ENVIRONMENTAL CHEMISTRY

LAB MANUAL

ES 252 (Environmental Chemistry Lab)

Spring Semester-2022

Course Instructor: Prof. VS Vamsi Botlaguduru

Environmental Science and Engineering Department

INDIAN INSTITUTE OF TECHNOLOGY-BOMBAY
 CONTENTS
Contents ..................................................................................................................................... ii

Experiment 1 pH Meter Calibration ..................................................................................... 4

Experiment 2 Alkalinity……………………………………………………………………..6

Experiment 3 Hardness ........................................................................................................... 8

Experiment 4 Sodium .......................................................................................................... 100

Experiment 5 Proximate Analysis of solid waste materials………………………………11

Experiment 6 Chloride……………………………………………………………………...13

Experiment 7 Ultimate analysis of MSW/Solid fuel Samples…………………….............14

Experiment 8 Heavy Metals Analysis in MSW/Fuels…………………………………….16

Experiment 9 Fluoride…………………………………………………………………..….18

Experiment 10 Sulphate…………………………………………………………………….20

Experiment 11 Phosphorous……………………………………………………………….21

Experiment 12 Total Organic Carbon…………………………………………….........….23

Experiment 13 Conductivity……………………………………………………………….25

Experiment 14 Residual Chlorine……………………………………………………..........26

Experiment 15 Biochemical Oxygen Demand…………………….………………………28

Experiment 16 Chemical Oxygen Demand………………………………….…………….31

Experiment 17 Nitrate Nitrogen……………………………………………………………33

Experiment 18 Ammonia Nitrogen…………………………………………….…………..34

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iii
 Experiment 1

pH Meter Calibration

Aim

To ensure calibration of pH meter and measurement of pH of given water sample.

Principle

Measurement of pH is one of the most important and frequently used tests in water chemistry.
pH is used in alkalinity and carbon dioxide measurements and many other acid-base
equilibria. At a given temperature the intensity of the acidic or basic character of a solution is
indicated by pH or hydrogen ion activity. pH as defined by Sorenson as −log [H+]. The basic
principle of electrometric pH measurement is determination of the activity of the hydrogen
ions by potentiometric measurement using a standard hydrogen electrode and a reference
electrode.

Apparatus

pH meter, Glassware

Reagents

 Standard solution (pH = 4.008): Weigh analytical grade 10.12 g potassium hydrogen
phthalate (KHC8H4O4) and dissolve in fresh 1000 mL distilled water.
 Standard solution (pH = 7.413): Weigh 1.179 g, potassium hydrogen phosphate (dried
at 110 - 130 °C for 2 hrs.) and 4.302 g disodium hydrogen phosphate. Dissolve these
into 1000 mL distilled water.

Procedure

Calibration of pH meter

1. Select the ‘CAL’ mode for calibration.

2. Rinse the electrode with de-ionized water and blot dry using a piece of tissue.
3. Place the electrode in the solution of buffer displayed on the meter, allow the pH value
to stabilize and then, enter the value by pressing ‘CFM’. Remove the electrode from
the buffer.
4. Rinse the electrode with de-ionized water and blot dry using a piece of tissue.
 5. Place the electrode in the solution of next buffer displayed on the meter, allow the pH
value to stabilize and then, enter the value by pressing ‘CFM’. Remove the electrode
from the buffer.
6. Repeat the above steps for all the pH standards.
7. After entering all the standards, again press ‘CAL’ to exit the calibration mode.

pH Measurement of samples

1. Make sure that the meter is set to the pH mode.

2. Place the electrode in the sample to be tested.
3. The pH of the solution appears in the display. Allow the display to stabilize before
taking your reading.
4. Note down the pH and temperature reading.
5. Rinse the pH electrode and place it back in the storage solution.

5
 Experiment 2

Alkalinity
Aim
To determine the alkalinity of a given water sample

Principle
Alkalinity is a measure of the capability of water to absorb H+ ions without significant change
of pH. It is the quantitative capacity of water to react with a strong acid to designated pH. In
other words, alkalinity is a measure of the acid buffering capacity of water. It is the sum of all
the titratable bases.
Alkalinity of a sample of water is due to the presence of hydroxide ion OH –, bicarbonate ion
HCO3– and carbonate ion CO32– or the mixture of two ions present in water. The possibility of
OH– and HCO3– ions together is not possible since they combine together to form CO32–– ions.

The alkalinity due to different ions can be estimated separately by titration against standard
acid solution, using selective indicators like phenolphthalein and methyl orange.
i) OH– + H+ → H2O
ii) CO32– + H+ → HCO3–
iii) HCO3– + H+ → H2O + CO2
The neutralization reaction upto phenolphthalein end point shows the completion of reactions
(i) and (ii) (OH– and CO32–) and (CO32– and HCO3–) only. The amount of acid used thus
corresponds to complete neutralization of OH– plus half neutralization of CO32–.
The titration of water sample using methyl orange indicator marks the completion of the
reactions (i), (ii) and (iii). The amount of acid used after phenolphthalein end point
corresponds to one half of normal carbonate and all the bicarbonates. Total amount of acid
used represent the total alkalinity due to all ions present in water sample.

End points:
When alkalinity is entirely due to hydroxide, carbonate or bicarbonate content, the pH at the
equivalence point of titration is determined by concentration of CO2 at that stage. CO2
concentration depends upon the total carbonate species present and any loss that may have
introduced during titration.

The following pH values are suggested as the equivalence points for the corresponding
alkalinity concentration as mg CaCO3 per litre.

6
 End-point pH
Test condition
Total Phenolphthalein
Alkalinity mg CaCO3/L:
30 4.9 8.3
150 4.6 8.3
500 4.3 8.3
Silicates, phosphates known or suspected 4.5 8.3
Routine or automated analyses 4.5 8.3
Industrial waste or complex system 4.5 8.3

Reagents

 Sodium carbonate solution, 0.05N: Dry 3-5 g primary standard Na2CO3 at 250oC for 4
hrs and cool in a desiccator. Weigh 2.5 ± 0.2g, transfer it into a 1 L flask and dilute it
to mark with distilled water.
 Standard sulphuric or hydrochloric acid, 0.1N: Dilute 3 ml of conc. H2SO4 or 8.3 ml of
conc. HCl to 1L distilled water. Standardize against 0.05N Na2CO3 solution, calculate
normality using the following formula:

Where,
A=gm Sodium Carbonate weighed into 1L flask,
B=ml Sodium Carbonate used for titration
C=ml of acid used
1ml of 0.1N solution=5.0 mg CaCO3
 Standard solution of HCl of H2SO4, 0.02N: dilute 200ml of 0.1N standard acid to 1L
 Methyl orange indicator
 Phenolphthalein indicator (alcoholic)

Procedure:
Select the appropriate sample size and normality of the titrant. Add 0.1 ml (2 drops) indicator
solution and titrate over a white surface till a persistent colour change, a characteristic of the
equivalence point is obtained.
Calculation
Alkalinity can be calculated using formula:

Where,
A=ml standard acid used N= normality of the standard acidReport pH of the end point as
follows: The alkalinity to pH------ = ---------mg CaCO3 /L.

7
 Experiment no. 3

Hardness

Aim
To determine the total hardness of given water sample by EDTA Titrimetric method.

Principle
Total hardness is defined as the sum of the calcium and magnesium concentrations, both
expressed as calcium carbonate, in mg/L. The hardness is measured using the EDTA titration
method which measures the calcium and magnesium ions in the water sample.
Ethylenediaminetetraacetic acid and its sodium salts (EDTA) form a chelated soluble complex
when added to a solution of certain metal cations. If a small amount of a dye such as
Eriochrome Black T or Calmagite is added to an aqueous solution containing calcium and
magnesium ions at a pH of 10.0 ± 0.1, the solution becomes wine red.
If EDTA is added as a titrant, the calcium and magnesium will be complexed, and when all of
the magnesium and calcium has been complexed the solution turns from wine red to blue,
marking the end point of the titration.

Reagents

 Buffer solution: Prepare either 1 or 2

1. Dissolve 16.9 g ammonium chloride (NH4Cl) in 143 mL conc ammonium
hydroxide (NH4OH). Add 1.25 g magnesium salt of EDTA and dilute to 250 mL
with distilled water.
2. If the magnesium salt of EDTA is unavailable, dissolve 1.179 g disodium salt of
EDTA and 780 mg magnesium sulfate (MgSO4⋅7H2O) or 644 mg magnesium
chloride (MgCl2⋅6H2O) in 50 mL distilled water. Add this solution to 16.9 g
NH4Cl and 143 mL conc NH4OH with mixing and dilute to 250 mL with distilled
water.
 Indicator Eriochrome Black T
Dissolve 0.5g dye in 100 mL triethanolamine or 2 ethylene glycol monomethyl ether.
 Standard EDTA titrant, 0.01M:
Weigh 3.723g di-sodium salt of EDTA, dihydrate, dissolve in distilled water and
dilute to 1000 mL.
 Standard Calcium Solution:
Weigh 1.000g anhydrous CaCO3 in a 500 mL flask. Add 1 part distilled water + 1 part
HCl till all CaCO3 is dissolved. Add 200 mL distilled water and boil for a few minutes to

8
 expel CO2. Cool and add a few drops of methyl red indicator and adjust to the
intermediate orange colour by adding 3N NH4OH or 1 + 1 HCl, as required. Transfer
quantitatively and dilute to 1000 mL with distilled water.
1 mL = 1mg CaCO3.

Procedure
Titration of sample
1. Select a sample volume that requires less than 15 mL EDTA. Dilute 25.0 mL sample
to about 50 mL with distilled water.
2. Add 1 to 2 mL buffer solution. Usually 1 mL will be sufficient to give a pH of 10.0
to 10.1.
3. Add 1 to 2 drops indicator. Add standard EDTA titrant, with continuous stirring,
until the last
reddish tinge disappears. At the end point the solution normally is blue.

Standardization
Standardize the EDTA titrant against standard calcium solution using the above procedure.

Calculation:

where
A = mL titration for sample and
B = mg CaCO3 equivalent to 1.00 mL EDTA titrant.

Summarize the major points based on literature survey in this chapter. Maximum of one page.

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 Experiment no. 4

Sodium

Aim

To determine the sodium content in a given water sample by flame emission photometric
method.

Principle

Trace amounts of sodium can be determined by flame emission photometry at 589 nm.
Sample is nebulized into a gas flame under carefully controlled, reproducible excitation
conditions. The sodium resonant spectral line at 589 nm is isolated by interference filters or
by prisms or gratings. Emission light intensity is measured by a phototube, or photodiode. The
light intensity at 589 nm is approximately proportional to the sodium concentration.
The calibration curve may be linear but has a tendency to level off or even reverse at higher
concentrations. Work in the linear to near-linear range.

Apparatus

Flame photometer, glassware

Reagents

 Deionized water: Use DI water to prepare all reagents and calibration standards, and
as dilution water.
 Stock sodium solution: Dissolve 2.542 g NaCl dried at 140°C and dilute to 1000 mL
with water; 1.00 mL = 1.00 mg Na (1000 ppm)
 Intermediate sodium solution: Dilute 10.00 mL stock sodium solution with water to
100.0 mL; 1.00 mL = 0.10 mg Na = 100 μg Na. Use this intermediate solution to
prepare calibration curve in sodium range of 1 to 10 mg/L.
 Standard sodium solution: Dilute 10.00 mL intermediate sodium solution with water
to
100 mL; 1.00 mL = 10.0 μg Na. Use this solution to prepare calibration curve in 0.1 to
1.0 mg/L range.

Procedure

1. Prepare a blank and sodium calibration standard in any of the following applicable
ranges: 0 to 1.0, 0 to 10, or 0 to 100 mg/L.
2. Aspirate calibration standards and samples enough times to secure a reliable average
reading for each starting from the highest calibration standard. Construct a calibration
curve from the sodium standards.
3. Determine sodium concentration of sample from the calibration curve.

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Calculation

For direct reference to the calibration curve

mg Na/L in sample = (mg Na/L in portion) × D
where D= Dilution ratio

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 Experiment no. 5

Proximate Analysis of solid waste materials

Aim

To perform proximate analysis of solid waste/sludge samples.

Principle

Proximate analysis is the quantitative measurement of “moisture content, volatiles matter,

fixed carbon and ash content” of a solid waste material.
Sludge is semi-solid slurry which can be produced as sewage sludge from wastewater
treatment processes or as a settled suspension obtained from conventional drinking water
treatment and numerous other industrial processes. Sludge constituents are organic and
inorganic compounds, including traces of heavy metals such as chromium, zinc, mercury,
lead, nickel, cadmium and copper. Generally, sewage sludge of a municipal wastewater
treatment plant is made of approximately 80% moisture and 20% dry matter.
Sludge sample is kept for drying in an oven at temperatures no greater than 45°C for 24 to 48
h of duration before the proximate analysis. However, in this method there is a risk for
microbial growth during drying. After drying, proximate composition can be determined.

Apparatus:
Hot air oven, mixer grinder, muffle furnace, analytical balance accurate, desiccator, crucibles,
tongs and heat resistant gloves.

Procedure

1. Sample preparation
In this procedure, drying, size reduction, obtaining samples with a uniform particle size is
done to obtain representative of the sample.
2. Moisture content (MC)
1-2 g of prepared solid waste sample is taken in dry and weighed crucible and kept for
drying in a hot air oven at 103-105˚C for 1 h and cooled in a desiccator. The difference in
the weight of solid weight material represents the moisture content. Report the result in %
weight.
3. Volatile matter (VM)
Take approximately 1.0 g of moisture free sample in a clean dry crucible with lid. For
biomass sample like wood, heat the sample in a muffle furnace at temperature 550 - 600˚C
for 4 h. For coal sample, it is heated at 950˚C for 7 min duration. Cool the sample in a
desiccator and record the weight. Loss in weight is due to volatile matter content. Report it
as % weight (dry basis).
4. Ash content

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 Take approximately 1.0 g of moisture free sample in a clean dry open crucible. Heat the
sample in a muffle furnace at 700 - 750˚C until all the carbonaceous matter burns away.
Cool the crucible and record the weight which represents the ash content. Report the result
in % weight (dry basis).
5. Fixed carbon (FC)
It is the solid residue that remains after a solid waste material is heated and the volatile
matter expelled. The fixed carbon content of a solid waste material is determined by
subtracting the percentage moisture, volatile matter and ash content from a sample.

Calculation

Where,

FC = Fixed carbon content

MC = Moisture content
VM = Volatile matter

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 Experiment no. 6
Chloride

Aim

To determine the chloride content of a given water sample by argentometric method.

Principle

Chloride ion is one of the major inorganic anions in water and wastewater. Argentometric
titration method (also called as Mohr’s method) is used for determination of chloride
concentration in aqueous solution. In a neutral or slightly alkaline solution, potassium
chromate can indicate the end point of the silver nitrate titration of chloride. Silver chloride is
precipitated quantitatively before red silver chromate is formed.

Apparatus

Erlenmeyer flask, Burette

Reagents

 Potassium chromate indicator solution: Dissolve 50 g K2CrO4 in a little distilled water.

Add AgNO3 solution until a definite red precipitate is formed. Let stand 12 h, filter,
and dilute to1 L with distilled water.
 Standard silver nitrate titrant, 0.0141M (0.0141N): Dissolve 2.395 g AgNO3 in
distilled water and dilute to 1000 mL. Standardize against NaCl by the procedure
described below. Store in a brown bottle. 1.00 mL = 500 μg Cl–.
 Standard sodium chloride, 0.0141M (0.0141N): Dissolve 824.0 mg NaCl (dried at
140°C) in distilled water and dilute to 1000 mL; 1.00 mL = 500 μg Cl–.

Procedure

1. Use a 100-mL sample or a suitable portion diluted to 100 mL.

2. Directly titrate samples in the pH range 7 to 10. Adjust sample pH to 7 to 10 with
H2SO4 or NaOH if it is not in this range.
3. Add 1.0 mL K2CrO4 indicator solution.
4. Titrate with standard AgNO3 titrant to a pinkish yellow end point. Be consistent in
end-point recognition.
5. Standardize AgNO3 titrant and establish reagent blank value by the titration method
outlined above. A blank of 0.2 to 0.3 mL is usual.

14
Calculation

where:

A = mL titration for sample

B = mL titration for blank
N = normality of AgNO3.

mg NaCl/L = (mg Cl–/L) × 1.65

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 Experiment 7

Ultimate Analysis of MSW/ Solid Fuel Samples

Aim
To perform ultimate analysis of MSW/ solid fuel samples.
Theory
Ultimate analysis refers to an analysis of solid waste to determine the proportion of carbon,
hydrogen, oxygen, nitrogen and sulphur, and the analysis is done to make mass balance
calculation for a chemical or thermal process. Besides, it is necessary to determine ash
fraction because of its potentially harmful environmental effects, brought about by the
presence of toxic metals such as cadmium, chromium, mercury, nickel, lead, tin and zinc.
Note that other metals (e.g., iron, magnesium, etc.) may also be present but they are
considered as non-toxic.
In this experiment we will be also using biomass fuels which are derived from biological
material. The biological material takes carbon from the atmosphere and returns it when it is
burnt. Biomass derives from burning crops, manure, garbage and most commonly, wood.
Burning wood, for the production of energy has been common practice both domestically and
industrially for heating and cooking, or to produce steam and electricity for industrial use.
Bio-fuels are an alternative to fossil fuels and are derived mainly from plants and animals,
which do not increase the carbon dioxide level in the atmosphere.
Many manufacturing plants and research laboratories are involved in the characterization of
these bio-fuel products to establish their viability for production, carbon dioxide emission
levels, impact on water resources, energy balance and efficiency. A common method to
characterize these materials is by identifying their energetic value via elemental composition
Principle
In the combustion process (furnace at 1000o C), carbon is converted to carbon dioxide;
hydrogen to water; nitrogen to nitrogen gas/ oxides of nitrogen and sulphur to sulphur
dioxide. If other elements such as chlorine are present, they will also be converted to
combustion products, such as hydrogen chloride. A variety of absorbents are used to remove
these additional combustion products as well as some of the principal elements, sulphur for
example, if no determination of these additional elements is required. The combustion
products are swept out of the combustion chamber by inert carrier gas such as helium and
passed over heated (about 600oC) high purity copper. This copper can be situated at the base
of the combustion chamber or in a separate furnace. The function of this copper is to remove
any oxygen not consumed in the initial combustion and to convert any oxides of nitrogen to

16
nitrogen gas. The gases are then passed through the absorbent traps in order to leave only
carbon dioxide, water, nitrogen and sulphur dioxide.
Detection of the gases can be carried out in a variety of ways including (i) a GC separation
followed by quantification using thermal conductivity detection (ii) a partial separation by GC
(‘frontal chromatography’) followed by thermal conductivity detection (CHN but not S) (iii) a
series of separate infra-red and thermal conductivity cells for detection of individual
compounds. Quantification of the elements requires calibration for each element by using
high purity ‘micro-analytical standard’ compounds such as acetanilide and benzoic acid.
Oxygen Analysis
As there is no satisfactory method for the direct determination of oxygen, it is calculated by
difference, that is, sum total of carbon, hydrogen, nitrogen and organic sulphur and deducted
from 100 is taken as oxygen or
Oxygen % = 100 - (C% + H% + N% + organic sulphur %).
Procedure
1. Sample Preparation

Bulk samples are dried, crushed and homogenized using a mortar and pestle or electric
mill, and weighed into a sample cup (crucible). The crucibles are then closed (referred to
as “wrapping” the sample) and placed in the autosampler for instrumental analysis.
2. Drying and Homogenizing the Sample
1. Break clumpy sample into pieces.
2. Dry sample for at least 12 hr. until sample is completely dry (may require additional
freeze-drying time, up to 3 hr.). The sample is dry when there is no more condensate
in the cold part of the tube.
3. Wipe an agate mortar and pestle with ethanol and allow to dry completely before
grinding each sample. Be sure the mortar and pestle are completely dry; ethanol
reagent residue can cause false C and H results.
4. Grind and homogenize sediment using the cleaned and dry agate mortar and pestle.
5. Fill a sample bottle with powdered homogenized sediment.
6. Place sample bottle into the desiccator.
Many mineral species are hygroscopic (tend to absorb water), so the sample bottles
containing the dried samples should be kept in the desiccator until used.

17
 Experiment no. 8

Heavy Metals Analysis in MSW/ Fuels

Aim:
To determine the trace metal concentration in a given sample by ICP-AES method.

Principle:
Trace element determination by ICP-AES using the microwave digestion method gives
reliable results. The method of microwave digestion provides the best digestion without the
loss of target metal concentration. The method needs to be programmed according to the
samples. Advantage of the method is to avoid loss of volatile metal like Sn, As, Hg, Pb and
also lesser quantity of sample is needed.
Sampling:
Sample amount: 0.5 g
Reagents:
a. Food mix

7 ml of HNO3 , 70% AR grade + 1 ml of H2O2 , 30% AR grade.

b. Sediments

9 ml of HNO3 , 70% AR grade + 3 ml of HCI, 37% AR grade.

Procedure:
1. Weigh the sample to the accurate amount.
2. Add the acids; if a part of the samples stays on the inner wall of the TFM vessel wet it
by adding acids drop by drop, then gently swirl the solution to homogenize the sample
with the acids.
3. Close the vessel and introduce into the rotor segment, tighten preferably by using the
torque wrench.
4. Insert the samples into microwave cavity.
5. Run the microwave according to the temperature program to completion.
6. Cool the rotor and wait until to reach room temperature.
7. Open the vessel and transfer the solution to marked container.

Temperature program:
a. Food mix

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Step Run Time Temperature Microwave Power
1 10 mins. 200°C Upto 90%
2 20 mins. 200°C Upto 90%
b. Sediment

Step Run Time Temperature Microwave Power

1 5 mins 175°C Upto 90%
2 15 min 175°C Upto 90%

Calculation:
Concentration(mg/L)

Reference:
FSSAI Manual of Methods of Analysis of Foods Metal. Lab Manual 9. 2012
http://www.fssai.gov.in/Portals/0/Pdf/15Manuals/METALS.pdf
Sandroni, V., Smith, C.M.M., (2002) Microwave digestion of sludge, soil and sediment
samples for metal analysis by inductively coupled plasma-atomic emission spectrometry,
Anal. Chim. Acta, 468, 335–344.

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 Experiment 9

Fluoride

Aim
To determine fluoride content by SPADNS method

Principle
The SPADNS colorimetric method is based on the reaction between fluoride and a zirconium-
dye lake. Fluoride reacts with the dye lake, dissociating a portion of it into a colorless
complex anion (ZrF62–); and the dye. As the amount of fluoride increases, the color produced
becomes progressively lighter. If the proportion of acid in the reagent is increased, the
reaction can be made almost instantaneous.

Apparatus
Spectrophotometer, for use at 570 nm.

Reagents

 Stock fluoride solution: Dissolve 221.0 mg anhydrous sodium fluoride, NaF, in

distilledwater and dilute to 1000 mL; 1.00 mL = 100 μg F–.
 Standard fluoride solution: Dilute 100 mL stock fluoride solution to 1000 mL with
distilledwater; 1.00 mL = 10.0 μg F–.
 SPADNS solution: Dissolve 958 mg SPADNS, sodium2-(parasulfophenylazo)-
1,8dihydroxy-3, 6-naphthalene disulfonate, also called 4, 5-dihydroxy-3-
(parasulfophenylazo)-2,7-naphthalenedisulfonic acid trisodium salt, in distilled water
and dilute to 500 mL. This solution is stable for at least 1 year if protected from direct
sunlight.
 Zirconyl-acid reagent: Dissolve 133 mg zirconyl chloride octahydrate, ZrOCl2⋅8H2O,
in about 25 mL distilled water. Add 350 mL conc HCl and dilute to 500 mL with
distilled
water.
 Acid zirconyl-SPADNS reagent: Mix equal volumes of SPADNS solution and
zirconyl-acid reagent. The combined reagent is stable for at least 2 years.
 Reference solution: Add 10 mL SPADNS solution to 100 mL distilled water. Dilute 7
mL conc HCl to 10 mL and add to the diluted SPADNS solution. The resulting
solution, used for setting the instrument reference point (zero), is stable for at least 1
year. Alternatively, use a prepared standard of 0 mg F–/L as a reference.

20
  Sodium arsenite solution: Dissolve 5.0 g NaAsO2 and dilute to 1 L with distilled
water.

Procedure

Preparation of standard curve

1. Prepare fluoride standards in the range of 0 to 1.40 mg F–/L by diluting appropriate
quantities of standard fluoride solution to 50 mL with distilled water.
2. Pipet 5.00 mL each of SPADNS solution and zirconyl-acid reagent, or 10.00 mL
mixed acid-zirconyl-SPADNS reagent, to each standard and mix well. Avoid
contamination.
3. Set photometer to zero absorbance with the reference solution and obtain absorbance
readings of standards.
4. Plot a curve of the milligrams fluoride-absorbance relationship.

Sample pretreatment
1. If the sample contains residual chlorine, remove it by adding 1 drop (0.05 mL)
NaAsO2 solution/ 0.1 mg residual chlorine and mix.

Color development
1. Use a 50.0-mL sample or a portion diluted to 50 mL with distilled water. Adjust
sample temperature to that used for the standard curve.
2. Add 5.00 mL each of SPADNS solution and zirconyl-acid reagent, or 10.00 mL acid-
zirconyl-SPADNS reagent; mix well and read absorbance, first setting the reference
point of the photometer as above. If the absorbance falls beyond the range of the
standard curve, repeat using a diluted sample.

Calculation

where:
A = μg F– determined from plotted curve,
B = final volume of diluted sample, mL, and
C = volume of diluted sample used for color development, mL.

21
 Experiment 10

Sulphate
Aim

To determine the sulphate content of a given water sample by Turbidimetric method.

Principle

Sulphate ion (SO42–) is precipitated in an acetic acid medium with barium chloride (BaCl2)
toform barium sulphate (BaSO4) crystals of uniform size. Light absorbance of the BaSO4
suspension is measured by a photometer and the SO42– concentration is determined by
comparison of the reading with a standard curve.

Apparatus

Turbidimeter, magnetic stirrer, stopwatch

Reagents

 Buffer solution A: Dissolve 30 g magnesium chloride, MgCl2⋅6H2O, 5 g sodium

acetate, CH3COONa⋅3H2O, 1.0 g potassium nitrate, KNO3, and 20 mL acetic acid,
CH3COOH (99%), in 500 mL distilled water and make up to 1000 mL.
 Buffer solution B (required when the sample SO42– concentration is less than 10
mg/L): Dissolve 30 g MgCl2⋅6H2O, 5g CH3COONa⋅3H2O, 1.0 g KNO3, 0.111 g
sodium sulphate, Na2SO4, and 20 mL acetic acid (99%) in 500 mL distilled water and
make up to 1000 mL.
 Barium chloride: BaCl2, crystals, 20 to 30 mesh. In standardization, uniform turbidity
is produced with this mesh range and the appropriate buffer.
 Standard sulphate solution: Prepare a standard sulphate solution as described in 1) or
2) below; 1.00 mL = 100 μg SO42–.
1) Dilute 10.4 mL standard 0.02N H2SO4 titrant specified in Alkalinity.
2) Dissolve 0.1479 g anhydrous Na2SO4 in distilled water and dilute to 1000 mL.

Procedure

1. Formation of barium sulphate turbidity:

Measure 100 mL sample or a suitable portion made up to 100 mL, into a 250-mL
Erlenmeyer flask. Add 20 mL buffer solution and mix in stirring apparatus. While
stirring, add a spoonful of BaCl2 crystals and begin timing immediately. Stir for 60 ± 2 s
at constant speed.
2. Measurement of barium sulphate turbidity:
After stirring period has ended, pour solution into absorption cell of turbidimeter and
measure turbidity at 5 ± 0.5 min.
3. Preparation of calibration curve:

22
 Estimate SO42– concentration in sample by comparing turbidity reading with a
calibration curve prepared by carrying SO42– standards through the entire procedure.
Space standards at 5-mg/L increments in the 0 to 40 mg/L SO42– range.Above 40 mg/L
accuracy decreases and BaSO4 suspensions lose stability. Check reliability of calibration
curve by running a standard with every three or four samples.
4. Correction for sample colour and turbidity:
Correct for sample colour and turbidity by running blanks to which BaCl2 is not added.

Calculation

23
 Experiment 11

Phosphorus
Aim

To determine the phosphorus content by stannous chloride method.

Principle

Phosphorus occurs in natural waters and in wastewaters as orthophosphates, condensed

phosphates (pyro-, meta-, and other polyphosphates), and organically bound phosphates.
Molybdophosphoric acid is formed and reduced by stannous chloride to intensely colored
molybdenum blue. This method is sensitive and makes feasible measurements down to 7 μg
P/L by use of increased light path length. The minimum detectable concentration I is about 3
μg P/L.

Apparatus

Spectrophotometer, to be used at 690 nm.

Reagents

 Phenolphthalein indicator aqueous solution.

 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.
 Standard phosphate solution: Dissolve in distilled water 219.5 mg anhydrous KH2PO4
and dilute to 1000 mL; 1.00 mL = 50.0 μg PO43–P.

Procedure

Preliminary sample treatment

1. To 100 mL sample containing not more than 200 μg P and free from colour and
turbidity, add 0.05 mL (1 drop) phenolphthalein indicator.
2. If sample turns pink, add strong acid solution drop wise to discharge the colour.

Colour development
1. Add, with thorough mixing after each addition, 4.0 mL molybdate reagent and 0.5 mL
(10 drops) stannous chloride reagent to each standard and sample.
2. 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.

24
Colour measurement
1. 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.
2. Always run a blank on reagents and distilled water. Because the colour at first
develops progressively and later fades, maintain equal timing conditions for samples
and standards.
3. Prepare at least one standard with each set of samples or once each day that tests are
made.
4. The calibration curve may deviate from a straight line at the upper concentrations of
the 0.3 to 2.0-mg/L range.

Calculation

25
 Experiment 12

Total Organic Carbon

Total Organic Carbon (TOC) is a sum measure of the concentration of all organic carbon
atoms covalently bonded in the organic molecules of a given sample of water. TOC is
typically measured in Parts per Million (ppm or mg/L), although some industries require more
refined measurements expressed in parts per Parts per Billion (ppb or μg/L), or even Parts per
Trillion (ppt). TOC in source waters comes from decaying natural organic matter (NOM) as
well as synthetic sources. Humic acid, fulvic acid, amines and urea are examples of NOM.
Some Detergents, pesticides, fertilizers, herbicides, chemicals and chlorinated organics are the
examples of synthetic sources. Before source water is treated for disinfection, TOC provides
an estimate of the amount of NOM in the water source. As a sum measurement, Total Organic
Carbon does not identify specific organic contaminants. It will, however, detect the presence
of all carbon-bearing molecules, thus identifying the presence of any organic contaminants,
regardless of molecular make-up.

Measurement of TOC:
TOC is measured using TOC analyzer. Since all TOC analyzers only actually measure total
carbon, TOC analysis always requires some accounting for the inorganic carbon that is always
present. One analysis technique involves a two-stage process commonly referred to as TC-IC.
It measures the amount of inorganic carbon (IC) evolved from an acidified aliquot of a sample
and also the amount of total carbon (TC) present in the sample. TOC is calculated by
subtraction of the IC value from the TC the sample. Another variant employs acidification of
the sample to evolve carbon dioxide and measuring it as inorganic carbon (IC), then oxidizing
and measuring the remaining non-purgeable organic carbon (NPOC). This is called TIC-
NPOC analysis. A more common method directly measures TOC in the sample by again
acidifying the sample it to a pH value of two or less to release the IC gas but in this case to the
air not for measurement. The remaining non-purgeable CO2 gas (NPOC) contained in the
liquid aliquot is then oxidized releasing the gases. These gases are then sent to the detector for
measurement.

Importance of monitoring TOC:

TOC detection is an important measurement because of the effects it may have on the
environment, human health, and manufacturing processes. TOC is a highly sensitive,
nonspecific measurement of all organics present in a sample. It, therefore, can be used to
regulate the organic chemical discharge to the environment in a manufacturing plant. In
addition, low TOC can confirm the absence of potentially harmful organic chemicals in water
used to manufacture pharmaceutical products. TOC is also of interest in the field of potable
water purification due to disinfection of byproducts. Inorganic carbon poses little to no threat.
Early detection of high organic loads in influent enable plant operators to optimize processing
for improved system efficiency. Analyzing TOC levels in effluent demonstrates compliant
levels of organics prior to discharging treated wastewater to surface waters. Industrial
discharge is carefully regulated, and excessive organics in industrial wastewater can result in
fines, citations and, in extreme cases, even plant closure. Organic carbon readily binds with
other elements in the environment, producing any of a large number of compounds, many of

26
which are harmful to the environment or threaten the public health. US EPA regulations
require that industries limit their environmental discharge of organic carbon, and violations
are often accompanied by severe financial penalties. In severe cases, losing an NPDES permit
can lead to plant closure. Many industries monitor TOC as a means of validating sanitary
conditions. This is especially critical in industries that use high purity water as an ingredient
in pharmaceutical products (water for injection), and as a rinsing or cooling agent to protect
expensive industrial systems. In process industries TOC analysis provides cleaning validation
and detects organic contaminants in process water. The presence of organic material in pure
water systems can indicate a failure in filtration systems, storage methods, compromised seals
or other system component failure. Monitoring total organic carbon is vital in many
industries, including Power Generation, Production of Pharmaceuticals, Semiconductor
Manufacturing, and any industry where ultrapure water (De-Ionized, or DI Water) is produced
or consumed.

Additional info:
(Source:http://cn.mt.com/dam/mt_ext_files/Editorial/Generic/2/Datasheet_TOC550_An
alyzer_Editorial-Generic_1159441587374_files/ml0095e.pdf)
The Thornton 550 TOC Analyzer measures Total Organic Carbon based on differential
conductivity. The sample water enters the analyzer and passes through a pressure regulator,
which controls sample pressure to downstream components. Here the sample splits into two
flow paths, where a portion of the flow is directed to the by-pass line where
resistivity/conductivity and temperature are measured. These values are represented on the
LCD display. The remainder of the sample is directed through a second conductivity sensor,
measuring the sample conductivity prior to oxidation. This conductivity measurement
accounts for background ions and inorganic carbon (CO2) in the water stream. Next, the
sample enters the oxidation chamber. As the sample moves through the oxidation chamber, it
is subjected to high intensity ultraviolet radiation at 185 nanometers, effectively oxidizing the
sample. After oxidation, the sample passes through a third conductivity sensor where the
conductivity and temperature are measured again to determine the level of Total Organic
Carbon (TOC). The measurement and sample flow are continuous; therefore, measurement
update time is minimized, providing rapid response.

Figure. Schematic of TOC analyzer using UV oxidation and conductivity sensors for
detection (figure taken from Mettler-Toledo Thornton, Inc. MA, USA)

27
 Experiment 13

Conductivity
Aim

To determine the conductivity of given water sample.

Principle

Conductivity, k, is a measure of the ability of an aqueous solution to carry an electric current.

This ability depends on the presence of ions; on their total concentration, mobility, valence
and on the temperature of measurement.Conductivity is customarily reported in micromhos
per centimeter (μmho/cm). Freshly distilled water has a conductivity of 0.5-2 μmho/cm.

Apparatus

Conductivity meter

Reagents

 Conductivity water: Pass distilled water through a mixed bed deionizer and discard
first 1000 mL of water. Conductivity of this water should be less than 1 μmho/cm.
 Standard potassium chloride solution, KCl, 0.0100M:
Dissolve 745.6 mg anhydrous KCl in conductivity water and dilute to 1000 mL at
25°C.
This is the standard reference solution, which at 25°C has a conductivity of
1412 μmhos/cm.

Procedure

Conductivity measurement:
Thoroughly rinse the conductivity electrode cell with one or more portions of sample. Adjust
temperature of a final portion to about 25°C. Measure sample conductivity and note
temperature to ±0.1°C.

Calculation

Conductivity k at 25°C

Where
km = measured conductivity in units of μmho/cm at t°C, t = temperature of measurement

28
 Experiment 14

Residual Chlorine
Aim

To determine residual chlorine content of a given sample by DPD ferrous titrimetric method.

Principle

Chlorine applied to water undergoes hydrolysis to form free chlorine. With ammonia, chlorine
reacts to form chloramines: monochloramine, dichloramine, and nitrogen trichloride. Free
chlorine (hypochlorite ions and hypochlorous acid) and chloramines stoichiometrically
liberate
iodine from potassium iodide at pH 4 or less. Iodine is titrated with FAS. N,N-diethyl-p-
phenylenediamine (DPD) is used as an indicator in the titrimetric procedure with ferrous
ammonium sulfate (FAS).
In the absence of iodide ion, free chlorine reacts instantly with DPD indicator to produce a red
color. Subsequent addition of a small amount of iodide ion acts catalytically to cause
monochloramine to produce color. Addition of iodide ion to excess evokes a rapid response
from dichloramine. In the presence of iodide ion, part of the nitrogen trichloride (NCl3) is
included with dichloramine and part with free chlorine. Approximately 18 μg Cl as Cl2/L is
the minimum detectable limit by this method.

Reagents

 Phosphate buffer solution: Dissolve 24 g anhydrous disodium hydrogen phosphate,

Na2HPO4, and 46 g anhydrous potassium dihydrogen phosphate, KH2PO4, in distilled
water.
 Dissolve 800 mg disodium EDTA in 100 mL distilled water. Combine these two
solutions and dilute to 1 liter with distilled water. Add 20 mg. HgCl2 as a preservative.
 N,N-Diethyl-p-phenylenediamine (DPD) indicator solution: Dissolve 1 g DPD oxalate
or 1.5 g p-amino-N,N-diethylaniline sulfate in chlorine free distilled water containing
8 mL of 1 + 3 H2SO4 and 200 mg disodium EDTA and dilute to 1 L.
 Standard ferrous ammonium sulfate (FAS) titrant: Dissolve 1.106 g
Fe(NH4)2(SO4)2,6H2O in distilled water containing 1 mL of 1 + 3 H2SO4 and make up
to 1 L with distilled water.
 Sulfuric acid solution (1 + 3): Slowly add one part of H2SO4 (sp. gr. 1.84) to three
parts of distilled water.
 Potassium iodide, KI, crystals OR Potassium iodide solution: Dissolve 500 mg KI and
dilute to 100 mL, using distilled water.

Procedure

1. Standardization of FAS:
To 80 mL distilled water, add with constant stirring, 1 mL conc H2SO4, 10.00 mL 0.1
N K2Cr2O7, and 1 g KI keep for 6 min in dark. Titrate immediately with 0.1N Na2S2O3

29
 titrant until the yellow color of the liberated iodine is discharged. Add 1 mL starch
indicator solution and continue titrating until the blue color disappears.
2. For Residual chlorine
The quantities given below are suitable for concentrations of total chlorine up to 5
mg/L. Mix usual volumes (5 mL) of buffer reagent and DPD indicator solution(or
usual amount of DPD powder) with distilled water before adding sufficient sample to
bring total volume to 100 mL.
 Free chlorine—Titrate rapidly with standard FAS titrant until red color is
discharged (Reading A).
 Monochloramine—Add one very small crystal of KI (about 0.5 mg) or 0.1 mL
(2 drops) KI solution and mix. Continue titrating until red color is discharged
again (Reading B).
 Dichloramine—Add several crystals KI (about 1 g) and mix to dissolve. Let
stand for 2min and continue titrating until red color is discharged (Reading C).
3. Total Chlorine (Combined + Free): Add full amount of KI (1 g) at the start into 5 ml
each of buffer reagent and DPD indicator. Add 100 ml of sample, and titrate after 2
min until red colour is discharged.
4. Nitrogen trichloride: Place one very small crystal of KI (about 0.5 mg) or 0.1 mL KI
solution in a titration flask. Add 100 mL sample and mix. Add contents to a second
flask containing 5 mL each of buffer reagent and DPD indicator solution. Titrate
rapidly with standard FAS titrant until red color is discharged (Reading N).

Calculations:

For a 100-mL sample, 1.00 mL standard FAS titrant used = 1.00 mg Cl (in form of Cl2/L).

Free Chlorine = Reading A

Total Chlorine = Reading C
Combined Chlorine = Reading (C-A)

Where,
NH2Cl = monochloramine
NHCl2 = dichloramine
NCl3 = trichloramine

30
 Experiment 15

Biochemical Oxygen Demand (BOD)

Aim

BOD Determination of given waste water.

Principle

Biochemical Oxygen Demand (BOD) refers to the amount of oxygen that would be consumed
if all the organics in one litre of water were oxidized by microorganisms. The BOD
determination is an empirical test that measures the oxygen equivalent for the biochemical
degradation of organic materials (carbonaceous demand). The oxygen used to oxidize may
measure the oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless
this oxidation is prevented by an inhibitor. The first step in measuring BOD is to obtain equal
volumes of water from the area to be tested and dilute each specimen with a known volume of
distilled water which has been thoroughly shaken to insure oxygen saturation. The method
consists of filling with sample, to overflowing, an airtight bottle of the specified size and
incubating it at the specified temperature for 5 d. Dissolved oxygen is measured initially and
after incubation, and the BOD is computed from the difference between initial and final DO.
Because the initial DO is determined shortly after the dilution is made, all oxygen uptake
occurring after this measurement is included in the BOD measurement. In this experiment, the
initial and final DO is measured by Winkler’s method with azide modification. This method is
based on the addition of divalent manganese solution, followed by strong alkali, to the sample
in a glass-stoppered bottle. DO rapidly oxidizes an equivalent amount of the dispersed
divalent manganous hydroxide precipitate to hydroxides of higher valency states. In the
presence of iodide ions in an acidic solution, the oxidized manganese reverts to the divalent
state, with the liberation of iodine equivalent to the original DO content. The iodine is then
titrated with a standard solution of thiosulfate. The titration end point can be detected visually,
with a starch indicator. This method can be used on a routine basis to provide accurate
estimates for DO in the microgram-per-liter range.

Apparatus

Incubation bottles: 300-mL glass bottles having a ground-glass stopper and a flared mouth.
Air incubator or water bath: thermostatically controlled at 20 ±1°C.

Reagents

For BOD
 Phosphate buffer: Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4⋅7H2O,
and 1.7 g NH4Cl in about 500 mL distilled water and dilute to 1 L. The pH should be
7.2
 Magnesium sulfate: Dissolve 22.5 g MgSO4⋅7H2O in distilled water and dilute to 1 L.
 Calcium chloride: Dissolve 27.5 g CaCl2 in distilled water and dilute to 1 L.

31
  Ferric chloride solution: Dissolve 0.25 g FeCl3⋅6H2O in distilled water and dilute to 1
L.
 Acid and alkali solutions, 1N, for neutralization of caustic or acidic waste samples.
 Sodium sulfite solution: Dissolve 1.575 g Na2SO3 in 1000 mL distilled water.
 Dilution water: Place desired volume of water in a suitable bottle and add 1 mL each
of phosphate buffer, MgSO4, CaCl2, and FeCl3 solutions/L of water.

For DO measurement
 Manganous sulfate solution: Dissolve 480 g MnSO4⋅4H2O, 400 g MnSO4⋅2H2O, or
364 g MnSO4⋅H2O in distilled water, filter, and dilute to 1 L. The MnSO4 solution
should not give a color with starch when added to an acidified potassium iodide (KI)
solution.
 Alkali-iodide-azide reagent: For saturated or less-than-saturated samples—Dissolve
500 g NaOH (or 700 g KOH) and135 g NaI (or 150 g KI) in distilled water and dilute
to 1 L. Add 10 g NaN3 dissolved in 40 mL distilled water.
 Sulfuric acid, H2SO4, conc: One milliliter is equivalent to about 3 mL alkali-iodide-
azide reagent.
 Starch: Dissolve 2 g laboratory-grade soluble starch in 100 mL hot distilled water.
 Standard sodium thiosulfate titrant: Dissolve 6.205 g Na2S2O3⋅5H2O in distilled water.
Add 1.5 mL 6N NaOH or 0.4 g solid NaOH and dilute to 1000 mL. Standardize with
bi-iodate solution.
 Standard potassium bi-iodate solution, 0.0021M: Dissolve 812.4 mg KH(IO3)2 in
distilled water and dilute to 1000 mL.

Procedure

For BOD
1. Seeding: It is necessary to have a present a population of microorganisms capable of
oxidizing the biodegradable organic matter in the sample. Domestic waste water,
unchlorinated or otherwise disinfected effluents from biological waste treatment plants
and surface water receiving waste water discharge contain satisfactory microbial
populations.For the sample, which do not contain sufficient microbial population the
dilution water is seeded by adding a population of microorganisms. The preferred seed
is effluent from the biological treatment system processing the waste, if there is not
available, used supernatant from domestic waste water after settling at 20oC for at
least 1 hour but no longer than 36 hrs.
2. Dilution technique: Dilution that result in a residual DO of at least 1 mg/L and DO
uptake of at least 2 mg/L, after 5 days of incubation produce the most efficient results.
Make several dilutions of prepared sample to obtain DO in this range. Prepare dilution
either in the graduated cylinders and then transfer to BOD bottle or directly in BOD
bottles. Use the dilution water for this purpose. Measure DO by Iodometric method.
3. Dilution water blank: Use dilution water blank as a rough check on the quality of
unseeded dilution water and cleanliness of the incubation bottles. Determine the initial
and final DO. Treat this same as other samples.
4. Incubation: Incubate at 20 ± 1oC BOD bottles containing desired dilutions and dilution
water blank. Determine final DO. After 5 days of incubation, determine the DO in
sample and dilution blanks.

For DO measurement

32
 1. Standardization of sodium thiosulfate —Dissolve approximately 2 g KI, in an
Erlenmeyer flask with 100 mL distilled water. Add 1 mL 6N H2SO4 or a few drops of
conc H2SO4 and 20 mL standard bi-iodate solution. Dilute to 200 mL and titrate
liberated iodine with thiosulfate titrant, adding starch toward end of titration, when a
pale straw color is reached. When the solutions are of equal strength, 20.00 mL 0.025M
Na2S2O3 should be required. If not, adjust the Na2S2O3 solution to 0.025M.
2. To the sample collected in a 250- to 300-mL bottle, add 1 mL MnSO4 solution,
followed by 1 mL alkali-iodide-azide reagent. Hold pipet tips just above liquid surface
when adding reagents.
3. Stopper carefully to exclude air bubbles and mix by inverting bottle a few times.
4. When precipitate has settled sufficiently (to approximately half the bottle volume) to
leave clear supernate above the manganese hydroxide floc, add 1.0 mL conc H2SO4.
Restopper and mix by inverting several times until dissolution is complete.
5. Titrate a volume corresponding to 200 mL original sample after correction for sample
loss by displacement with reagents. Thus, for a total of 2 mL (1 mL each) of MnSO4
and alkali-iodide-azide reagents in a 300-mL bottle, titrate 200 × 300/(300 − 2) = 201
mL.
6. Titrate with 0.025M Na2S2O3 solution to a pale straw color.
7. Add a few drops of starch solution and continue titration to first disappearance of blue
color.
8. If end point is overrun, back-titrate with 0.0021M bi-iodate solution added dropwise.
Correct for amount of bi-iodate solution or sample.

Calculation

When dilution water is not seeded

BOD, mg/L = (D1-D2)/P

When dilution water is seeded,

BOD (mg/L) = [(D1-D2)-(B1-B2)]/P

Where,
D1= DO of the diluted sample immediately after preparation
D2= DO of diluted sample after 5 days incubation at 20oC
P= decimal volumetric fraction of sample used
B1= DO of seed control before incubation
B2=DO of seed control after incubation
f =ratio of seed in sample to seed in control= (% of seed in D1)*(% of seed in B1)
D1, D2, B1 and B2 are in mg/L.

33
 Experiment 16

Chemical Oxygen Demand (COD)

Aim

To determine the chemical oxygen demand of given water sample by closed reflux titrimetric
method.

Principle

Chemical oxygen demand (COD) is defined as the amount of a specified oxidant that reacts
with the sample under controlled conditions. The quantity of oxidant consumed is expressed
in terms of its oxygen equivalence.

The COD method determines the quantity of oxygen required to oxidize the organic matter in
a waste sample, under specific conditions of oxidizing agent, temperature, and time. Most
types of organic matters are oxidized by a boiling mixture of chromic and sulfuric acids. A
sample is refluxed in strongly acid solution with an excess of potassium dichromate. After
digestion, the remaining unreduced potassium dichromate is titrated with ferrous ammonium
sulfate. The amount of potassium dichromate consumed is determined and the amount of
oxidizable organic matter is calculated in terms of oxygen equivalent.
The reaction may be represented as follows:

6 Fe2+ + Cr2O7- + 14 H+ → 6 Fe3+ + 2 Cr3+ +7H2O

The end point is determined using oxidation reduction indicator i.e. Ferroin indicator
(Ferrous- 1, 10-phenanthroline sulfate) to indicate when all the dichromate ions have been
reduced by ferrous ions. It gives a sharp brown colour change that is easily detected. When
reduction of Cr6+ to Cr3+ is complete, Fe2+ reacts to form the ferroin complex and the solution
color changes sharply from greenish-blue to orange-brown, signaling the end point.

Silver sulfate is used as a catalyst and mercuric sulfate is added to remove chloride
interference. The excess dichromate is titrated with standard ferrous ammonium sulfate, using
ortho-phenanthroline ferrous complex as an indicator.

This procedure is applicable to COD values between 40 and 400 mg/L. Obtain higher values
by dilution. Alternatively, use higher concentrations of dichromate digestion solution to
determine greater COD values.
Apparatus

 Digestion vessels: Preferably use borosilicate culture tubes with TFE-lined screw caps.
 Block heater to operate at 150 ± 2°C, with holes to accommodate digestion vessels.
 Titration apparatus

Reagents

34
  Sulfuric acid reagent: Add Ag2SO4 reagent or technical grade, crystal or powder to
conc.H2SO4 at the rate of 5.5 g Ag2SO4 /Kg H2SO4. Let stand 1 to 2 day to dissolve
the Ag2SO4.
 Potassium dichromate, K2Cr2O7, 0.0167M: Add 4.903 g K2Cr2O7 (previously dried at
103°C for 2 hr) to about 500 mL distilled water, 167 mL conc. H2SO4 and 33.3 g
HgSO4. Dissolve them and cool to room temperature and dilute to 1000mL.
 Ferrous ammonium sulfate, 0.10M: dissolve 39.2 g FAS in distilled water, add 20 ml
conc. H2SO4, cool it and dilute to 1000ml.
 Ferroin indicator solution: Dissolve 1.485 g 1, 10-phenanthroline monohydrate and
695mg FeSO4.7H2O in distilled water and dilute to 100ml.
 Sulfamic acid: It is required only if the interference of nitrites is to be eliminated. Add
10 mg Sulfamic acid for each mg nitrite-N present in the sample volume used. Add
the same amount of sulfamic acid to the reflux vessel containing the distilled water
blank.

Procedure

Standardization of FAS
1. Pipet 5.00 mL digestion solution into a small beaker. Add 10 mL distilled water to
substitute for sample. Cool to room temperature.
2. Add 1 to 2 drops diluted ferroin indicator and titrate with FAS titrant.

COD digestion
1. Wash culture tubes and caps with 20% H2SO4 before first use to prevent
contamination.
2. In a COD vial, add 2.5 mL sample or distilled water (in case of blank), then add 1.5
mL K2Cr2O7 +3.5 mL sulfuric acid reagent. Tightly cap tubes, and invert each several
times to mix completely.
3. Place tubes in block digester preheated to 150°C and reflux for 2 h behind a protective
shield.
4. Cool to room temperature and transfer contents to a larger container for titrating.
5. Add 0.05 to 0.10 mL (1 to 2 drops) ferroin indicator and titrate with standardized
0.10M FAS. The end point is a sharp color change from blue-green to reddish brown.
6. In the same manner reflux and titrate a blank containing the reagents and a volume of
distilled water equal to that of the sample.

Calculation
Molarity of FAS solution

For COD

35
where:
A = mL FAS used for blank,
B = mL FAS used for sample,
M = molarity of FAS, and
8000 = milliequivalent weight of oxygen × 1000 mL/L.

36
 Experiment 17

Nitrate Nitrogen
Aim

To measure Nitrate-N content of a given sample by UV Spectrophotometric Screening

method.

Principle

Measurement of UV absorption at 220 nm enables rapid determination of NO3–. Because

dissolved organic matter also may absorb at 220 nm and NO3– does not absorb at 275 nm, a
second measurement made at 275 nm may be used to correct the NO3-- value. Acidification
with 1N HCl is designed to prevent interference from hydroxide or carbonate. The NO3–
calibration curve follows Beer’s law up to 11 mg N/L.

Apparatus

Spectrophotometer, for use at 220 nm and 275 nm.

Reagents

 Nitrate-free water: Use redistilled or distilled, deionized water of highest purity to

prepare all solutions and dilutions.
 Stock nitrate solution: Dry potassium nitrate (KNO3) in an oven at 105°C for 24 h.
Dissolve 0.7218 g in water and dilute to 1000 mL; 1.00 mL = 100 μg NO3–-N.
 Intermediate nitrate solution: Dilute 100 mL stock nitrate solution to 1000 mL with
water; 1.00 mL = 10.0 μg NO3–-N.
 Hydrochloric acid solution, HCl.

Procedure

1. To 50 mL clear sample, filtered if necessary, add 1 mL HCl solution and mix

thoroughly.
2. Prepare NO3– calibration standards in the range 0 to 7 mg NO3–-N/L by diluting to
50 mL sample volume. Treat NO3 – standards in same manner as samples.
3. Read absorbance at wavelength of 220 nm to obtain NO3– reading and a wavelength
of 275 nm to determine interference due to dissolved organic matter.
4. Construct a standard curve by plotting absorbance due to NO3– against NO3 –-N
concentration of standard obtain sample concentrations directly from standard curve.

Calculation

A NO3– = A220 – 2 (A275)

Where:
A220 = Absorbance at 220 nm

37
 A275 = Absorbance at 275 nm

Note: If correction value is more than 10% of the reading at 220 nm, do not use this method.

38
 Experiment 18

Ammonia Nitrogen
Aim

To measure ammonia nitrogen of a given sample by Phenate method.

Principle

An intensely blue compound, indophenol, is formed by the reaction of ammonia,

hypochlorite, and phenol catalyzed by sodium nitroprusside. Complexing magnesium and
calcium with citrate eliminates interference produced by precipitation of these ions at high
pH.

Apparatus

Spectrophotometer, for use at 640 nm.

Reagents

 Phenol solution: Mix 11.1 mL liquified phenol with 95% v/v ethyl alcohol to a final
volume of 100 mL. (CAUTION: Be extremely careful when handling Phenol)
 Sodium nitroprusside: Dissolve 0.5 g sodium nitroprusside in 100 mL deionized water.
Store in amber bottle.
 Alkaline citrate: Dissolve 200 g trisodium citrate and 10 g sodium hydroxide in
deionized water. Dilute to 1000 mL.
 Sodium hypochlorite: commercial solution, about 5%.
 Oxidizing solution: Mix 100 mL alkaline citrate solution with 25 mL sodium
hypochlorite.
 Stock ammonium solution: Dissolve 3.819 g anhydrous NH4Cl (dried at 100°C) in
water, and dilute to 1000 mL; 1.00 mL = 1.00 mg N = 1.22 mg NH3.

Procedure

1. Take 25-mL sample in a 50-mL erlenmeyer flask and add, 1 mL phenol solution, 1 mL
sodium nitroprusside solution, and 2.5 mL oxidizing solution.
2. Cover samples with plastic wrap or paraffin wrapper film.
3. Let color develop at room temperature (22 to 27°C) in subdued light for at least 1 h.
Color is stable for 24 h.
4. Measure absorbance at 640 nm.
5. Treat standards the same as samples.

39
Calculations

Prepare a standard curve by plotting absorbance readings of standards against ammonia

concentrations of standards. Compute sample concentration by comparing sample absorbance
with the standard curve.

40