= molarity of FAS
Milliequivalents of oxygen per ml =
Actual COD = COD x Dilution Factor =

g/ml

Apparatus:
1

Burette and Burette stand.

2 x 250 ml conical flask (Erlenmeyer Flask).

Reflux flask.

100 ml measuring cylinder.

10 ml cylinder measuring cylinder.

Distilled water.

Reagents:
1

Potassium Dichromate (K2Cr2O7)(0.25N).

Mohrs Salt: Ferrous Ammonium Sulphate (FAS)(0.25N).

Mercuric Sulphate.

Silver Sulphate.

Phenanthroline Ferrous Sulphate Indicator Solution (Ferroin Indicator).

Concentrated Sulphuric Acid.

Procedure:

25 ml of sample was taken into a refluxing flask and 0.25 gm of HgSO 4 is added to
the solution.

37.5 ml of COD acid (Ag2SO4 H2SO4) is added slowly. The solution is swirled
slowly.

12.5 ml of K2Cr2O7 solution is then added.

Thorough mixing of the solution is ensured by swirling the flask in a water bath to
recover any volatile substances that may have escaped from the liquid phase.

The solution is then refluxed for 2 hours.

A blank run (using 25 ml distilled water instead of sample) is simultaneously

conducted with the same procedure.

After cooling, the solution is transferred to an Erlenmeyer flask. The reflux flask is
rinsed pouring the rinsing water to the Erlenmeyer flask.

The solution is diluted to about 150ml and about 3 drops of ferroin indicator is added.

The solution is titrated against the Mohrs salt and the titre value required for the
colour change from blue-green to reddish brown is noted.

The procedure is repeated for the blank run.

Discussions:

K2Cr2O7 acts as a strong oxidising agent and oxidizes the organic and inorganic
matter in the waste water
Cr2O72- + 14H+ + 6e-

2Cr3+ + 7H2O

If Chlorides are present in the sample it will interfere with the oxidation of the
organic matter. To ensure non-interference of Chlorides, Mercuric Sulphate is added
which will form complex of Mercuric Chloride. An amount of 10gm of Mercuric
Sulphate is required for 1gm of Chloride to form complex.

H2SO4is added to the mixture so that the mercury is completely dissolved. Besides it
assists in oxidizing the nitrogen compounds in the sample and the increased heat will
accelerate the reaction rate.

Silver Sulphate catalyses the reaction and also assists in the oxidation of the nitrogen
compounds.

Mercury Sulphate is added first in order to allow the chlorine atoms to combine with
mercury. If Silver Sulphate is added first, the Chlorine would bind with Silver, it will
take some time for the Chlorine to detach from the Silver and bind to Mercury present
in Mercury Sulphate. Thus, it is best to add Mercury Sulphate first.

The titre volume of the sample gives the volume of FAS required to react with the
excess K2Cr2O7in the solution. Similarly, the titre volume for the blank (distilled
water) gives the volume of FAS required to react with the excess K 2Cr2O7 in the
blank.
Cr2O72- + FeSO4(NH4)2SO4

Cr3+ + NH4++ Fe3+

Result:
Chemical oxygen demand of the given sample of water = 20.8 g/ml

DETERMINATION OF ORGANIC
CARBON CONTENT IN SOILS

Exp No:
Date: xx/xx/2015
DETERMINATION OF ORGANIC CARBON CONTENT IN SOILS

Aim:
To determine the percentage of organic carbon present in soil.
Introduction:
Soil carbon is the generic name for carbon held within the soil, primarily in association with
its organic content. Soil carbon is the largest terrestrial pool of carbon. Humans increasingly
influence the size of this pool. Soil carbon plays a key role in the carbon cycle and thus is
important in global climate models.

Although the figure is frequently being revised upwards with new discoveries, over 2,700 Giga
tonnes (Gt) of carbon is stored in soils worldwide, which is well above the combined total of
atmosphere (780 Gt) or biomass (575 Gt), most of which is wood. Carbon

Observations:
Burette Reading (ml)
Sl
no.

Weight of sample
(g)

Volume of FAS
Initial

SAMPLE 1
1

0.2

SAMPLE 2
2

0.2

BLANK
3

Final

(ml)

is taken out of the atmosphere by plant photosynthesis; about 60 Gt annually is incorporated into
various types of soil organic matter (SOM) including surface litter; about 60 Gt annually is
respired or oxidized from soil.
Soil carbon is the last major pool of the carbon cycle. The carbon that is fixed by plants is
transferred to the soil via dead plant matter including dead roots, leaves and fruiting bodies. This
dead organic matter creates a substrate which decomposes and respires back to the atmosphere as
carbon dioxide or methane depending on the availability of oxygen in the soil. Soil carbon is
also oxidized by combustion and returned to the atmosphere as carbon dioxide. Soil carbon is
primarily composed of biomass and non-biomass sources. Biomass carbon includes various
bacteria and fungi. Non-biomass carbon sources or substrates reflect the chemical composition of
plant biomass and primarily include cellulose, starch, lignin and other diverse organic carbon
compounds. Some of the substrate carbon binds to the mineral soil, becoming encapsulated in
soil aggregates (singular masses of coherent soil particles, or peds) or chemical complexional.
Biomass feeds on the substrate carbon compounds at different rates.
Some of the carbon compounds are easily digested and respired by the microbes resulting in a
relatively short residence time. Others, like lignin, humic acid or substrate encapsulated in soil
aggregates are very difficult for the biomass to absorb and have long residence times.
Principle:
Organic Carbon is determined by sulphuric acid and aqueous potassium dichromate
mixture. After the complete oxidation, the unused or residual dichromate is titrated against
Ferrous Ammonium Sulphate (FAS). The used dichromate which is the difference between the
original and the residual dichromate, gives a measure of the organic carbon content of the soil.
2K2Cr2O7 + 8H2SO4

2K2SO4 + 2Cr2(SO4)3 + 6[O]

Calculations:
Sample 1

% Organic Carbon Content

=
=

Sample 2

% Organic Carbon Content

=
=

Titration method:
The reagents used are
(a)

(b)
(c)

K2Cr2O7 solution (1 M)
Dissolve 49.024 grams of dry potassium dichromate in 800 ml of distilled
water and dilute it to 1 litre
Concentrated H2SO4
(FAS)
Dissolve 78.390 grams of FAS powder in 50 ml of concentrated H 2SO4
and dilute it to 1 litre with distilled water. Standardise it by adding ferroin
indicator.

Formulae used:

% Organic Carbon Content

Procedure:
1. Weigh 0.2 gram of ground soil.
2. Add 5 ml of K2Cr2O7 and 7.5 ml of Conc. H2SO4 to the sample.
3. Place the container in a pre-heated block at 1450C 1500C for exactly 30 minutes.

4. After 30 minutes, remove it and allow it to cool.

5. Transfer the digest to a 100 ml conical flask and add 0.3 ml of ferroin indicator.
6. Using the stirrer, titrate the flask using the FAS solution. The end point is the colour
change from green to brown.

Inference:
The exchange of carbon between soils and the atmosphere is a significant part of the world
carbon cycle, which is extensive both spatially and temporally. Carbon, as it relates to the
organic matter of soils, is a major component of soil and catchment health. Several factors affect
the variation that exists in soil organic matter and soil carbonthe most significant has, in
contemporary times, been the influence of humans and agricultural systems. There are clear
benefits for catchment health by focusing on soil carbon efforts would need to be extensive and
economical for the collective benefit to be realized.
Result:
The percentage of organic carbon content in the soil
Sample 1 =

Sample 2 =

EXP.NO:

DETERMINATION OF GROWTH CURVE USING E. coli

AIM
To determine the growth curve studies using yeast and E. coli.
APPARATUS REQUIRED
Autoclave, Laminar chamber, Weighing machine
CHEMICALS REQUIRED
Peptone

5 g/lit

NaCl

5 g/lit

Beef Extract

1.5 g/lit

Yeast Extract

1.5 g/lit

Malt Extract

1.0 g/lit

pH

7.4

0.2

AUTOCLAVE CONDITIONS
Pressure

15 psi

Temperature

120 0 C

Pressure release time =

30 min

THEORY
Batch culture is a closed culture system which contains an initial limited amount of nutrient. The
inoculated culture will pass through a number of phases. After inoculation, there is a period
during which it appears that no growth takes place. This period is referred to as lag phase and

may be considered as time of adaptation. In the commercial phase, length of lag phase should be
reduced as much as possible and this may be achieved by using suitable inoculums. Following a
period during which the growth rate of the cell gradually increases.
OBSERVATIONS

Culture (min of incubation)

0
30
60
90
120
180
240
300
360
1380
1440

Absorbance OD(600nm)

Table 1.1: Data for adsorbance and time of incubation

The cells grow under constant maximum rate and this period is known as log or exponential
phase.

The population is most nearly uniform in terms of chemical composition of cells,

metabolic activity and other physiological characteristics. They tend toward cessation of growth
can be attributed to a variety of substances. Particularly the exhaustion of some nutrients and
production of toxic products during growth. The population remains constant for the time since
the reproduction rate is balanced by an equivalent death rate. This is referred to as stationary
phase. During the death phase, number of viable cells decreases exponentially. Essentially, the
inverse of growth during log phase. A variety of condition contributes to cell death but the most
important are depletion of essential nutrients and accumulation of inhibitory products such as
acid.
Six characteristic phases of the growth cycle are recognized.
1. Lag Phase: Immediately after inoculation of the cells into fresh medium, the population
remains temporarily unchanged. Although there is no apparent cell division occurring, the
cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and
increasing in metabolic activity. The length of the lag phase is apparently dependent on a
wide variety of factors including the size of the inoculum; time necessary to recover from
physical damage or shock in the transfer; time required for synthesis of essential
coenzymes or division factors; and time required for synthesis of new (inducible)
enzymes that are necessary to metabolize the substrates present in the medium.
2. Acceleration Phase: Once after the cells have adapted to the new environment, cell
division occurs at increasing frequency until maximum growth rate reached.
3. Exponential (log) Phase: The exponential phase of growth is a pattern of balanced
growth wherein all the cells are dividing regularly by binary fission, and are growing by
geometric progression. The cells divide at a constant rate depending upon the
composition of the growth medium and the conditions of incubation. The rate of
exponential growth of a bacterial culture is expressed as generation time, also
the doubling time of the bacterial population. Generation time (G) is defined as the time
(t) per generation (n = number of generations). Hence, G=t/n is the equation from which
calculations of generation time (below) derive.

Bacteria growth rate vs time

4. Deceleration Phase: When level of substrate decreases, it eventually become limiting

and no longer sustain maximum growth rate.
5. Stationary Phase: Exponential growth cannot be continued forever in a batch
culture. Population growth is limited by one of three factors: 1. exhaustion of available
nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of
space, in this case called a lack of "biological space". During the stationary phase, if
viable cells are being counted, it cannot be determined whether some cells are dying and
an equal number of cells are dividing, or the population of cells has simply stopped
growing and dividing. The stationary phase, like the lag phase, is not necessarily a period
of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so
during the stationary phase of the growth cycle. It is during the stationary phase that
spore-forming bacteria have to induce or unmask the activity of dozens of genes that may
be involved in sporulation process.
6. Death Phase: If incubation continues after the population reaches stationary phase, a
death phase follows, in which the viable cell population declines. (Note, if counting by
turbidimetric measurements or microscopic counts, the death phase cannot be observed.).
During the death phase, the number of viable cells decreases geometrically
(exponentially), essentially the reverse of growth during the log phase.
PROCEDURE
1.
2.
3.
4.
5.

Prepare the nutrient broth and sterilize it in the autoclave.

After sterilizing aseptically transfer 5ml of broth culture to 15ml of nutrient broth.
Determine the initial OD at 600nm.
Place the inoculated culture flask in the shaker at 37C for 16 hr for incubation.
Take 5ml of culture from the incubated flask and inoculate it into 100ml of sterile

nutrient broth aseptically.

6. Determine the OD of the sample at 600nm.
7. Repeat the step 6 at each 30 min interval for a period of 8hrs.

A: Acceleration Phase
Figure : Bacteria growth curve

B: Deceleration Phase

Experiment Number:

Date:

SEPARATION OF COMPOUNDS BY PAPER CHROMATOGRAPHY

AIM
To perform paper chromatography to separate the unknown compounds in a mixture and to
determine the Rf values of compounds.

THEORY
Chromatography is the physical separation of components of a mixture on the basis of
their differing affinities for a stationary phase vs. a mobile phase. It is one of the most common
chemical and biochemical separation techniques. In a variety of chromatography methods,
chemists use liquid solvents moving compounds over paper, thin film, or materials held in a
column. The material in the column can be a solid, a gel, or a polymer. We can analyze the
compounds as they come out of the column (at different times) or remove the compound from
the paper and then analyze it. Paper chromatography (PC) is a technique similar to the widelyused thin-layer chromatography (TLC).
In typical thin-layer chromatography, silica gel a material commonly used as a
stationary phase is coated onto a glass or plastic sheet. In paper chromatography, the mixture is
spotted onto a sheet of paper. Both of these techniques can be used to identify an unknown
sample in solution. Paper chromatography works because of the capillary action of water, or
solvent, in paper. In this experiment you will put a paper spotted with unknown and known
solutions into a jar with a small amount of a solvent.
Capillary action allows us to separate compounds due to the attraction, or affinity, of the
compounds for the mobile phase (the solvent) vs. the stationary phase (the paper or thin film).
Compounds which are not soluble in the solvent or are very attracted to the stationary phase stay
in place. Compounds those are very soluble and less attracted to the stationary phase travel up
the stationary phase with the solvent. Some compounds are somewhat soluble and somewhat
attracted to the stationary phase. They travel up the paper, but at a slower rate.
Terms those are commonly used in Chromatography:
1. Mobile phase: This is the solvent. It may be a single compound or a mixture of solvents,
often with different polarities.

2. Stationary phase: This is the chromatographic medium in our experiment, the paper.

Chromatogram distance for Rf calculation

3. Baseline (origin): This is where the mixture of compounds are loaded on the
chromatography paper. Using a pencil draw a baseline on the paper (approximately 1.5-2
cm from the bottom of the paper) where mixtures can be loaded. The baseline must be
above the liquid level, so that the solvent does not dissolve the compounds into the liquid
phase in the chamber.
4. Solvent front: This is the highest point that the solvent traveled during developing.
Note: you need to mark where the solvent front reaches before the solvent evaporates.
5. Retention factor (Rf): This is the ratio of how far the solute (metal ions) travels on the
chromatograph vs. how far the solvent front travels.

Rf=

The Rf is a property characteristic of a substance (intensive property) and will be the same if the
solvent and stationary phase are the same.

APPARATUS REQUIRED

a weigh scale

a beaker

measuring jar

pan or porcelain plates, for resting the plates on and for putting in the oven.

a syringe, 10cc minimum or a micropipette

Chromatography filter paper

Solvents(mobile phase)

a pencil

a spatula

PROCEDURE
1. In a piece of chromatography filter paper, draw one line 2cm from one of the longer
edges. Mark the line with a pencil dot at equal divisions.

SAMPLE DATA TABLE:

Sample

1
2
3
4
5

CALCULATIONS:

dspot

d solvent front

(cm)

(cm)

Spot colour

Rf

Compound

2. Fill your capillary pipet by dipping the end into the solutions of ions(Cr 6+,Cu2+and
unknown solution)
3. Apply a single drop of the solution on to the filter paper.
4. Pour the solvent mixture into a depth of 1cm (about 20mL) in the bottom of a 600mL
beaker.
5. Fold the filter paper and staple to make a cylindrical shape and place it in the beaker.
6. Cover the beaker carefully with a watch glass. Do not disturb the beaker while the
chromatograms are developing.
7. When the solvent front has nearly reached the top of the paper, carefully remove the
watch glass and the paper. Place the wet paper on a clean surface and immediately mark
the solvent front with a pencil. Allow the filter paper to dry.
8. Label your chromatogram, calculate the Rf for each spot and draw a picture of it in your
notebook.

RESULTS
Separation of the compounds of the mixture using proper solvent system was done and Rf values
of the compound in the mixture was determined.
Rf values of compounds used in the experiment are given below:
Sl No
1

Compound

Rf value

2
3
4
5

PRECAUTIONS TO BE TAKEN

Use precautions working with acids, bases and solvents. Wear gloves while handling the solvents
and wash your hands properly after the experiment.

Experiment No:

Date:

SEPARATION OF COMPOUND BY THIN LAYER CHROMATOGRAPHY

(TLC)
AIM
To perform thin layer chromatography to separate compounds from a mixture using a proper
solvent system and to determine the Rf values of the compounds in the mixture.
THEORY
Chromatography is the separation of two or more compounds or ions by the distribution
between two phases, one which is moving and the other which is stationary. These two phases can be
solid-liquid, liquid-liquid or gas-liquid. Thin Layer Chromatography (TLC) is an extremely useful
technique for monitoring reactions. It is also used to determine the proper solvent system for
performing separations using column chromatography. TLC uses a stationary phase, usually alumina
or silica, that is highly polar (standard) or non-polar (reverse phase), and a mobile phase, some
solvent whose polarity you will choose. You will apply your reaction mixture in solution to the plate
then "run" the plate by allowing a solvent (or combination of solvents) to move up the plate by
capillary action.
Depending on the polarity of the components of the mixture, different compounds will travel
different distances up the plate. More polar compounds will "stick" to the polar silica gel and travel
short distances on the plate, while non-polar substances will diffuse into the solvent and travel large
distances on the plate. The measure of the distance a compound travels is called Retention factor.
This number, between zero and one, is determined by measuring the distance the compound moved
from the baseline (where it was originally spotted) divided by the distance the solvent moved from
the baseline. An unknown mixture is pushed passed what is called the stationary phase in a

continuous stream, it can be a solid in the case of TLC, or a liquid as is the case in many forms of
Gas Chromatography.
The exact nature of the stationary phase is not so important, what is important is that the
individual components of the unknown mixture interact with the stationary phase in some way.
Ones that interact more strongly and prefer to stay associated with the stationary phase will move
slowly and will be held back. Species that interact weakly move more swiftly through the stream.
In this manner the different components of the mixture can be separated out by the amount of
time it takes for them to elute or move through a column of stationary phase.

A measure of how strongly or weakly a compound is retained is the Retention Factor (RF) or
Retention Time. In TLC the RF of a compound can be used to identify it from tabulated values.
The retention factor is the ratio of the distance traveled by the compound up the plate versus the
distance traveled by the solvent front. The RF for a given compound is (relatively) unique as it
depends upon the structure and chemistry of that compound.
Your compounds will travel different distances up the plate depending on the solvent you
choose. In non-polar solvents like pentane and hexane, most polar compounds will not move, while
non-polar compounds will travel some distance up the plate. In contrast, polar solvents will usually
move non-polar compounds to the solvent front and push the polar compounds off of the baseline. A
good solvent system is one that moves all components of your mixture off the baseline, but does not
put anything on the solvent front - R f values between 0.15 and 0.85. This is not always possible, but
should be your goal when running a TLC. (For column chromatography the correct solvent system
should give an Rf between 0.2 and 0.3.) Here's a list of some standard solvents and their polarity
Very polar additives:
Methanol > Ethanol > Isopropanol
Moderately polar additives:
Acetonitrile > Ethyl Acetate > Chloroform > Dichloromethane > Diethyl Ether > Toluene
Non-polar additives:
Cyclohexane, Petroleum Ether, Hexane, Pentane

APPARATUS REQUIRED

a weigh scale

a beaker

measuring jar

pan or porcelain plates, for resting the plates on and for putting in the oven.

a syringe, 10cc minimum or a micropipette

Chromatogram distance for Rf calculation

glass slides, you can also use sheets of tin or plastic, basically anything stiff that won't
interact with water.

Silica Gel

an oven

solvents (mobile phase)

a pencil

a spatula

PROCEDURE
1. Weigh out required amount of silica, make a paste using water.
2. The stationary phase (here it is silica) is applied onto a clean and dried plate uniformly
and then allowed to dry and stabilize. Dont pour or dispense it all at once as the layer
will form a hill instead, and will be too thick. The thickness of the layer is important, less
than 1mm when dry is preferable, so be careful not to overdo it.
3. Just leave the slides in a calm place for about 15 minutes, or until they are dried (white
and smooth).
4. With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots.
5. Then, samples solutions are applied on the spots marked on the line in equal distances.
6. Fill TLC chamber (glass beaker) with 1-2 mL of the desired solvent system.
7. The plate prepared with sample spotting is placed in TLC chamber so that the side of the
plate with the sample line is facing the mobile phase. The plate is then immersed, such
that the sample spots are well above the level of mobile phase, but not immersed in the
solvent. Then the chamber is closed with a lid.

SAMPLE DATA TABLE

Sample

1
2
3
4
5

CALCULATIONS

dspot

d solvent front

(cm)

(cm)

Spot colour

Rf

Compound

8. Allow sufficient time for the development of spots. Then remove the plates and allow
them to dry.
9. Mark the solvent front and spots immediately with a pencil.
10. Label your TLC, calculate the Rf for each spot and draw a picture of it in your notebook.

RESULTS
Separation of the compound of the mixture using proper solvent system was done and Rf values
of the compounds in the mixture was determined.

Sl No
1

Compound

Rf value

2
3
4
5

PRECAUTIONS TO BE TAKEN
Silica Gel is hygroscopic, and its fine particles can be harmful if inhaled. So, be careful
while handling and wear gloves and a mask while handling this stuff.
When using organic solvents ensure appropriate safety measures are in place. Such as proper
ventilation, safety glasses, etc.