Indian Institute of Technology Delhi

Lab Handouts

pH

Aim:
To determine the pH of the given sample.
Theory:
pH of aqueous solutions can be defined as negative logarithm of hydrogen ion concentration. pH values
ranging from 0 to 7 are acidic, and from 7 to 14 are alkaline. The effect of pH on the chemical and
biological properties of liquids makes its determination very important. It is used in several calculations
in analytical work. The pH determination is usually done by electrometric method which is the most
accurate method and free of interference.
Environmental Significance:
The pH is one of the basic water and wastewater characteristics. It expresses the intensity of acid or
alkaline conditions by indicating the hydrogen ion activity. Some of the processes in water quality
engineering that require pH monitoring and control are the following: disinfection, coagulation,
softening, biological treatment etc. Natural waters usually have pH values close to neutral.
Determination of pH is one of the important objectives in biological treatment of the wastewater. In
anaerobic treatment, if the pH goes below 5 due to excess accumulation of acids, the process is severely
affected. Shifting of pH beyond 5 to 10 upsets the aerobic treatment of the wastewater. In these
circumstances, the pH is generally adjusted by addition of suitable acid or alkali to optimize the
treatment of the wastewater. pH value or range is of immense importance for any chemical reaction. A
chemical shall be highly effective at a particular pH. Chemical coagulation, disinfection, water softening
and corrosion control are governed by pH adjustment. Dewatering of sludges, oxidation of cynides and
reduction of hexavalent chromium into trivalent chromium also need a favorable pH range. It is used in
the calculation of carbonate, bicarbonate, CO2 corrosion, stability index and acid base equilibrium.
Lower value of pH below 4 will produce sour taste and higher value above 8.5 a bitter taste. Higher
values of pH hasten the scale formation in water heating apparatus and also reduce the germicidal
potential of chlorine. High pH induces the formation of trihalomethanes, which are causing cancer in
human beings.
Standard:
pH of drinking water should be between 6.5 and 8.5
Requirements:
pH Meter, Flask, Magnetic Stirrer, Funnel, Beaker, Wash Bottle
Electrometric Method:
The pH is determined by measurement of the electromotive force of a cell comprising an indicator
electrode (an electrode responsive to hydrogen ions such as glass electrode) immersed in the test solution
and a reference electrode or a combined electrode. The contact between the test solution and the
reference electrode is usually achieved by means of a liquid junction. The emf of this cell is measured
with a pH meter. This is a high impedance electrometer calibrated in terms of pH. Reagents: Calibrates
the electrode system against standard buffer solution of known pH. Buffer tablets having pH 4.0, 7.0
and 10 are available.
Reagents:
pH 4, pH7, pH 10 Buffer
Solutions:
Dissolve buffer tablet of pH 4 in 100 ml of distilled water to get pH 4 buffer solution. Similarly other
buffer solutions can be prepared by dissolving corresponding buffer tablets.
Procedure:
1. Prepare buffers of pH 4, 7 and 10.
2. Rinse the electrode thoroughly with distilled water.
3. Standardize pH meter electrode with any two of these buffers.
4. Dip the electrode in the sample for test.
5. Let the reading on screen stabilize; note it as pH & temperature of your sample.
Results:
Sample pH Temperature(0C)
 Turbidity

Aim: To determine the turbidity of a given water sample

Introduction: Turbidity is the technical term referring to the cloudiness of a solution and it is a
qualitative characteristic which is imparted by solid particles obstructing the transmittance of light
through a water sample. Turbidity often indicates the presence of dispersed and suspended solids like
clay, organic matter, silt, algae and other microorganisms. So in short turbidity is an expression of the
optical property that causes light to be scattered and absorbed rather than transmitted in straight lines
through the sample

Environmental significance: The filter may go out of operation, if excess turbidity exists. Knowledge
of the turbidity variation in raw water supplies is useful to determine whether a supply requires special
treatment by chemical coagulation and filtration before it may be used for a public water supply.
Turbidity measurements are used to determine the effectiveness of treatment produced with different
chemicals and the dosages needed. Turbidity measurements help to gauge the amount of chemicals
needed from day-to-day operation of water treatment works. Measurement of turbidity in settled water
prior to filtration is useful in controlling chemical dosages so as to prevent excessive loading of rapid
sand filters. Turbidity measurements of the filtered water are needed to check on faulty filter operation.
Turbidity measurements are useful to determine the optimum dosage of coagulants to treat domestic and
industrial wastewaters. Turbidity determination is used to evaluate the performance of water treatment
plants.
Apparatus: Nephelometric Turbidity Meter
Chemical: Formazin Polymer standards
Procedure:
1. For testing the given water sample first the reagents are to be prepared. Then the turbidity meter is
required to be calibrated.
2. To the sample cells, add sample water up to the horizontal mark, wipe gently with soft tissue and
place it in the turbidity meter. Cover the sample cell with the light shield.
3. Check for the reading in the turbidity meter. Wait until you get a stable reading.
Observation Table:

Sample No. Turbidity (NTU)

 Conductivity

Aim: To determine the conductivity of given water sample

Introduction: Conductivity of a substance is defined as 'the ability or power to conduct or transmit heat,
electricity or sound'. When an electrical potential difference is placed across a conductor, its movable
charges flow, giving rise to an electric current. This property is called conductivity. Since the charge on
ions in solution facilitates the conductance of electrical current, the conductivity of a solution is
proportional to its ion concentration.
Environmental significance:
• Electrical conductivity measurements are often employed to monitor desalination
plants. It is useful to assess the source of pollution.

• In coastal regions, conductivity data can be used to decide the extent of intrusion
of sea water into ground water.

• Conductivity data is useful in determining the suitability of water and wastewater

for disposal on land. Irrigation waters up to 2 millisiemens / cm conductance have
been found to be suitable for irrigation depending on soils and climatic
characteristics.

• It is also used indirectly to fine out inorganic dissolved solids.

Apparatus: Conductivity Meter with Electrode

Procedure:

1. Before starting the experiment, switch on the instrument for at least 30 minutes, so that the
instrument stabilizes.
2. The Mode button is pressed to select the parameter to be measured. The Display can show
conductivity reading in microsiemens or in millisiemens.

3. Rinse the electrode thoroughly with deionized water and carefully wipe with a tissue
paper.
4. Measure 200 mL of water sample and transfer it to a beaker.
5. Dip the electrode into the sample solution taken in a beaker and wait for a steady reading.
Make sure that the instrument is giving stable reading.
6. Note down the reading in the display directly, which is expressed in microsiemens
or millisiemens
Observation Table:

Sample No. Conductivity (µS/cm)

 DETERMINATION OF SOLIDS

Total Suspended Solids

Aim:
To determine the Total Suspended Solids (TSS) present in water sample & wastewater.
Theory:
The term TSS applies to the dry weight of the material that is removed from a measured volume of water
sample by filtration through a standard filter. To achieve reproducibility and comparability of the results
close attention to procedural details, especially filter characteristics and time and temperature of drying
is required.
Environmental Significance:
• TSS can cut down light transmission through the water and so lower the rate of photosynthesis
in aquatic flora.
• In less turbulent parts of the river, some of the solids may sediment out, smothering life of the
riverbed.
• The TSS determination is extremely valuable in the analysis of polluted water.
• It is a major parameter used to evaluate the strength of domestic wastewater and to determine
the efficiency of treatment units.
Apparatus:
Suction Flask, Filtration Apparatus, Aluminum Weighing Dishes, Desiccator, Beakers, Weighing
Balance, Hot Air Oven, Filter Paper, Graduated Cylinder etc.
Procedure:
1. Measure the empty weight of filter paper and note down reading (Wi).
2. Mix Sample well and pour into a graduated cylinder to the selected volume (V).
3. Apply suction to filter flask and seat filter with a small amount of distilled water.
4. Pour selected volume into filtration apparatus.
5. Wash with 3-successive 10-mL volume of distilled water, allowing complete drainage between
washing, and continue suction for about 3 minutes after filtration is completed.
6. Carefully remove the filter paper from the filtration apparatus and transfer it to a weighing dish as a
support.
7. Dry for at least 1 hour at 103-105°C in a hot air oven.
8. Cool in desiccator and weigh filter paper and record the reading (Wf)
9. Repeat cycle of drying, cooling, desiccating and weighing until a constant weight is obtained, or
weight change is less than 4% of previous weight or 0.5 mg, whichever is less. Duplicate determination
should be within 5% of their average
Observation

Initial Weight of Filter Paper in gm, (Wi) =

Final Weight of Filter Paper in gm, (Wf) =
Volume of Sample in ml, (V) =
Calculation
𝑊𝑖−𝑊𝑓×1000000
Suspended solids in mg/L = 𝑉

Result
TSS of given sample =
Total dissolved solids
Aim:
To determine the Dissolved solids, present in water sample & waste water.
Theory:
A well-mixed, measured portion of sample is filtered through a standard glass fibre filter and the filtrate
portion is evaporated to dryness at 180 ± 2 °C and that gives the amount of total dissolved solids. The
reason for higher temperature used is to remove all mechanically occluded water. Where organic matter
is generally very low in concentration, the losses due to higher drying temperature will be negligible.
Environmental Significance:
• A high content of dissolved solids elevates the density of water, influences osmoregulation of
freshwater organisms, reduces solubility of gases (like oxygen) and reduces utility of water for
drinking, irrigation and industrial purposes.
• TDS concentration beyond 500 mg/L, decreases palatability and may cause gastrointestinal
irritation.
Apparatus:
Suction Flask, Filtration Apparatus, porcelain dish, Desiccator, Beakers, Weighing Balance, Hot Air
Oven, Filter Paper, Graduated Cylinder etc.
Procedure:
1. Take a clean porcelain dish/beaker which has been washed and dried in a hot air oven at 103-105 °C
for one hour.
2. Now weigh the empty porcelain dish/beaker in analytical balance (Wi)
3. Mix sample well and pour into a funnel with filter paper. Filter a known volume of a well-mixed
sample in the above beaker. (i.e. 100 mL, 200 mL or 500 mL) (V)
4. Note: Choose the sample volume to yield 10 to 200 mg of dried residue. If more than 10 minutes are
required for complete filtration, decrease the sample volume.
5. Evaporate the sample in a hot air oven at 180 ± 2 °C after the whole water is evaporated, cool the
evaporating dish/beaker in a desiccator and take the final weight (Wf).
6. Total solids = (Suspended solids) + (Dissolved solids).
Observations:
1. Initial Weight of crucible in gm, (Wi)
2. Final Weight of crucible in gm, (Wf)
3. Total Weight of crucible in gm, (W)
4. Volume of Sample in ml, (V)
Calculations:
𝑊𝑖−𝑊𝑓×1000000
Total Dissolved solids in mg/L = 𝑉

Results
TDS of given sample =
Total solids = TSS + TDS =
Volatile suspended solids & fixed suspended solids
Aim
To determine Volatile suspended solids & fixed suspended solids present in a given water sample
Procedure
1. Weigh filters (mass = B g).
2. Filter samples (50 ml).
3. Run each sample in duplicates. You will have a total of 4 samples.
4. Oven dry at 103o C for 30 min (please note that standard methods recommend 1 hour). At this stage
all water will be evaporated and only suspended solids will be retained on the filter. Weigh filters now
(mass =A g).
5. Calculate concentration of total suspended solids :
mg total suspended solids =1000*(A-B)/(sample volume in mL)
6. Weight crucibles (mass=C) and then weight crucible and filter (total mass=E).
7. Put crucibles with filter paper in muffle furnace and Ignite at 550o C for 15 min. At this stage all
volatile components of solids will be volatilized and only fixed inert solid materials will be left in
crucible. Weigh crucible after proper cooling (mass=D g).
8. Calculate concentration of volatile suspended solids (F) : mg volatile suspended solids/L =1000*(E-
D)/(sample volume in mL)
9. Calculate concentration of fixed suspended solids (G) : mg volatile suspended solids/L =1000*(D-
C)/(sample volume in mL)

Result
Volatile suspended solids present in a given sample =
Fixed suspended solids present in a given sample =
 Acidity and Alkalinity

Aim: To determine acidity and alkalinity of the given water sample by Titration Method.

Theory: Acidity is the capacity of water to neutralize bases. Acidity of a sample might be thought of
as the amount of strong acid present in a weak acid or weak acid salt solution. In similar manner,
alkalinity can be defined as the capacity of water to neutralize acids. Alkalinitycan be conceptualized as
the amount of strong base present in a weak acid or weak acid salt solution. Alkalinity and acidity
related to water and wastewater treatment are normally based on the carbonic acid system. Acidity and
alkalinity measurements can be correlated with the three equivalence points associated with carbonic
acid system.

1. Alkalinity
Alkalinity measured to the phenolphthalein end point around pH 8.3 (bicarbonate equivalencepoint) is
known as phenolphthalein alkalinity. If the titration is continued till the pH of sampleis lowered to 4.5
(carbonic acid equivalent point), the amount of acid required to lower the pHof sample represents total
alkalinity. If initial pH of the sample is below carbonic acid equivalence point, no alkalinity is present
in the sample.

2. Acidity
Acidity measured to the methyl orange end point (carbonic acid equivalence point) is termed as mineral
acidity. Total acidity can be measured if titration of the sample is continued to the phenolphthalein end
point. If the initial pH of the sample is above carbonate equivalence point,there is no acidity in the
system.

Procedure:

1. Alkalinity
• Measure the initial pH of the sample.
• Take 50 mL of sample in conical flask. Add 2-3 drops of phenolphthalein indicator. (Ifno pink
color appears, add methyl orange indicator.)
• Titrate against 0.02 N H2SO4 till pink color disappears. Note down the burette reading in mL
(V1).
• Add 2-3 drops of methyl orange indicator. Continue titration till faint orange color changes to
yellow. Note down the final burette reading in mL (V2). Measure the final pH of the sample.
𝑃ℎ𝑒𝑛𝑜𝑙𝑝ℎ𝑡ℎ𝑎𝑙𝑒𝑖𝑛 𝑎𝑙𝑘𝑎𝑙𝑖𝑛𝑖𝑡𝑦 (𝑚𝑔𝐿 𝑎𝑠 𝐶𝑎𝐶𝑂3) = 𝑉1×𝑁×50×1000
Sample volume in ml

𝑉2 × 𝑁 × 50 × 1000
𝑇𝑜𝑡𝑎𝑙 𝑎𝑙𝑘𝑎𝑙𝑖𝑛𝑖𝑡𝑦 (𝑚𝑔⁄𝐿 𝑎𝑠 𝐶𝑎𝐶𝑂3) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑚𝐿
where, N is the normality of the titrant.
 • Find out hydroxide, carbonate and bicarbonate alkalinity in the sample. Refer the tablebelow.
Result of Hydroxide Carbonate Bicarbonate
Titration Alkalinity Alkalinity Alkalinity
P=0 0 0 T(initial pH < 8.3)
P=0.5T 0 2P 0
P=T T 0 0
P<0.5T 0 2P T – 2P
P>0.5T 2P - T 2(T-P) 0

2. Acidity
• Measure the initial pH of the sample.
• Take 50 mL of sample in conical flask. Add 2-3 drops of methyl orange indicator.
• Titrate against 0.02 N NaOH till faint orange color develops. Note down the burettereading
in mL (V1).
• Add 2-3 drops of phenolphthalein indicator. Continue titration till pink color appears.Note
down the final burette reading in mL (V2). Measure the final pH of the sample.
𝑉1 × 𝑁 × 50 × 1000
𝑀𝑖𝑛𝑒𝑟𝑎𝑙 𝑎𝑐𝑖𝑑𝑖𝑡𝑦 (𝑚𝑔⁄𝐿 𝑎𝑠 𝐶𝑎𝐶𝑂3 ) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑚𝐿

𝑉2 × 𝑁 × 50 × 1000
𝑇𝑜𝑡𝑎𝑙 𝑎𝑐𝑖𝑑𝑖𝑡𝑦 (𝑚𝑔⁄𝐿 𝑎𝑠 𝐶𝑎𝐶𝑂3 ) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑚𝐿

where, N is the normality of the titrant.

 Biochemical Oxygen Demand

Theory: BOD test measures the amount of oxygen utilized during a specified incubation periodfor the
biochemical degradation of organic material. Dissolved oxygen (DO) in the sample is measured
initially and after incubation, and the BOD is computed from the difference betweeninitial and final
DO. Winkler or iodometric method is the most precise and reliable titrimetric procedure for DO
analysis. In this method, divalent manganese solution is added to the sample,followed by strong alkali.
DO in the sample oxidizes the manganese (II) to manganese (IV). The oxidized manganese is reduced
to divalent state in the presence of iodide ions in an acidicsolution. During this step, iodine is liberated
and the amount of iodine formed is equivalent tothe DO consumed. This liberated iodine is then titrated
with a standard solution of sodium thiosulfate and starch solution is used as the indicator.

Procedure:

• Prepare dilution water by aerating the distilled water. Add 2 mL/L of phosphate buffer,
magnesium sulfate, calcium chloride and ferric chloride solutions to the dilution water.
• Prepare 2, 5 and 7.5% dilutions of the given sample using dilution water. Avoid the entry of air
during dilution.
• Fill BOD bottles with the diluted samples. Prevent turbulence and formation of bubbleswhile
filling and allow the bottle to overflow for some time. Place the stopper and ensure water seal.
• Prepare two sets of each dilution (for 0-day and 5-day DO measurement) and two blank(only
dilution water) BOD bottles.
• Measure the DO in all 0-day BOD bottles.
• Store 5-day BOD bottles in the incubator at 20 ºC. Measure the DO in these bottles after5 days.
Determination of DO in the sample
• Take the BOD bottle to be analyzed for DO. Add 2 mL of manganous sulfate solution,followed
by 2 mL of alkali-iodide-azide reagent. While adding the reagent, dip the pipette tip below the
water surface. Care should be taken to eliminate the entry of air bubbles. Place the stopper
immediately and mix well for 20s by inverting the bottle.
• Allow the precipitate to settle. Formation of brown precipitate indicates the presence ofDO in
the sample. If there is no DO in the sample, white precipitate will be formed.
• When the precipitate has settled down to approximately half the bottle volume, add 2 mL of
conc. H2SO4 to the bottle.
• Close the bottle and mix the contents by inverting the bottle till the precipitate is completely
dissolved.
• Transfer 203 mL of sample from the BOD bottle to a conical flask. Titrate the sample with
0.025 N sodium thiosulfate solution till the color of sample turns to pale straw color.
• Add a few drops of starch indicator and the solution will turn into blue color. Continueto titrate
till the solution becomes colorless. Note down the volume of titrant consumed.

Dissolved oxygen in the sample, mg/L = mL of 0.025N sodium thiosulfate consumed

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐷𝑂 − 𝐹𝑖𝑛𝑎𝑙 𝐷𝑂
𝐵𝑂𝐷, 𝑚𝑔⁄𝐿 =
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟

𝑚𝐿 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 = 300
 Chemical Oxygen Demand

Theory: Chemical oxygen demand (COD) is defined as the amount of a specified oxidant thatreacts
with the sample under controlled conditions. COD often is used as a measurement of pollutants in
wastewater and natural waters. Because of its unique chemical properties, the dichromate ion

(Cr2O72–) is the specified oxidant and it is reduced to the chromic ion (Cr3+) inthese tests. Both organic
and inorganic components of a sample are subject to oxidation, but inmost cases the organic component
predominates and is of the greater interest. COD is a definedtest; the extent of sample oxidation can be
affected by digestion time, reagent strength, and sample COD concentration.

Procedure:

1. Homogenize the sample. Add 2.5 mL of sample (or diluted sample) in the tube and add
1.5 mL of digestion solution.
2. Carefully run 3.5 mL of sulfuric acid reagent down inside of vessel so an acid layer is
formed under the sample-digestion solution layer and tightly cap tubes, and invert each
several times to mix completely.
3. Repeat the same for blank also.
4. Place tubes in block digester preheated to 150°C and reflux for 2 h.
5. Cool to room temperature. Transfer contents to a conical flask for titrating.
6. Add 1-2 drops of Ferroin indicator and titrate with 0.25 N FAS solution.
7. The end point is a sharp color change from blue-green to reddish brown, although the
blue-green may reappear within minutes.
8. Titrate the blank sample in the same manner.
(𝐴 − 𝐵) × 𝑁 × 8000
𝐶𝑂𝐷 𝑎𝑠 𝑚𝑔 𝑂2⁄𝐿 =
𝑚𝐿 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

A- mL FAS used for blank

B- mL FAS used for
sample

N- Normality of FAS
 Chloride

Aim: Determination of chloride ions in water and wastewater samples using Argentometric Method

Principle:

In Mohr method, the sample is titrated with silver nitrate solution and chloride ion is precipitated as
white silver chloride.

𝐴𝑔+ + 𝐶𝑙− ⇋ 𝐴𝑔𝐶𝑙(𝑠), 𝐾𝑠𝑝 = 3 × 10−10

In order to visualize the end point, potassium chromate is used as indicator. When all the chloride ions
are precipitated as silver chloride, the excess silver ions and chromate ions supplied by indicator form
silver chromate. When the solubility product of silver chromate is exceeded, it forms a reddish brown
precipitate.

2𝐴𝑔+ + 𝐶𝑟𝑂4 2− ⇋ 𝐴𝑔2𝐶𝑟𝑂4(𝑠), 𝐾𝑠𝑝 = 5 × 10−12

Apparatus: Burette; pipettes; volumetric flask; conical flasks; measuring cylinders.

Procedure:

• Measure the initial pH of the sample.

• Take 50 mL of sample in conical flask. Add 1 mL of K2CrO4 indicator solution.
• Titrate with 0.0141 N AgNO3 titrant till the solution turns pinkish yellow. Note downthe
burette reading in mL (V1).
• Repeat the same for blank. Note down the burette reading in mL (V2).

(𝑉1 − 𝑉2) × 𝑁 × 35.45 × 1000

𝐶ℎ𝑙𝑜𝑟𝑖𝑑𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝑔⁄𝐿) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑚𝐿
where, N is the normality of the titrant.
 Hardness

Principle:

Hardness is caused by polyvalent metallic ions, predominantly, divalent calcium and magnesium ions.
When alkalinity < total hardness, alkalinity is considered to be carbonate hardness. Carbonate hardness
is also called temporary hardness as it can be removed by prolonged boiling. The amount of hardness
in excess of carbonate hardness is called non- carbonate hardness or permanent hardness. This is mainly
caused by sulfate, chloride and nitrate ions. In lime soda softening, calcium and magnesium ions are
precipitated by adding lime (Ca(OH)2) and soda ash (Na2CO3) to the water.

Hardness in water is measured by EDTA titrimetric method. Chelating agents such as EDTA forms
stable complex ions with hardness causing divalent ions. Eriochrome black T is used asthe indicator.

Procedure:

Total hardness
• Take 20 mL of sample in a conical flask.
• Add 2 mL of ammonia buffer and a pinch of Eriochrome black T indicator.
• Titrate the sample with 0.01 N EDTA till the wine red color changes to blue. Note down
the volume of the sample (V).
𝑉 × 𝑁 × 50 × 1000
𝑇𝑜𝑡𝑎𝑙 ℎ𝑎𝑟𝑑𝑛𝑒𝑠𝑠 (𝑚𝑔⁄𝐿 𝑎𝑠 𝐶𝑎𝐶𝑂3) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑚𝐿
where, N is the normality of the titrant.
Calcium hardness
• Take 50 mL of sample in a conical flask.
• Add 2 mL of 1 N NaOH and thoroughly mix. After that, add a pinch of muroxide to the
sample.
• Titrate with 0.01 N EDTA solution till pink color changes to purple. Note down the
volume of the sample (V).
• Repeat the same with blank sample for color comparison.
𝑉 × 𝑁 × 50 × 1000
𝐶𝑎𝑙𝑐𝑖𝑢𝑚 ℎ𝑎𝑟𝑑𝑛𝑒𝑠𝑠 (𝑚𝑔⁄𝐿 𝑎𝑠 𝐶𝑎𝐶𝑂3) =
𝑆𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑖𝑛 𝑚𝐿
where, N is the normality of the titrant.
 Multiple tube fermentation technique

Principle:

Coliform group bacteria are all facultative anaerobic, gram-negative, non-spore-forming, rod-shaped
bacteria that ferment lactose with gas and acid formation within 48 h at 35°C. The presence of these
bacteria can be determined by multiple tube fermentation technique or membrane filter technique. In
multiple tube fermentation technique, the results are expressed in terms of the Most Probable Number
(MPN) of organisms present. Schematic outline of presumptive, confirmed and completed phases for
coliform detection is given below.

For the routine examination of public water supplies the object of the total coliform test is to determine
the efficiency of treatment plant operation and the integrity of the distribution system. It is also used
as a screen for the presence of fecal contamination. The object of the examination of non-potable water
generally is to estimate the density of bacterial contamination, determine a source of pollution, enforce
water quality standards, or trace the survival of microorganisms.
Procedure:

Determination of total coliforms:

• Add lauryl tryptose broth to water, mix thoroughly and heat to dissolve. Prepare double
strength broth to add in 10 mL sample tube.
• Arrange 5 fermentation tubes with Durham tubes per dilution of 10, 1 and 0.1 mL
sample. Pack the fermentation tubes, broth and dilution water.
• Sterilize the prepared broth, dilution water and glassware in an autoclave at 121-124ºC
for 15 min. After sterilization, pH of the broth should be 6.8 ± 0.2.
• Prepare appropriate serial dilutions of given wastewater sample. Shake sample and
dilutions vigorously. Add required amount of sample to fermentation tubes. Maintain
the total volume of 10, 1 and 0.1 mL tubes to be 20, 10 and 10 mL, respectively. Mix
test portions in the medium by gentle agitation.
• After inoculation, incubate the tubes at 35±0.5 ºC. After 24±2 h, examine each tube for
growth and gas production. If no gas is observed, reincubate the tubes for another 24 h.
Production of gas in the tubes within 48 ± 3h constitutes a positive presumptive
reaction.
• Compute MPN/100 mL based on the following methods.

(i) According to Table 9221:IV in Standard methods

(ii) According to Thomas formula
(iii) 𝑀𝑃𝑁100 𝑚𝐿 = 𝑛𝑜. 𝑜𝑓 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑡𝑢𝑏𝑒𝑠×100
√(𝑚𝐿 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 −𝑣𝑒 𝑡𝑢𝑏𝑒𝑠×𝑚𝐿 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑎𝑙𝑙 𝑡𝑢𝑏𝑒𝑠)

• A-1 broth is used for the direct isolation of fecal coliforms.

• Repeat the same procedure for total coliforms using A-1 broth. Inoculate the tubes.
• Incubate for 3h at 35 ± 0.5°C and then at 44.5 ± 0.2°C for 21± 2 h.
• Gas production in any A-1 broth culture within 24 h or less is a positive reaction
indicating the presence of fecal coliforms. Calculate MPN from the number of positive
A-1 broth tubes.
Heterotrophic plate count for completed phase:

• Add required amount of Endo agar in water, mix thoroughly and heat to boiling to
dissolve. Sterilize by autoclaving for 15 min at 121°C. Temper agar after sterilization
and pour into petri plates. pH should be 7.1 ± 0.2 after sterilization.
• Perform serial dilutions of the sample. Inoculate the petri plates by spread plate method.
Incubate the inverted plates at 35 ± 0.5 °C for 24 ± 2 h.
• Typical colonies showing pink to dark red with a green metallic surface sheen after 24
h indicates coliform colonies.
 Residual chlorine

Principle:

Chlorination is a widely used disinfection method. Chlorine applied to water initially undergoes
hydrolysis to form free chlorine. Free chlorine reacts readily with ammonia and other nitrogenous
compounds to form combined chlorine. Both free and combined chlorine may be present
simultaneously. In breakpoint chlorination experiment, chlorine dose to be applied after which free
chlorine will be present in treated water is determined.

In DPD method for residual chlorine analysis, N,N-diethyl-p-phenylenediamine (DPD) is added to

sample containing free residual chlorine which leads to formation of red color. Addition of iodide
causes mono and dichloramines to produce iodine. As a result, more DPD is oxidized to form additional
red color. The sample is titrated with FAS till red color disappears. Thus free and combined chlorine
in the sample can be determined.

Procedure:

Determination of free chlorine

• Add 5 mL each of buffer reagent and DPD indicator solution in a conical flask and mix.
• Add 100 mL of sample (or diluted sample) to the conical flask.
• Titrate immediately with standard FAS solution until red color disappears. Note the
burette reading (V1).
Free chlorine, mg Cl2/L = mL of titrant (V1)

Determination of total chlorine

• Add 5 mL each of buffer reagent and DPD indicator solution in a conical flask and mix.
• Add 100 mL of sample (or diluted sample) to the conical flask. Add around 1g of KI
crystals and mix to dissolve. Wait for 2 min.
• Titrate with standard FAS solution until red color disappears. Note the burette reading
(V2).
Total chlorine, mg Cl2/L = mL of titrant (V2)
Combined chlorine, mg Cl2/L = V2 - V1
 Sulfate

Theory: Sulfate ions in water supplies are of important concern as the excess sulfate ions leadto scale
formation in boilers and heat exchangers. In wastewater, the reduction of sulfate to hydrogen sulfide
causes odour problems and crown corrosion in sewers. Sulfate content in thesludge fed to anaerobic
digestion should be monitored as the hydrogen sulfide in the biogas can affect gas engine performance.
Moreover, sulfate reducing bacteria can dominate methanogens, thereby affecting the performance of
the digester.

In turbidimetric method, sulfate in the sample is precipitated as barium sulfate by adding barium
chloride to the solution. Formation of colloidal barium sulfate is enhanced in the presence of an acidic
buffer.

𝐵𝑎2+ + 𝑆𝑂42− ⇋ 𝐵𝑎𝑆𝑂4, 𝐾𝑠𝑝 = 1 × 10−10

Procedure:

• Prepare 0, 5, 10, 15, 20, 25, 30, 35 and 40 mg/L standard sulfate solutions.
• Take 50 mL of standard solution in a conical flask.
• Add 20 mL of buffer solution to the standard solution and mix thoroughly. While
mixing, add 0.1-0.2 g of BaCl2 crystals. Mix for 1 min and then wait for 5 min.
• After 5 min., measure the turbidity of prepared standard solution.
• Plot the calibration curve using turbidity vs concentration data.
• Take 50 mL of sample in a conical flask. If the sample contains suspended particles,
filter the sample.
• Follow the same procedure as that of standard solutions and measure the turbidity of
the sample.
• From the calibration curve, find out the concentration of sulfate in the sample.
• Analyze the turbidity of blank to correct for sample color and turbidity.

Note: The time after preparation of each solution at which the turbidity is measured is critical.Follow
the same waiting period for all samples.
 Phosphate
Aim: Determination of phosphate present in water and wastewater sample by Stannous Chloride
Method
Principle: Stannous chloride method is a colorimetric method, in which ammonium molybdate reacts
with phosphate to form ammonium moly diphosphate, which is reduced by stannous chloride to
molybdenum blue
Apparatus: Spectrophotometer, standard flask, glass pipette, pipette bulbs, beakers & measuring
cylinder.
Reagents:
Standard phosphate solution: Prepare standard solution by dissolving 219.5 mg anhydrous KH2PO4 in
distilled water and dilute to 1000 mL. The concentration of standard phosphate solution is 50 mg PO4-
/L
Phenolphthalein indicator aqueous solution
Strong acid solution: 300 mL conc H2SO4 was added slowly to 600 mL distilled water, cooled, then 4.0
mL conc HNO3 was added before dilution to 1 L
Ammonium molybdate reagent: Dissolve 25 g (NH4)6Mo7O24.4H2O in 175 mL distilled water.
Cautiously add 280 mL conc H2SO4 to 400 mL distilled water. Cool, add molybdate solution, and dilute
to 1 L.
Stannous chloride reagent: Dissolve 2.5 g fresh SnCl2.2H2O in 100 mL glycerol. Heat in a water bath
and stir with a glass rod to hasten dissolution. This reagent is stable and requires neither preservatives
nor special storage.
Procedure
1) Preliminary sample treatment: To 100 mL sample containing not more than 200 g P and free
from colour and turbidity, add 0.05 mL (1drop) phenolphthalein indicator. If sample turns pink,
add strong acid solution dropwise to discharge the colour. If more than 0.25 mL (5 drops) is
required, take a smaller sample, and dilute to 100 mL with distilled water after first discharging
the pink colour with acid.
2) Add, with thorough mixing after each addition, 4.0 mL molybdate reagent and 0.5 mL (10 drops)
stannous chloride reagent. Rate of colour development and intensity of colour depend on
temperature of the final solution, each 1°C increase producing about 1% increase in colour.
Hence, hold samples, standards, and reagents within 2°C of one another and in the temperature
range between 20 and 30°C.
3) After 10 min, but before 12 min, using the same specific interval for all determinations, measure
colour photometrically at 690 nm and compare with a calibration curve, using a distilled water
blank
4) Preparation of Standard Curve: Prepare a calibration curve by using suitable volumes of standard
phosphate solution and proceeding as in 2 & 3. Set Spectrophotometer to zero absorbance with
the blank solution and obtain absorbance readings of standards. Plot a standard curve as
concentration on the X-axis and absorbance on the Y-axis. It a best-fit linear model to the data.
Find out the linear equation (Y= C+MX) and R2 value from the graph. Express equation as:
Absorbance (Abs)= A+ B× Phosphate concentration (in mg/L).
References

1. APHA, AWWA, WEF, Standard Methods for the Examination of Water and
Wastewater, 2012.
2. Sawyer, C. N., McCarty, P. L., & Parkin, G. F., Chemistry for environmental
engineering and science. Boston: McGraw-Hill, 2003.
3. Benefield, L. D., Judkins, J. F., and Weand, B. L., Process Chemistry for Water and
Wastewater Treatment, Prentice-Hall, Englewood Cliffs, New Jersey, 1982.