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VII SEMESTER
06 CVL 77
ENVIRONMENTAL ENGINEERING
LABORATORY MANUAL
BATCH :____________________________
INSTRUCTIONS
1. This laboratory manual is ONLY for reference for those using the environmental
engineering laboratory.
3. To Note:
(a) In water and wastewater analysis, the results are usually reported in terms of
mg/l of some particular ion, element or compound. It is most convenient to have
the standard titrating agent of such strength, that 1mL is equivalent to 1mg of
material being measured. Thus 1 litre of the standard solution is usually
equivalent to 1g of the standard substance.
(b) Rules listed by Worthing and Geffner are to be followed while plotting graphs.
4. Users may refer the following for writing the discussion after each experiment:
(a) “Standard Methods for the Examination of Water and Waste Water”, American
Public Health Association, 1015, 15th Street, N.W., Washington D.C., 2005.
(b) “Chemistry for Environmental Engineers”, Sawyer and McCarty, Tata Mc-Graw
Hill.
(c) “Manual of Standards of Quality for Drinking Water Supplies”, Indian Council
of Medical Research, New Delhi.
(d) “International Standards for Drinking Water” — World Health Organisation.
(e) “IS 2490 - 1981, IS 3306 - 1974, IS 3307 - 1977, IS 7968 - 1976, IS 2296 –
1974”, Bureau of Indian Standards, New Delhi.
Determination of Alkalinity.
03 Determination of Acidity.
Determination of pH.
Determination of COD.
06
Determination of Percentage of Available Chlorine in Bleaching Powder.
11 MPN Determination.
Preview
Aim
Theory
One of the most important characteristics of sewage is the solid content. Sewage
normally contains 99 % water and 1% solids. Solids test is an important parameter as it is
used in:
1. Design of biological units and water pollution control methods, which depend on the
concentration of organic solids.
2. In Grit chambers and sedimentation tank design which depends on the quantity of
inorganic and organic settleable solids respectively.
3. Dissolved inorganic solids are to be considered in the design of land treatment of
Sewage.
Total solids: Analytically the total solids content of a wastewater is defined as all the
matter that remains as residue on evaporation at 103°C and subsequent drying. These
solids are very important as they indicate the strength of sewage and amount of treatment
required.
Dissolved solids are those which remain dissolved in sewage just as salt in water and are
generally organic in nature causing putrefaction and thus increasing the strength of
sewage and creating nuisance if disposed off untreated. Colloidal solids are finely divided
solids remaining either in solution or in suspension.
Suspended solids are those, which are in suspension and are floating in sewage.
• Settleable solids, which settle down at the bottom of an Imhoff cone and are an
approximate measure of the quality of sludge that will be removed by sedimentation.
• Non settleable solids are those, colloidal in nature and can be removed by Chemical
treatment.
Fixed solids: Fixed solids are also called inorganic solids consisting of minerals and salts,
which include sand, gravel, debris, dissolved salts etc. They constitute about 55°/o of the
total solids and are not harmful in sewage treatment operations and require only
mechanical appliances such as screens for their removal.
Volatile solids: They are also called organic solids or ignited solids, consisting of organic
matter such as carbohydrates, fats, oils etc and constitute about 45% of the total solids
and commonly used in the analysis of wastewater sludge's to measure their biological
stability.
Procedure
a] Total solids
1. Ignite the clean evaporating dishes in the muffle furnace for 30 minutes at 550°C and
cool in a desiccator.
2. Note down the empty weight of the dish (W1).
3. Pour a measured portion (50 to 100 ml) of the well-mixed sample into the dish and
evaporate the contents by placing the dish on a steam bath.
4. Transfer the dish to an oven maintained at either 103–105°C or 179–181°C and dry it
for 1 hour.
5. Allow the dish to cool briefly in air before placing it, while still warm in a desiccator to
complete cooling in a dry atmosphere.
6. Weigh the dish as soon as it has completely cooled (W2).
7. Weight of residue = WT = (W2 – W1) mg. [W2 and W1 should be expressed in mg.]
1. Keep the same dish (WT) used for determining total residue in a muffle furnace for 1
hour at 550°C.
2. Allow the dish to partially cool in air until most of the heat has dissipated, then transfer
to a desiccator for final cooling in a dry atmosphere.
3. Weigh the dish as soon as it has cooled (W3).
4. Weight of total fixed residue = WF = (W3 – W1) mg. [W3 and W1 should be expressed
in mg.]
1. Filter a measured portion of the mixed sample (50 or 100 ml) through a Whatmann
filter paper (No 44).
2. Heat and collect the filtrate in a previously prepared and weighed evaporating dish at
103 °C for 1 to 1.5 hours in a hot air oven and weigh the residue.
3. Allow the dish to cool briefly in air before placing it, while still warm in a desiccator to
complete cooling in a dry atmosphere.
4. Weigh the dish as soon as it has completely cooled (W5).
5. Weight of total dissolved solids = WD = (W5 – W4) mg.
Where,
W4 = Weight of empty evaporating dish in mg.
W5 = Weight of empty evaporating dish in mg + Residue left after evaporating the
filtrate in mg.
1. Fill an imhoff cone (lit capacity, graduated up to 40ml) to the litre mark with a
thoroughly mixed sample.
2. Allow to settle for 45 minutes.
3. Gently stir the sides of the cone with a rod a few times to ensure that all solids
adhering to the sides is loosened.
4. Allow to settle for further 15 minutes.
5. Record the volume of settleable matter in the cone as ml/l.
Conclusion
ELECTRICAL CONDUCTIVITY
Aim
Apparatus
Reagents
1] Conductivity water
2] Standard potassium chloride solution [0.01M KCl]
Theory
Procedure
Conclusion/Result
Aim
Reagents
Theory
If water containing chlorides is titrated with silver nitrate solution, chlorides are
precipitated as white silver chloride. Potassium chromate is used as indicator, which
supplies chromate ions. As the concentration of chloride ions approaches extinction,
silver ion concentration increases to a level at which reddish brown precipitate of silver
chromate is formed indicating the end point.
Procedure
1. Pipette out 50m1 of the given water sample into a clean conical flask.
2. Add 3 drops of Potassium chromate indicator. The solution turns yellow in colour.
3. Titrate this with 0.0141 N AgNO3 to a stable reddish brown or brick red precipitate.
Observations
Calculation
Where,
A = Volume of Silver nitrate consumed.
B = Blank Correction
N = Normality of AgNO3 (0.0141)
Conclusion/Result
SULPHATE
Aim
To determine the concentration of sulphate in water by Turbidimetric method.
Apparatus
Nephelometer, Magnetic stirrer, Stopwatch, Measuring spoon 0.2 to 0.3 ml capacity,
Nessler tubes
Reagents
Barium chloride: crystals, 20-30mesh, Standard sulphate solution, Buffer solution A
and Buffer solution B: (required when the sample sulphate (SO4--) is less than 10mg/L).
Principle
The turbidimetric method of measuring sulphate is based upon the fact that Sulphate ion
is precipitated in an acetic acid medium with Barium chloride (BaCl2) so as to form
Barium sulphate (BaSO4) crystals of uniform size and that this tendency is enhanced in
presence of a sodium chloride—hydrochloric acid solution containing glycerol and other
organic compounds. The absorbance of the barium sulphate solution is measured by a
nephelometer or turbidimeter and the sulphate iron concentration, determined by
comparison of the reading with a standard curve.
Procedure
1. Take suitable volume of sample and dilute to 100mL into a 250 ml Erlenmeyer flask.
2. Add 20 ml buffer solution, mix well by placing on a magnetic stirrer.
3. Keep the flask constantly stirred with the help of stirrer. Add 1-spatula BaCl2 crystals
with stirring and begin timing immediately.
4. Continue stirring at constant speed exactly for 1 minute after addition of BaCl2
5. After stirring pour some of the solution into the absorption cell of the photometer, and
measure the turbidity at 30 second intervals for four minutes.
6. Usually maximum turbidity occurs within two minutes and the reading remains
constant thereafter for 3 to 10 minutes. So, take reading with maximum turbidity
occurring in within four minutes.
7. Prepare a calibration curve. The standards are prepared at 5 mg/l increments in the 0–
40 mg/l sulphate range and their turbidity or absorbance read alternatively using a
spectrophotometer at 420nm with a light path of 2.5 to 10 cm.
8. Absorbance versus sulphate concentration is plotted and a curve is obtained.
9. Finding the absorbance for a given sample, the concentration of sulphates in the
solution is determined with the help of calibration curve.
Observations
Sample Size Turbidity/ Absorbance
0 [BLANK]
Unknown Sample
Calculation
Conclusion/Result
ALKALINITY
Aim
Reagents
1. 0.02N H2SO4
2. Methyl orange indicator
3. Phenolphthalein indicator
4. Sodium thio sulphate 0.1 N
Theory
Procedure
Phenolphthalein Alkalinity
Observations
Calculation
Where,
A = Volume of H 2 SO 4 consumed.
N = Normality of H2SO4 (0.02)
Result:
Procedure
Total Alkalinity
1. Take 25m1 of sample (Water) in a conical flask, add 2-3 drops of Methyl-orange
indicator.
2. Titrate the contents in the conical flask with 0.02N H2SO4. The end point is pale
yellow to pale pink.
Observations
Calculation
Result:
Aim
To determine the mineral acidity and total acidity of the given water sample.
Apparatus
Burette, pipette, conical flask, measuring jar.
Reagents
Theory
Procedure
Mineral Acidity
1. Pipette out 25ml of the given water sample into a clean conical flask. Add
3 drops of methyl orange indicator to it. The solution becomes pink in colour.
2. Titrate this solution against 0.02 N NaOH solution taken in the burette till
the colour changes from pink to pale yellow.
3. Not e do wn t h e b ur et t e r e adi n g w hi ch i n di c at e s t he v ol u m e of N a OH
ru n d ow n (consumed). Repeat the experiment until at least 2 concurrent readings
are obtained.
4. The End Point is Pink to pale yellow.
Observation
Calculation
P = [A × N × 50 × 1000] / ml of sample = mg/l as CaCO3
Where,
A = Volume of NaOH consumed.
N = Normality of NaOH (0.02)
Result:
Procedure
Total Acidity
5. Pipette out 25ml of the given water sample into a clean conical flask.
7. Titrate this solution against 0.02 N NaOH solution taken in the burette till the
colour changes to pink.
8. Note down the burette reading which indicates the volume of NaOH run down
(consumed).
10. Repeat the experiment until at least 2 concurrent readings are obtained.
Observations
Calculation
Result:
pH
Aim
Reagents
Theory
Procedure
1. By pH Paper Method
Tear off a piece of pH paper from the pH paper book. Dip this paper in a test
tube filled with the given water sample. Allow this paper to dry and
develop color. Compare the colour with the standard colour printed on the
cover of the pH paper book and record the pH. This method is an
approximate method only.
1) Check the inside of the electrode of the pH Meter to ascertain whether it contains
sufficient level of saturated KOH solution. The two electrodes are always
immersed in distilled water.
2) Set the temperature to 25°C (room temperature). Switch on the pH Meter and
allow the meter to warm up for about 15 minutes.
3) Take out the electrodes from the distilled water, rinse it and clean with a tissue
paper and immerse them in standard buffer solution of say pH 4. Press the knob to
adjust it to read pH position using the calibrating knob.
4) Repeat the procedure with the second buffer solution. If the meter shows the
correct reading directly, the instrument is calibrated
5) Place the electrodes in the given water sample and record the pH directly.
Observation
HARDNESS TEST
Aim
To determine the Total, Permanent and temporary hardness of given water sample.
Apparatus
Burette, pipette, conical flask.
Reagents
Theory
Types of Hardness
Permanent hardness is caused due to the presence of sulphates, chlorides and nitrates of
calcium and magnesium. This cannot be removed by boiling but requires special
treatment such as demineralization, ion exchange etc.
Principle
A compound called Ethylene Di amine Tetra Acetic acid is made use of in this method.
In alkaline condition the Ca2+ and Mg2+ develops a wine red colour with eriochrome
black-T indicator. When EDTA is added as a titrant, the Calcium and Magnesium ions
get complexed resulting in a sharp change from wine red to blue, which indicates the end
point of the reaction.
Procedure
Total Hardness
1. Pipette out 25m1 of the given water sample into a conical flask.
2. Add 1m1 of ammonia buffer solution and 3 drops of Eriochrome black-T
indicator to it. The solution turns wine red in color.
3. Titrate this solution against 0.01 M EDTA taken in a burette till the color changes
from wine red to blue which indicates the end point.
4. Note down this burette reading.
Observation
1. Take about 50 ml of the given water sample, boil and cool it to remove temporary
hardness.
2. Transfer 25 ml of this solution into a clean conical flask.
3. Add l ml of ammonia buffer solution and 3 drops of Eriochrome black-T indicator
to it. The solution turns wine red in colour.
4. Titrate this solution against 0.01 M EDTA taken in a burette till the colour
changes from wine red to blue which indicates the end point.
5. Note down this burette reading which gives the volume of EDTA consumed.
Observation
Calculation
Conclusion
HARDNESS TEST
Aim
Reagents
Significance
Principle
When EDTA is added to water containing both Ca2+ and Mg2+, it combines with Ca2+ in
preference to Mg2+. When the pH is sufficiently high (12 to 13), Mg2+ is largely
precipated as hydroxide and Ca2+ can be determined directly using EDTA.
Procedure
Observation
Calculation
Calcium Hardness = [A × B × 1000] / ml of sample = mg/l as CaCO3
Where,
A = Volume of EDTA consumed.
B = 1 (mg of CaCO3 equivalent to 1ml of EDTA titrant)
Result
DISSOLVED OXYGEN
Aim
To determine the Dissolved Oxygen content present in the given wastewater sample.
Apparatus
BOD bottles, burette, pipette, conical flask, measuring jar.
Reagents
When MnS04 and alkali iodide azide are added to water containing DO, the oxygen
oxidizes Mn2+ to give a precipitate of Mn(OH)2. By the addition of H2SO4, free iodine is
liberated, which is converted to blue colour by the addition of starch.
Procedure
1. Fill up the BOD bottle with the given water sample up to the neck.
2. Add 2ml of MnSO4 and 2ml of alkali iodide azide solution to the BOD bottle.
Exclude the air bubbles and mix the contents of the bottle by inverting the bottle a
few times.
3. After the precipitate has settled to about 1/3rd the volume of the BOD bottle from
the bottom, add 2 ml of concentrated sulphuric acid. Once again mix the contents
gently till the suspension is completely dissolved and a uniform yellow colored
solution is obtained.
4. Take 203 ml of this solution in a conical flask and titrate with 0.025 N Na2S2O3 to
a pale straw color.
5. Add 1 ml of starch, which turns the solution blue in color. Continue the titration
till the blue color disappears. Note down the volume of Na2S203 consumed, which
gives the DO directly.
6. Hence the endpoint changes colour from Yellow to pale straw to blue to colorless.
Note
Observation
Result
Aim
To determine the 5 day Bio-Chemical oxygen demand (BOD5) of the given sewage
sample with 2% dilute solution.
Apparatus
Reagents
Theory
BOD is the amount of oxygen required to stabilize the organic matter present in
wastewater. The degree of magnitude of BOD depends on the amount of organic matter
present in the wastewater and temperature during the experiment. The rate of oxygen
demand or the process of stabilization on the organic matter is very high or rapid in the
initial stage i.e. 1-7 days. This is mainly due to the oxidation of carbonaceous matter
present in sewage. The oxidation of the rest is done due to nitrification. The time required
for complete oxidation is infinite, but it is limited to 20 days. For practical purpose the
period of oxidation is taken as 5 days. Then 70%-80% of oxidation will be completed
within first 5 days itself.
Principle
The standard test temperature is 20°C. This temperature is taken as the temperature of
natural water. There are 2 methods for determining the BOD.
1. Direct method: In this method BOD is directly calculated. This method is applicable
only for wastewater whose BOD value is < 7mg/l.
Detailed Procedure
1. Place the desired volume of distilled water in a 5 litre flask (usually about 3 litres of
distilled water will be needed for each sample).
2. Add 1ml each of phosphate buffer, magnesium sulphate solution, calcium chloride
solution and ferric chloride solution for every litre of distilled water.
3. Seed the sample with 1–2 ml of settled domestic sewage.
4. Saturate the dilution water in the flask by aerating with a supply of clean compressed
air for at least 30 minutes.
Overview of Procedure
Calculations
Observations for D1
Observations for D2
Result
Aim
To determine the COD of the given sewage sample by Open Reflux method.
Apparatus
Reflux apparatus of capacity 500m1 with a condenser, Burner or hot plate, 250 or 500mL
Erlenmeyer flask with standard (24/40) tapered glass joints, Friedrich’s reflux condenser
(12 inch) with standard (24/40) tapered glass joints, Volumetric pipettes (10, 25, and
50ml capacity), Burette, 50ml with 0.1ml accuracy, Burette stand and clamp, Analytical
balance, accuracy 0.001gm, Spatula, Volumetric flasks (1000ml capacity), glass beads.
Reagents
0.25N Std Potassium-dichromate (0.04167 M), Conc. Sulphuric acid reagent, (0.25M)
Std Ferrous ammonium sulphate , Ferroin indicator, Mercuric sulphate crystals, analytical
grade and Standard Potassium hydrogen phthalate (KHP)
Principle
The organic matter gets oxidized completely by K2Cr2O7 in the presence of H2SO4 to
produce CO2 and H2O. The excess K2Cr2O7 remaining after the reaction is treated with
Ferrous Ammonium Sulphate .The dichromate consumed gives the oxygen required for
oxidation of the organic matter.
Procedure
Standardisation
Dilute 10 ml standard K2Cr2O7 to about 100 ml, add 30 ml conc.H2SO4, cool. Add 2
drops of ferroin indicator and titrate with FAS.
Estimation
Place 0.4 gm HgSO4 in a reflux flask. Add 20 m1 sample or an aliquot of sample diluted
to 20m1 with distilled water. Mix well and add glass beads followed by 10 m1 Std
K2Cr2O7. Add slowly 30 m1 H2SO4 containing Ag2SO4, mixing thoroughly. Connect the
flask to the condenser and mix the contents before heating. Reflux for a minimum period
of 2 hours, cool and wash down the condenser with distilled water. Dilute to about 150
m1, cool and titrate excess K2Cr2O7 with 0.1 N FAS using Ferroin indicator. Sharp colour
change from blue green to wine red indicates the end point. Reflux the blank in the same
manner using distilled water instead of the sample.
Calculation
Observation
Result
AVAILABLE CHLORINE
Aim
To determine the Percentage of Chlorine in Bleaching Powder.
Apparatus
Conical flask, burette, pipette, measuring jar, funnel.
Reagents
Bleaching powder, Potassium Iodide (KI), Glacial Acetic acid, Starch solution and 0.1 N
Sodium thio sulphate
Theory
Chlorine a strong oxidizing agent and a very effective disinfectant used for the
destruction of pathogenic bacteria. Chlorine is generally applied in the form of bleaching
powder for disinfection of water. Commercial bleaching powders generally contain 25 to
30% of available chlorine. This percentage is a very critical factor for effective
disinfection of water.
Procedure
1. Weigh 1gm of fresh bleaching powder and make it into a paste by adding a small
quantity of distilled water.
2. Add some more water, stir it and allow it to settle for a few minutes. Decant the
supernatant solution and dilute to 1000 ml with distilled water. This is called
chlorine water or standard chlorine solution
3. Pipette out 25 ml of this chlorine water into a conical flask. Add 1 pinch of KI
crystals and 5ml of glacial acetic acid. Wait for 15-20 minutes (contact period)
4. Titrate this solution with 0.025 N Sodium thio sulphate. The solution turns from
deep yellow to pale yellow.
5. Add 2 drops of starch solution. The solution becomes blue in color.
6. Continue the titration till the blue color just disappears. Note down the volume of
Sodium thio sulphate used.
Observation
Calculation
Percentage of chlorine in the given bleaching powder = [(V × N × 35.46 × 1000) / 25]
Note
Result
RESIDUAL CHLORINE
Aim
To determine the residual chlorine present in the given sample of water by Iodometric
method.
Apparatus
Conical flask, burette, pipette, measuring jar, funnel, beakers.
Reagents
0.1N Sodium thio sulphate, Glacial Acetic acid, KI solution and Starch solution indicator.
Principle
Chlorine present in water reacts with KI and iodine is liberated, when starch is used as
indicator, the presence of blue color produced indicates the presence of Iodine.
Procedure
Chlorine in aqueous solution is not stable, and the chlorine content of samples or
solutions, particularly weak solutions, will decrease rapidly. Exposure to sunlight or other
strong light or agitation will accelerate the reduction of chlorine. Therefore, chlorine
determination should be done immediately after sampling, avoiding excessive light and
agitation. The sample should not be stored for analyses of residual chlorine.
Observation
Calculation
Amount of residual chlorine = [V x N x 35.46 x 1000] / ml of sample = mg/l.
Where
V = volume of sodium thio sulphate run down.
N = Normality of sodium thio sulphate.
Result
CHLORINE DEMAND
Aim
Reagents
0.01N Sodium thio sulphate, KI solution, Glacial acetic acid and Starch solution
Theory
Chlorine is used as a disinfectant in water. The chlorine demand of water is the difference
between the amount of chlorine applied and the amount of free chlorine left after the
contact period as residue. The optimum dose of chlorine for given water is therefore
generally determined experimentally by adding varying amounts of chlorine to the given
sample and observing the residual chlorine left after a contact period of about 20 minutes.
The dose, which leaves a residual of 0.2 mg/l, is then selected. This total dose minus the
free residual will automatically represent the chlorine demand of water.
Procedure
1. Take 6 clean BOD bottles and add 200m1 of homogenous water sample to each.
2. Add 0, 2, 4, 6, 8 ml of chlorinated water (known strength) to each bottle respectively.
3. Put stopper and Shake well. Allow it to remain so for about 20 minutes. Chlorine
reacts with the organic matter and oxidizes it and also kills the bacteria present in the
sample
4. After contact period, add 1gm KI and 5ml glacial acetic acid to each of these bottles.
5. Titrate this solution with 0.025 N Sodium thio sulphate. The solution turns from deep
yellow to pale yellow.
6. Add 2 to 3 drops starch indicator. The appearance of blue color indicates the presence
of chlorine.
7. To estimate the amount of chlorine left in the sample as residue, it can be titrated
against 0.01 N Na2S203 till the end point is reached, which is indicated by the
disappearance of blue color.
8. The chlorine demand is given by the difference between the applied Chlorine and the
residual chlorine after the contact period.
Calculation
Sl.No BOD ml of chlorine mg of chlorine Residual Chlorine
bottle No added added to 1000 chlorine Demand [mg/l]
ml [mg/l]
Result
TURBIDITY
Aim
Apparatus
Nephelo turbiditymeter.
Reagents
Determine turbidity on the day the sample is taken. It storage is unavoidable, store the
samples in dark for up to 24 hr. Prolonged storage before measurement is not
recommended because irreversible changes in turbidity may occur. All samples should be
shaken vigorously before examination.
Theory
Suspension of particles in water interfering with the passage of light is called turbidity.
Turbid water is undesirable from aesthetic point of view in drinking water supplies and
may also affect products in industries. It also causes a number of problems in water
treatment plants. The permissible limits for drinking water is 5 to 10 ppm, measured on a
silica scale
Principle
When light is passed through a sample having suspended particles, some light is scattered
by the particles. The scattering of light is proportional to the turbidity.
Procedure
1. Switch on the Nephelo Meter and wait for 10 to 15 minutes for the instrument to warm
up.
2. Fill up the sample holder with distilled water, place it in the instrument & Close the
cover. Using the zero adjustment knob, adjust the reading to show zero.
3. Take out the distilled water sample and fill up the holder with the standard solution of
500 NTU from the already prepared standard stock solution. Place it in the instrument
and close the cover. Turn on the range switch to 1000 and adjust the instrument to read
50 (500 NTU) using the calibrating knob. Repeat the procedure with other standard
solutions to calibrate the instrument.
4. To know the turbidity of the given unknown sample, Keep it in the instrument and
record the turbidity reading directly in NTU.
Note
Strength Dilution
Result
Apparatus
Glass beakers, Jar test apparatus, Nephelometer, Pipette, pH meter.
Reagents
Alum
Theory
The public water supplies consists of very fine particles of clay, silt etc which are present
in a finely divided state and it is not possible to detain them in primary sedimentation
tanks unless they are designed for longer detention periods. The other way to remove
them is to increase their size so that they become settleable, by adding certain chemicals
called coagulants, which react with impurities in water and convert them into settleable
size. Some common coagulants used are Alum, Sodium aluminate, ferric chloride,
copperas, chlorinated copperas, ferric sulphate etc.
Procedure
Note
Strength of solution. 1 gm = 100m1
Observation
Jar ml of mg of Concentration
Description of
No coagulant coagulant mg/l
the sample added added
Conclusion
IRON
Aim
Apparatus
Nessler's tubes (100 ml), Conical flasks, Pipettes, Colorimeter.
Reagents
Principle
In the Phenanthroline method, the ferric form of iron is reduced to ferrous form by
boiling with hydrochloric acid and hydroxylamine hydrochloride. The reduced iron
chelates with 1,10 phenanthroline at pH 3.2 - 33 to form a complex of orange-red color.
The intensity of color is proportional to the concentration of iron and obeys Beer's law.
Procedure
1. Pipette 10, 20, 30 and 50m1 of standard iron solution into 100m1 conical flask
2. Add l ml of Hydroxyl amine hydrochloride solution and 1m1 of sodium acetate
solution to each flask.
3. Dilute each to about 75m1 with distilled water and Add 2ml of 1,10-phenanthroline
solution to each flask.
4. Make up the contents of each flask exactly to 100m1 by adding distilled water and
allow atleast 10-15 minutes for maximum color development.
5. For visual comparison pour the solution in 1.00 m1 Nessler tubes and keep them in a
stand. Mix the sample thoroughly and measure 50ml into a conical flask
8. Add 2 ml of concentrated HCl and 1 ml of Folderol amine solution and a few glass
beads. Boil the contents to half of the volume for dissolution of all the iron
9. Cool the flask to room temperature and transfer the solution to a 100 m1 Nessler tube
10. Add 10m1 of Ammonium acetate buffer solution and 2m1 Phenanthroline solution
and dilute to 100m1 mark with distilled water. If the sample contains interference of
heavy metals add 10m1 of Phenanthroline instead of 2m1
11. Mix thoroughly and allow atleast 10-15 minutes for maximum color development
12. For visual comparison, match the color of sample with that of the standards prepared
in steps 1-6 above
13. The matching color standard will give the concentration of iron in the sample
For photometric measurement, use light path at 510nm. Read standards against distilled
water set at zero absorbance and plot a calibration curve, including a blank. If samples
are coloured or turbid, carry a second set of samples through all steps of the procedure
without adding Phenanthroline.
Comments
Environmental Significance
Long time consumption of drinking water with a high concentration of iron can lead to
liver diseases. Iron rich water exposed to the air becomes turbid and highly unacceptable
from the aesthetic view point.
FLUORIDE
Aim
Apparatus
Distillation apparatus, Colorimetric, Nessler tubes [capacity-100ml], Pipette
Reagents
Polyethylene bottles are preferred for collecting and storing samples for fluoride analysis.
Glass bottles are satisfactory, provided that they have not previously contained high-
fluoride solutions. Always rinse the bottle with a portion of the sample.
Principle
This method is used for estimation of fluoride in natural water in the concentration range
0-1.4 mg/l. The test is based on the fact that fluoride ion combines with zirconium on to
form a stable complex ion, ZrF6 and this results in bleaching the reddish colour of
Zirconium and alizarin combination. The decrease in intensity of colour is directly
proportional to fluoride concentration.
Procedure
1. The sample should be free from chlorine, if chlorine present, it shall be dechlorinated
with a slight excess of sodium thio sulphate solution before use.
2. If the sample contains excess interfering ions, the sample should be appropriately
diluted and distillated before test.
3. Take 0, 1, 2, 6, 8, 14 ml of Standard sodium fluoride solution in six Nessler tubes.
[The range should be such that it is between 0 and 1.4 mg/l]
4. Add 5m1 of acid zirconium reagent in each Nessler tube.
5. Similarly add 5m1 of acid zirconium reagent into the Nessler tubes containing 100m1
of sample.
6. Mix thoroughly and compare the colors after standing for one hour.
Comments
Environmental Significance
Fluoride ions have dual significant in water supplies. High concentration of F- causes
dental fluorosis (disfigurement of the teeth) and skeletal fluorosis. At the same time, a
concentration less than 0.8mg/l results in ‘dental caries’. Hence, it is essential to maintain
the F- concentration between 0.8 to 1.0mg/l in drinking water.
Aim
To determine the most probable number (MPN) index of coliforms and E-coli organisms
in the given water sample by multiple tube fermentation tube technique.
Apparatus
Incubator, fermentation tubes, test tubes, EMB agar plate, Petri-dishes, microscopes,
Autoclave, Pipettes, Measuring jars, Inoculating equipments [platinum loop], Media
preparation utensils.
Reagents
Theory
Coliform group comprises of all the aerobic, facultative and anaerobic gram-negative
non-spore forming rod shaped bacteria that ferment lactose with gas formation within 48
hours at 35°C. The standard test for this group may be carried out either by multiple tube
fermentation technique or by membrane filter technique. Escherichia coli (E.coli) is used
as an indicator organism for the purpose of sanitary examination of water. The E.coli test
by multiple tube fermentation technique consists of 3 phases – presumptive, confirmed
and completed. The results are expressed in terms of MPN (Most Probable Number),
which is based on certain probability formulae. The estimate may give a value greater
than the actual number of coliform present. The accuracy of any single test depends on
the number of tubes fermented. This method helps in describing the sanitary quality of
water. The safety of the water is generally judged from the knowledge of sanitary
condition and mentioned by the number of samples yielding positive or negative results.
If more than 95% should yield negative results, the safety is usually assured.
Procedure
Observations
Computation of MPN
The number of positive finding of coliform group organisms resulting from the multiple
portion decimal dilution planting should be computed as the combination of positives and
recorded in terms of the Most Probable Number (MPN). The MPN may be estimated by
Thomas’ simple formula:
Result : MPN/100 ml =
Conclusion
NITRATES
Aim
To determine the concentration of Nitrate Nitrogen present in the given sample of given
water sample.
Apparatus
Reagents
Principle
Nitrate reacts with Phenol di sulphonic acid and produces a nitro-derivative which in
alkaline medium develops a yellow color. The color produced follows the Beer's law and
is directly proportional to the concentration of nitrate present in the sample.
Procedure
Interferences
Chlorides and nitrite are the two main sources of interferences. Pretreatment of sample is
necessary when the interfering radicals are present.
Result:
Conclusion:
Environmental Significance
In surface waters, nitrate may cause objectionable eutrophication. Nitrates are poisonous
to infants, animals including humans. In humans it can cause 'methemoglobinemia'.
SODIUM
Aim
To determine the concentration of Sodium in given water sample by Flame Photometer.
Apparatus
Flame photometer
Reagents
Stock sodium solution, Intermediate sodium solution, Standard sodium solution
Storage
Store samples containing low sodium concentrations or alkaline samples in polyethylene
bottles to eliminate the possibility of sample contamination due to leaching of the glass
container.
Principle of Quantification
Trace amounts of sodium can be determined by flame emission photometry at the
wavelength of 589 nm. The sample is sprayed into a gas flame and excitation is carried
out under carefully controlled and reproducible conditions. The calibration curve may be
linear but has a tendency to levels off at higher concentrations.
Procedure
Calculation
Where,
Observations
Result
POTASSIUM
Aim
Reagents
Stock Potassium solution, Intermediate potassium solution, Standard potassium solution
Storage
Do not store samples in soft-glass bottles because of the possibility of contamination
from leaching of the glass. Use acid washed polyethylene or borosilicate glass bottles.
Adjust sample to pH <2 with nitric acid. This will dissolve potassium salts and reduce
adsorption on vessel walls.
Principle of Quantification
Trace amounts of potassium can be determined in either a direct-reading of internal
standard type of flame photometer at a wavelength of 766.5 nm. The sample is sprayed
into a gas flame and excitation is carried out under carefully controlled and reproducible
conditions. The calibration curve may be linear but has a tendency to levels off at higher
concentrations.
Procedure
1. Follow instructions of flame photometer manufacturer for selecting proper photocell
wavelength, slit width adjustments, fuel gas and air pressure, steps for warm up,
correcting for interference and flame background, rinsing of burner, sample ignition
and emission intensity measurements.
2. Prepare a blank and potassium calibration standards, in any of the applicable ranges, 0-
100, 0-10, or 0-1 mg/l. Calibrate instrument, set instrument to zero absorbance with
blank solution containing no potassium.
3. Measure emission at 766.5 nm and prepare calibration curve.
4. Determine potassium concentration of the sample, or diluted sample, from the curve.
Calculation
mg K/l = mg K/l [from the calibration curve] x Dilution
Where,
Observations
Sample Size (ml) Absorbance
0 [BLANK]
Unknown Sample
Result