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# DETERMINATION OF SLUDGE VOLUME INDEX, MIXED LIQUOR SUSPENDED

## SOLIDS (MLSS) AND MIXED LIQUOR VOLATILE SUSPENDED SOLIDS (MLVSS)

INTRODUCTION:
In a biological treatment plant it is very important to monitor property of biological sludge by its
volume and weight. Samples could be drawn once a shift or once a day as desired.
PROCEDURE:
1.

## Draw the sample from aeration tank.

2.

Mix the sample thoroughly and transfer one litre of it in Imhoff cone/ measuring
cylinder.

3.

4.

5.

6.

## Pour off the supernatant.

7.

Transfer gradually the settled mass in a pre-weighed filter paper. As far as possible take
bigger size diameter (18.5 cm) filter paper.

8.

After complete filtration, transfer the filter paper to oven and dry it at 103 oC to
104oC.

9.

Dry the mass thoroughly and transfer the filter paper in to dessicator.

10.

## Weigh the filter paper together with sludge.

11.

Repeat the procedure of drying and weighing to a constant weight. Record the constant
weight for determining MLSS.

12.

Transfer the preweighed crucible with dried sludge filter paper to a muffle furnace
and heat till buff coloured residue is obtained. Cool the crucible, transfer it to a
dessicator and weigh it to know the weight of Ash to determine MLVSS.

CALCULATION:
volume occupied by sludge after 30 min settling
SVI

=
Volume of sample taken in Imhoff Cone/cylinder
(Y-X)

MLSS =
Volume of sample taken (1 litre)
Where,
Y
=
X
=
YX =

## Weight of filter paper with sludge (MLSS)

Weight of filter paper alone
Weight of sludge alone

MLVSS

(Y - X) - Z}
Volume of sample taken (1 litre)
Where,
Y
X
Z

=
=
=

## Weight of filter paper with sludge (MLSS)

Weight of filter paper alone
Weight of Ash or Residue left in crucible

## DETERMINATION OF FIXED AND VOLATILE SOLIDS

PRINCIPLE:
The residue is ignited to constant weight at 550 + 50oC. Remaining solids represents the fixed
total, dissolved, or suspended solids while the weight lost on ignition is the volatile solids. The
determination is useful in control of waste water treatment plant operation because it offers a
rough approximation of the amount of organic matter present in the solid fraction of waste water,
activity sludge, and industrial wastes.
INTERFERENCES:
Negative errors in the volatile solids may be produced by loss of volatile matter during drying.
Determination of low concentrations of volatile solids concentration may be subject to
considerable error. In such case, measure for suspect volatile components by another test, for
example, total organic carbon.
APPRATUS:
a.
b.
c.
d.
e.

Evaporating Dishes.
Drying oven.
Analytical balance.
Muffle furnace for the operation at 550 + 50O C.
Dessicator.

PROCEDURE:
a. Preparation of evaporating dish:
If the volatile solids are to be measured ignite clean evaporating dish at 550 + 50oC for 1 h in
a muffle Furnace. Store dish in dessicator until needed. Weight immediately before use.
b. Sample analysis:

Ignite the residue to constant weight in a muffle furnace at a temperature of 550 + 50oC

Have furnace up to temperature before inserting sample. Usually, 15 to 20 min ignition are
required. Let dish or filter dish cool partially in air until most of the heat has been dissipated.
Transfer to a dessicator for final cooling in a dry atmosphere. Do not overload on dessicator.
Weight dish or dish as soon as it has cooled to balance temperature. Repeat cycle of igniting,
cooling, desiccating, & weighing until a constant weight is obtained or weight loss is less
than 4% of previous weight.

CALCULATION:
(A B) 1000
Mg volatile solid/L

=
Sample volume, mL
(B C) 1000

Mg fixed solids/L

=
Sample volume, mL

Where:
A = Weight of residue + Dish before ignition, mg,
B = Weight of residue +Dish or filter after ignition, mg, and
C = Weight of dish or filter, mg

## DETERMINATION OF TOTAL SOLIDS

PRINCIPLE:
A well mixed sample is evaporated in a weighed dish and dried to constant weight in an oven at
103 to 105oC. The increase in weight over that of the empty dish represents the total solids. The
results may not represent the weight of actual dissolved and suspended solids in wastewater
sample.
INTERFERENCE:
Highly mineralized water with a significant concentration of calcium, magnesium, chloride,
and/or sulphate may be hygroscopic and may require prolonged drying, proper desiccation, and
rapid weighing, Exclude large, floating particles or submerged agglomerates of nonhomogeneous materials from the sample if it is determined that their inclusion is not desired in
final results. Disperse visible floating oil & grease with a blender before withdrawing a sample
portion for analysis. Because excessive residue in the dish may form a water trapping crust,
limit sample to no more than 200 mg residue.
APPARATUS:
a. Evaporating dishes
b. Muffle furnace for operation at 550 + 50oC.
c. Steam bath.
d. Desiccator.
e. Drying oven
f. Analytical balance.

PROCEDURE:
a. Preparation of evaporating dish:
If the volatile solids are to be measured ignite clean evaporating dish at 550 + 50oC for 1 h in
a muffle Furnace. If only total solids are to be measured, heat clean dish to 103 to 105 oC for
1 h. Store dish in dessicator until needed. Weigh immediately before use.
b. Sample analysis:
Choose sample volume that will yield a residue between 2.5 mg and 200 mg. Transfer a
measured volume of well mixed sample to preweighed dish and evaporate to dryness on a
steam bath or in a drying oven. If necessary, add successive sample portions to the same dish
after evaporation. When evaporating in a drying oven, lower temperature to approximately
2oC below boiling to prevent splattering. Dry evaporate sample for at least 1 hr in an oven at
103 to 105oC, cool dish in dessicator to balance temperature, and weigh. Repeat cycle of
drying, cool, desiccating, and weighing until weight loss is less than 4% of previous weight
or 0.5 mg, whichever is less.
CACULATION:
(A B) 1000
Mg total solids/ L =
Sample volume, mL
Where:
A = Weight of drying residue + dish, mg, and
B = Weight of dish, mg

## DETERMINATION OF TOTAL DISSOLVED SOLIDS

PRINCIPLE:
A well-mixed sample is filtered through a standard glass fiber filter, and the filtrate is evaporated
to dryness in a weighed dish and dried to constant weight at 180oC. The increase in the dish
weight represents the total dissolved solids.
INTERFERENCES:
Highly mineralized water with a considerable Calcium, Magnesium, Chloride, and/or Sulphate
content may be hygroscopic and require prolonged drying, proper dessication, and rapid
weighing. Samples high in bicarbonate require careful and possible prolonged drying at 180 oC to
insure complete conservation of bicarbonate to carbonate. Because excessive residue in the dish
may form a water trapping crust, limit sample to no more than 200 mg residue.
APPARATUS:
a. Evaporating dishes
b. Muffle furnace for operation at 550 + 50oC.
c. Steam bath.
d. Dessicator.
e. Drying oven
f. Analytical balance.
g. Glass - fiber filter disks* without or ganic binder.
h. Gooch crucible.
i. Drying oven

PROCEDURE:
a. Preparation of glass filter disk:
Insert disk with wrinkled side up into filtration apparatus. Apply vacuum and wash
disk with three successive 20- ml volumes of distilled water. Continue suction to
remove all traces of water. Discarded washing.
b. Preparation of evaporating dish:
If the volatile solids are to be measured ignite clean evaporating dish at 550 + 50oC
for 1 hr in a Muffle Furnace. If only total solids are to be measured, heat clean dish
to 180+2oC for1 hr. Store dish in dessicator until needed. Weigh it immediately
before use.
c. Selection of filter and sample sizes:
Choose sample volume to yield between 2.5 and 200mg dried residue, If more than 10
min are required to complete filtration, increase filter size or decrease sample volume but
not less than 2.5 mg residue.
d. Sample analysis:
Filter the measured volume of the well mixed sample through glass fiber filter paper,
wash with three successive 10ml volume of distilled water, allowing complete drainage
between washing, and continue suction for about 3 min after filtration is complete. Transfer
filtrate to a weighed evaporating dish and evaporate to dryness on a steam bath. If filtrate
volume exceeds dish capacity add successive portion to the same dish after evaporation.
Dry for at least 1h in an oven at 180+ 2oC, cool in a dessicator to balance temperature, and
weigh. Repeat drying cycle of drying, cooling, desiccating, and weighing until a constant

weight is obtained or until weight loss is less than 4% of previous weight or 0.5 mg,
whichever is less.

CALCULATION:
(A B) 1000
Mg total dissolved solids/L =
Sample volume, mL
Where:
A = weight of dried residue + dish, mg, and
B = weight of dish, mg

## DETERMINATION OF TOTAL SUSPENDED SOLIDS

PRINCIPLE:
A well-mixed sample is filtered through a standard glass-fiber filter, and the residue retained on
the filter is dried to a constant weight at 103 to 105 oC. The increase in weight of the filter paper
represents the total suspended solids.
INTERFERENCES:
Exclude large floating particles or submerged agglomerates of non-homogeneous materials from
the sample if it is determined that their inclusion is not desired in the final results. Because
excessive residue on the filter may form a water- entrapping crust, limit the sample size to that
yielding no more than 200mg residue. For sample high in dissolved solids thoroughly wash the
filter to ensure removal of the dissolved material. Prolonged filtration times resulting from filter
clogging may produce high results owing to excessive solids capture on the clogged filter.
APPARATUS:
a. Muffle furnance.
b. Drying oven.
c. Analytical balance.
d. Gooch crucible.
e. Glass-fiber filter disks.
f. Planchet,* aluminium or stainless steel, 65-mm diam.

10

PROCEDURE:
a. preparation of glass-fiber filter disk:
Insert disk with wrinkled side up in filtration. Apply vacuum and wash disk with three
successive 20-ml portion of distilled water. Continue suction to remove all traces of water,
and discard washing. Remove filter from filtration apparatus and transfer to an aluminium or
stainless steel plantchet as a support. Alternatively remove crucible and filter combination if
a Gooch crucible is used. Dry in an oven at 103 to 105 oC for 1 h. If volatile solid are to be
measured, ignite at 550+ 50oC for 15 min in a muffle furnace. Cool in dessicator to balance
temperature and weigh. Repeat cycle drying igniting, cooling, desiccating, and weighing
until weight loss is less than 0.5 mg between successive weighing. Store in desiccator until
needed. Weigh immediately before use.
b. Selection of filters and sample sizes:
Choose sample volume to yield between 2.5 and 200mg dried residue, if more than 10 min
are required to complete filtration, increase filter size or decrease sample volume but do not
less than 2.5 mg residue. For non-homogenous sample such as raw waste water, use a large
filter to permit filtering a representative sample.
c. Sample analysis:
Assemble filtering apparatus and filter and begin suction. Wet filter with a small volume of
distilled water to set it. Filter a measured volume of well mixed through the glass fiberfilter.Wash with three successive 10 ml volume of distilled water, allowing complete drainage
between washing and continue suction for about 3 min after filtration is complete. Carefully
remove filter from filtration apparatus and transfer to an aluminium or stainless steel
planchet as a support. Alternatively, remove the crucible adapter if a Gooch crucible is used.

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Dry for at least 1 h at 103 to105oC in an oven, cool in a desiccator to balance temperature,
and weigh. Repeat drying cycle of drying, cooling, desiccating, and weighing until a constant
weight is obtained or until weight loss is less than 4% of previous weight or 0.5 mg,
whichever is less.
CALCULATION:
(A B)

1000

## Mg total suspended solid/L =

Sample volume, mL

## A = weight of filter + dried residue, mg and

B = weight of filter, mg.

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## PROCEDURE FOR DETERMINATION OF TURBIDITY OF A WATER SAMPLE

USING A NEPHELOMETER (TURBIDITY METER)
INTRODUCTION:
Turbidity is due to presence of clay, silt and other suspended or colloidal matter in the samples.
Turbidity caused by the floating or settleable solids is known as apparent turbidity and is
removable by filtration. But, turbidity caused by colloidal particles exhibit Tyndall phenomenon
and this phenomenon is made use of to measure turbidity of the given sample.
CALIBRATION OF TERBIDITY METER:
1.

Ensure availability of 230 V Single Phase stabilised power supply from the mains.

2.

## Preparation of Standard Formazine:

a)Solution I: Weigh accurately 5 g of Hydrazine Sulphate (NH 2)2 H2SO4 and transfer into a 500
ml volumetric flask. Add distilled water to make up to the mark. Allow it to stand for four
hours.
b) Solution II: Weigh accurately 50 g of Hexa-methylene- tetra-amine.
APLAB TURBIDOMETER
Select the calibration graph for the desired range and place the filter frame in position. Fill
tube to the mark with test sample and place the plunger. Remove the bubbles under plunger if
any. Close the door of the apparatus and switch on the light. Balance the light intensity of
the central spot with the surrounding field by turning the dial. Samples having a turbidity
higher than 150 ppm may be tested by diluting the sample with water of very low Turbidity and
multiply results by dilution factor.
SIGNIFICANCE:

13

Turbidity

aesthetic

## sense and hence determines

potability of water.
*

## Efficiency of water treatment units is measured in terms of turbidity removal.

Turbidity determines the cost of chlorination. More the turbidity more is the cost.

If turbidity is more, light penetration will be less and algal development will be deaccelerated, which causes the reduction in the rate of production of D.O. in water.

14

## DETERMINATION OF SETTEABLE SOLIDS

GRAVIMETRIC METHOD
INTRODUCTION:
Settleable solids in surface and saline water as well as domestic and industrial wastes may be
determine and reported on either a volume (ml/ L) or a weight (mg/L) basis.

APPARATUS:

a. Muffle furnace.
b. Drying oven.
c. Analytical balance.
d. Gooch crucible.
e. Glass-fiber filter disks.
f. Plantchet,* aluminium or stainless steel, 65-mm dia.
g. Glass vessel with a minimum diameter of 9cm.

PROCEDURE:
a. Determination total suspended solid of well-mixed sample
b. Pour a well-mixed sample into a glass vessel of not less than 9cm dia using not less than
1 L and sufficient to give a depth of 20cm. Alternatively use a glass vessel of greater
diameter and large volume of sample. Let stand quiescent for 1 h. and, without disturbing

15

the settled or floating material, siphon 250ml from centre of container at a point halfway
between the surface of the settled material and the liquid surface. Determine the total
suspended solids (milligrams per litre) of this supernatant liquor. These are non-settle
able solids.

CALCULATION:
Mg settleable solids/L = mg of total suspended solide/L - mg of non settleable solid/L

16

## DETERMINATION OF COLOR OF A LIQUID SAMPLE

PRINCIPLE:
Color in water is due to natural metallic ion such as Mg, Fe and humus and peat materials,
planktons, weeds and industrial waste. Color is determined by visual comparison of the sample
with known concentration of colored solution. The platinum-cobalt method of measuring colour
is a standard method. The unit of colour being produced by one mg/litre in the form of the
chloro-platinum ion. The ratio of cobalt to platinum may be varied for matchin the colour in
special cases. The colour of water depends on pH. As the pH increases colour intensity also
increases. It is advisable to determine and record the pH.
REQUIREMENT:
Nessler's tube of 50 ml capacity and a pH meter.
PREPARATION OF STANDARD: (Potassium chloroplatinate (K2PtCl6)
Dissolve exactly 1.246 g of potassium chloro-platinate and 1.0 g cobaltous chloride hexahydrate (CoCl2, 6H2O) in distilled water. Add 100 ml conc. HCl and make up to 1000 ml with
distilled water. The above stock colour standard solution has a colour of 500 Hazen Units.
Prepare standards having colour concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70 by
diluting 0.5, 1.0, 1.5, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 and 7.0 ml stock color standard with
distilled water to 50 ml in nessler tubes.
contamination when not in use.
PROCEDURE:

17

## Protect standards against evaporation and

Estimation of colour of the intact sample is carried out by comparison of the color of the
sample taken in a nessler's tube with the standards already prepared and preserved by looking
vertically downward through the tubes towards against a pure white surface placed at such an
angle that the light is reflected upward through the column of liquid. If turbidity is present, filter
the sample and report the colour as apparent colour. If the colour
dilute the sample

with distilled water in known proportion until the colour is within the

range.
CALCULATION:
Color unit

where,
A

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## MEASUREMENT OF HOURLY, DAILY MINIMUM & DAILY MAXIMUM

TEMPERATURES
INTRODUCTION:
Vertical temperature gradient is an indicator of atmospheric stability affecting the plume rise.
MEASUREMENT:
Temperature measurements are made with the help of mercury bulb thermometer. An ordinary
thermometer indicate temperature values in different ranges such as 0 to 100 oC, and 0 to 50 oC
with different sensitivities. Temperature measurements are noted at different intervals of time
in a day. Hourly

## temperature measurements are generally recorded at a meteorological

station. Minimum and Maximum temperatures that occurred in 24 hours (full day) period are
manually observed from Maximum and Minimum Thermometers at fixed hour of the day
(0800 hour) and recorded and reported as minimum and maximum daily temperatures. The
thermometers markers are readjusted at the time of recording of the readings.

However, automatic instruments based on sensors and electronic data recorders are also available
which record temperature measurements with high sensitivities capable of recording
continuously through out the day.

19

DETERMINATION OF CONDUCTIVITY
INTRODUCTION:
Conductivity is a numerical expression of the ability of a sample in a solution to carry an electric
current and is linearly proportional to the conductance of the solution. It is measured by the use
of a conductivity meter.

APPARATUS:
a. Self-contained conductivity instrument.
b. Thermometer.
c. Conductivity cell.
REAGENT:
a. Conductivity water:
Pass the distilled water through a mixed-bed deionizer and discard first liter. Conductivity
should be less than 1mho/cm.

## b. Standard potassium chloride solution:

KCL, 0.0100 M: Dissolved 745.6 mg anhydrous KCL in conductivity water and dilution to
1000ml at 25oC has conductivity of 1413 mhos/cm. It is satisfactory for most sample when
the cell has a constant between 1 and 2. For other cell constant, use the stronger and weaker
solution store in a glass-stoppered borosilicate bottle.

20

PROCEDURE:
a. Determination of cell constant:
Rinse conductivity cell with at least three portion of 0.01 M KCL solution. Adjust
temperature of a fourth portion to 25.0 0.1 oC. Measure the sample resistance of this portion
and note temperature. Compute cell constant, C.
C = (0.001 413) (RKCL) [1 + 0.0191 ( t 25 ) ]
Where :
RKCL = measured resistance, ohms, and
t

= observed temperature, o C

b. Conductivity measurement :
Rinse cell with one or more portion of sample. Adjust temperature of final portion to 25.0
0.1oC. Measure sample resistance or conductivity And note temperature.
CALCULATION:
The temperature coefficient of most waters is only approximately the same as that of
Standard KCL Solution. The more temperature of measurement deviates from 25.0 oC, The
greater the uncertainty in applying the temperature correction. Report all conductivity at 25.0oC.
a. When the sample resistance is measured, conductivity at 25oC is:
(1 000 000) ( C )
K=

21

R m [ 1 + 0.0191 ( t 25 ) ]

Where:
K

Rm =

## Measured resistance of sample, ohms, and

temperature of measurement.

## b. When the sample conductivity is measured, conductivity at 25o C is:

( Km ) ( 1 000 000 ) ( C )
K=
1 + 0.0191 ( t 25 )

Where:
Km = measurement conductivity, mhos at to C, and other unit are defined as above.

22

DETERMINATION OF ACIDITY
The acidity of water is usually due to the presence of uncombined CO 2 and the mineral acids and
salts of strong acid and weak bases. It can be distinguished as follows:
a)

b)

pH of 8.3.

Accordingly,

if

indicators

are

employed w.r.t.

REAGENTS:
1.

## Standard 0.02 N NaOH:

Dissolve 0.8 g of NaOH crystals in one litre of distilled water. Standardize the solution
with 0.02 N sulphuric acid using methyl red or phenolpthalein as indicator.

2.

Phenolpthalein indicator:
Dissolve 0.5 g phenolpthalein in 100 ml of 50% ethanol.

3.

## Methyl Orange indicator:

Dissolve 50 mg methyl orange in 100 ml of distilled water.

PROCEDURE:
Take 50 ml sample in conical flask. Add to it one drop of methyl orange indicator. If it gives
red colour, it means mineral acidity is available. Titrate it with 0.02 N NaOH to yellow end
point. Note ml of NaOH used.
If yellow colour forms on addition of methyl orange, the methyl orange acidity is absent.

23

In another flask take 50 ml sample. Add 0.5 ml phenolphthalein indicator. If it does not give
any colour, titrate with 0.02 N NaOH to pink end point. Note the ml of solution used.
If phenolphthalein gives a pink colour on addition in the sample, acidity is absent.

CALCULATION:
V, NaOH (Methyl Orange) x 50 x 1000 x N
Mineral acidity mg/l =
(as CaCO3)

ml of sample (50)
V, NaOH (Phenolphthalein) x 50 x 1000 x N

## Total acidity mg/l

(as CaCO3)

=
ml of sample (50)

24

DETERMINATION OF ALKALINITY
Alkalinity of water is due to hydroxides, carbonates and bicarbonates of elements such as
calcium, magnesium, sodium, potassium or ammonia and it is expressed in CaCO3 scale. It is a
quantitative capacity of water to neutralize a strong acid to a designed pH as below:
Alkalinity mg /lit

End point pH

30

5.1

150

4.8

500

4.5

3.7

REAGENTS:
1.

## Sodium Carbonate solution approximately 0.05 N:

Dry 3 to 5 g primary standard Na2CO3 at 2500C for 4 hours and cool in a dessicator.
Weight 2.5 g and dilute to one litre with distilled water.

2.

## 0.1 N H2SO4 or HCl:

Prepare stock solution of 0.1 N Sulphuric acid or Hydrochoric acid by diluting 3.0
ml concentrated H2SO4 or 8.3 ml concentrated HCl to one litre with distilled water.

Standardisation
Standardize against 40 ml 0.05N Na2CO3 solution with about 60 ml water, in a beaker
by titrating potentiometrically to pH of about 5. Boil for 3 to 5 minutes under a watch
glass cover. Cool to room temperature, rinse the cover glass into the beaker, and
titrate with 0.1 N acid using bromo-cresol green as indicator (0.1 N solution = 5 mg
CaCO3 /ml).

25

AxB
Normality,

N=
53 x C

3.

## Standard Sulfuric acid or Hydrochloric acid, 0.02 N:

Dilute 200 ml of the 0.1 N standard acid to one litre with distilled water. Standardize
by titrating 15 ml 0.05 N Na2CO3 as above (0.02 N solution = 1 mg CaCO3 /ml).

4.

## Mixed indicator or Methyl Orange indicator:

Mixed Indicator:
Dissolve 20 mg Methyl red and 100 mg bromocresol green in 100 ml 95% ethyl or
isopropyl alcohol.
Methyl Orange indicator:
Dissolve 50 mg of methyl orange in 100 ml distilled water.

PROCEDURE:
Take 50 ml of sample in a conical flask. If sample is turbid filter through filter paper. Add
one drop of mixed indicator in the sample. Keep blank distilled water with the same quantity of
indicator for comparison. Titrate with standard 0.02N H2SO4. End point is green to pink.
Potentiometric titration:
Take appropriate quantity of sample in a beaker. Immerse the pH meter electrode in it and
check the pH. Go on titrating the sample with standard acid (0.02 N or 0.1 N) to the end point
pH. Record the ml of acid used.

26

CALCULATIONS:
V x N x 50 x 1000
Total alkalinity mg/l =
(as CaCO3)

ml of sample

Where,
V

ml of H2SO4.

Normality of H2SO4.

50

## Equivalent weight of CaCO3.

SIGNIFICANCE
Information about alkalinity is useful in variety of sanitary engineering practice such as
chemical coagulation, water softening, corrosion and corrosion
treatment and biological treatment.

27

control,

Industrial

waste

## DETERMINATION OF AMMONIACAL NITROGEN

PRINCIPLE:
An intensely blue compound,indophenol,is formed by the reaction of ammonia, hypochlorite,
and phenol catalysed by amanganous salt.
INTERFERENCE:
Alkalinity over 500 mg as CaCO3/l, acidity over 100mg as CaCO3/L, and turbidity
interfere.Remove these interferences by preliminary distillation.
2) APPARATUS
a.Colorimetric equipment:
One of the following is required:
1) Spectrophotometer, for use at 630 nm with a light path of approximately 1 cm.
2) Filter photometer, equipped with a red-orange filter having a maximum transmittance near
630 nm and providing a light path of approximately 1 cm.
b) Magnetic stirrer.
3) REAGENTS
a. Ammonia-free water:
Use for making all reagents.
b.Hypochlorous acid reagent:
To 40 ml water add 10 ml 5%NaOCl solution prepared from commercial bleach. Adjust pH to
6.5 to7.0 with HCl. Prepare this unstable reagent weekly.
c. Manganous sulphate solution,0.003M:
Dissolve 50mg MnSO4.H2O in 100mL water.
d. Phenate reagent:
Dissolve 2.5 g NaOH and 10 g phenol, C6H5OH, in 100mL water.
because this reagent darkens on standing, prepare weekly.(Caution :Handle phenol with
care.)

28

## e. Stock ammonium solution:

dissolve 381.9 mg anhydrous NH4Cl, dried at 100oC, in water,
and dilute to1000mL; 1.00mL=100g N =122g NH3 .
f. Standard ammonium solution:
Dlute 5.00mL stock ammonium solution to 1000 mL with water; 1.00mL= 0.500 g N
=0.607mg NH3
4) PROCEDURE
Procedure Treatment of sample:
To a 10.0mL sample in a 50-mL beaker, add 1 drop (0.05 mL) MnSO 4 solution. Place on a
magnetic stirrer and add 0.5mL hypochlorous acid reagent. Immediately add, a drop at a time,
0.6 mL phenate reagent. Add reagent without delay using a bulb pipet or a buret for convenient
delivery. Mark pipet for hypochlorous acid at the 0.5mL level and deliver the phenate reagent
from a pipet or buret that has been calibrated by counting the number of drops to 0.6 mL. Stir
vigorously during addition of reagents. Because color intensity is affected by age of reagents,
carry a blank and a standard through the procedure with each batch of samples.
Measure absorbance using reagent blank to zero the spectrophotometer. Color formation is
complete in 10 min and is stable for at least 24 h. although the blue color has a maximum
absorbance at 630 nm, satisfactory measurements can be made in the 600- to 660-nm region.
Preparation of standards:
Prepare a calibration curve in the NH3-N range of 0.1 to 5 g, treating standards exactly as the
sample. Beers Law governs.
5. CALCULATION
Calculate ammonia concentration as follows:
mg NH3-N/L (11.1 mL final volume) =A x B X D
CxS
E

29

Where
A= absorbance of sample,
B = NH3-N in standard, g,
C= absorbance of standard,
S= volume of sample used, mL,
D= volume of total distillate collected, mL, including acid absorbent, neutralizing agent, and
ammonia-free water added, and

## E= volume of distillate used for color development, mL.

The ratio D/E applies only to distilled samples.

30

DETERMINATION OF CHLORIDES
ARGENTOMETRIC METHOD -4500 Cl- B
( Titrimetric )
PRINCIPLE:
In neutral or slightly alkaline solution, potassium chromate indicates the end point of AgNO 3
titration of chloride. AgCl2

## formed.Chloride occurs in all natural waters in varying conditions. Chloride content of

water can be determined by the use of potassium chromate and silver nitrate. If AgNO 3 is
added to water containing chlorides and chromate, Ag ++ will precipitated quantitatively with
chlorides present in the sample before red silver chromate is formed. Thus when a reddish
color of Ag2CrO4 develops in solution it is assumed that all the chlorides is precipitated.
Therefore amount of AgNO3 required to produce reddish coloration in the sample to which
K2CrO4 is previously added, indicates the amount of chloride present in the sample.
REAGENTS:
1.

Chloride-free water:

2.

## Potassium Chromate indicator solution:

Dissolve 50 g of potassium chromate in a little distilled water. Add silver nitrate
solution until a definite red ppt is formed. Let stand for 12 hrs, filter and dilute to 100 ml
with distilled water.

3.

## Standard Sodium Chloride solution (0.0141):

Dissolve 824 mg NaCl, dried at 140 oC, in distilled water and dilute to exactly 1000 ml (1
ml = 500 ug Cl-).

31

4.

## Standard AgNO3 (0.0141 N) solution, titrant:

Dissolve 2.395 g of silver nitrate in 1000 ml of distilled water. Standardize against 10
ml of std. 0.0141 N NaCl solution, using potassium chromate as an indicator.
10 x 0.0141
Normality of AgNO3 =
ml of AgNO3

5.

## Aluminium Hydroxide solution:

Dissolve 125 gm of aluminium potassium sulfate or aluminium ammonium sulfate in 1
litre distilled water. Warm to 60 oC and add 55 ml of concentrated Ammonium
Hydroxide.

6.

## Special reagents for pretreatment:

a)

Sulfuric acid

(1:1)

b)

Hydrogen peroxide

30 %

c)

Sodium Hydroxide

1N

PROCEDURE:
Sample preparation:
a)

If the sample is highly colored, add 3 ml Al (OH) 3 suspension, mix, let settle and
filter.

b)

If sulfide, sulfite or thiosulphate is present, add one ml H2O2 and stir for one minute.

32

Titration:
Titrate samples in the pH range 7 to 10. Adjust pH in this range with H 2SO4 or NaOH solution.
Take 100 ml of sample or diluted sample. Add 1 ml K 2CrO4 indicator solution. Titrate with
standard AgNO3 solution to a brick red or pinkish yellow end point and establish the reagent
blank (distilled water) value by the titration method given above. A blank of 0.2 to 0.3 ml is
usual for the method.
CALCULATION:
(A - B) x N x 35.45 x 103
Cl- mg/litre

=
ml sample

Where,
A

## ml titration for blank

Normality of AgNO3

33

DETERMINATION OF FLUORIDE
(BY SPADNS METHOD 4500-F D)
PRINCIPLE:
The SPADNS colorimetric method is based on the reaction between fluoride and a zirconiumdye lake. Fluoride reacts with the dye lake, dissociating a portion of it into a colorless complex
anion (ZrF6-2); and the dye. As the amount of fluoride increases, the color produced becomes
progressively lighter.The reaction rate between fluoride and zirconium ions

is influenced

greatly by the acidity of the reaction mixture. If the proportion of acid in the reagent is increased,
the reaction can be made almost instantaneous under such conditions. However, the effect of
various ions differs from that in the conventional alizarin methods. The selection of the dye
for this rapid fluoride method is governed largely by the resulting tolerance to these ions.
INTERFERENCE:
Whenever any one substance is present in sufficient quantity to produce an error of 0.1 mg/l or
whenever the total interfering effect is in doubt, distill the sample. Also distill colored or turbid
samples. In some instances, simple dilution or adding appropriate amounts of interfering
substances to the standards may be used to compensate for the interference effect. If
alkalinity is the only interference, neutralize it with either hydrochloric or nitric acid. Chlorine
interferes and provision for its removal is made.
Volumetric

## measurement of sample and reagent is extremely important to analytical

accuracy. Use samples and standards at the same temperature or at least within 2 oC. Maintain
constant temperature throughout the color development period. Prepare different calibration
curve for different temperature ranges.
APPARATUS:
Spectrophotometer, for use at 570 nm, providing 1 cm light path.

34

REAGENTS:
1.

## Stock Fluoride solution:

Dissolve 221.0 mg anhydrous sodium fluoride in 1000 ml distilled water; 1 ml = 100 ug
F-.

2.

## Standard Fluoride solution:

Dilute 100 ml, stock fluoride solution to 1000 ml with distilled water; 1 ml = 10 ug F-.

3.

## SPADNS Solution or Reference Solution:

Dissolve 958 mg SPADNS, Sodium 2-paranaphthalene disulfonate, also

called

## 4,5-dihydroxy- 3- parasulfophenylazo- 2,7-

naphthalene disulfonic acid trisodium salt, in distilled water and dilute to 500 ml. It is
stable for one year if protected from direct sunlight.
4.

Zirconyl-acid reagent:
Dissolve 133 mg zirconyl chloride octahydrate in 25 ml distilled

5.

## Mixed Acid-Zirconyl SPADNS Reagent:

Mix equal volumes of Zirconyl acid reagent and SPADNS reagent. The mixed reagent
is stable for atleast two years.

PROCEDURE:
Preparation of a Standard Curve:
1.

Prepare fluoride standards in the range of 0 to 1.4 mg/l of Fquantities of standard fluoride solution

by diluting appropriate

## temperature of the standards at the time of making the standard curve.

35

the

2.

Add 10 ml of mixed Acid Zirconyl and SPADNS reagent to each of the standards and
mix well.

3.

## Set absorbance to zero on the spectrophotometer at 570 nm using reference solution as

blank.

4.

Obtain absorbance readings with all the standards and plot a curve of Micro gram of
fluoride Vs. Absorbance.

COLOUR DEVELOPMENT:
Use a 50 ml sample or a portion diluted to 50 ml with distilled water. Adjust sample
temperature to that prevalent at the time of making the Standard curve. Add 10 ml of
mixed Acid Zirconyl and SPADNS reagent. Mix well. Set the absorbance to zero with
the reference solution and read absorbance with the sample at 570 nm.
CALCULATIONS:
g of F- read from curve
Mg/l of the fluoride

B
X

ml of sample
Where,
B

36

## DETERMINATION OF TOTAL HARDNESS

EDTA METHOD
PRINCIPLE:
Hardness is generally caused by calcium and magnesium ion present in water. Polyvalent ions of
some other metals like strontium, iron aluminium, zinc and manganese etc. are also capable of
precipitating the soap and thus contributing to the hardness. However , the concentration of these
ions is very low in natural waters, therefore, hardness is generally measured as concentration of
only calcium and magnesium as calcium carbonate, which are far higher in quantities over
hardness producing ions.
Calcium and magnesium form a complex of wine red colour with Eriochrome Black T at pH of
10.0 0.1. The EDTA has got a stronger affinity toword Ca ++ and Mg++ and, therefore, by
addition of EDTA, the former complex is broken down and anew complex of blue colour is
formed.
REAGENT:
A. EDTA solution, 0.01 M
Dissolved 3.723 g of disodium salt of EDTA in distilled water to prepare 1 litre of solution.
Store in Polyethylene or Pyrex bottle.

B. Buffer solution
a) Dissolved 16.9 g ammonium chloride ( NH 4CL) in 143 ml of concentrated ammonium
hydroxide ( NH4OH).
b) Dissolved 1.179 g of disodium EDTA AND 0.780 G OF MgSO4. 7 H2O in50ml distilled water.
Mix both (a) and (b) solutions and dilute to 250 ml with distilled water.

## C. Eriochrome Black T ( Solochrome Black T ) indicator

37

Mix 0.40 g of Eriochrome Black T, with 100g Nacl (A.R.) and grind.

## D. Sodium sulphide solution

Dissolved 5.0 g of Na2S.9H2O or 3.7 g in 100 ml of distilled water. Tightly close the bottle to
prevent oxidation.
PROCEDURE:
1. Take 50ml of sample in a conical flask. If sample is having higher calcium, take a smaller
volume and dilute to 50 ml.
2. Add 1 ml of buffer solution.
3. If the sample is having higher amount of heavy metals add 1 ml of Na2S solution.
4. Add 100 200 mg of Eriochrome Black T indicator; the solution wine red.
5. Titrate the contents against EDTA solution. At the end point colour change from wine red to
blue.
CALCULATION:
ml EDTA used 1000
Hardness as mg/L CaCO3 =
ml of sample

## DETERMINATION DISSOLVED OXYGEN

38

PRINCIPLE:
The presence of certain oxidizing and reducing material may effectively interfere with the
determination of oxygen by converting iodide ion to iodine or vice- versa. The azide
modification remove the interference of such substances especially nitrite. Nitrite is destroyed by
sodium azide ( NaNa). The method, therefore, is suitable particularly in polluted water,
biologically treated water`and`in`BOD`sample.
REAGENT:
a. Sodium thiosulphate, 0.025 N
Dissolved 24.82 g of Na2S2O3.5H20 in boiled distilled water and make up the volume to 1
litre. Add 0.4 g of borax or a pallet of NaOH as stabilizer. This is 0.1 N stock solution Dilute
it to 4 times with boiled distilled water to prepare 0.025 N solution ( 250 100ml ).Keep in a
boron glass stoppered bottle.

## b. Alkali iodide azide solution

1.Dissolved 500 g of NaOH or 700 g of KOH and 150 g of KI in distilled water to make 1
litre of solution.
2. Dissolved 10 g of NaNa in 40 ml of distilled water. Mix the two solution 1 & 2.

## c. Manganous sulphate solution

Dissolved 100 g of KOH and 50 g of KI in 200ml of boiled distilled water and filter.

d. Starch solution
Dissolved 1 g of starch in 100 ml of warm ( 80o C 90o C ) distilled water and filter

## e. Sulphuric Acid (H2SO4) conc.

39

PROCEDURE:
1. Fill the sample in a glass stoppered bottle ( BOD bottle ) of known volume (100
300ml), carefully, avoiding any kind of bubbling and trapping of the air bubbles in the
bottles after place the stopper.
2. Pour 1 ml of each MnSO4 and alkaline KI solution (in case, the volume of the sample is
about 300 ml, instead of 1 ml of reagent add 2 ml solution of each), well below the
surface from he walls. The reagents can also be poured at the bottom of the bottle with
the help of special pipette syringes to ensure better mixing of the reagents with the
sample Use always, separate pipettes pipettes for these two reagent. A precipitate.will
appear.
3. Place the stopper and shake the contents well by inverting the bottle repeatedly. Keep the
bottle for some time to settle down the precipitate. If the titration is to be prolonged for
few day, keep the sample at this stage with precipitate.
4. Add 1 2 ml of concentrated H2SO4 and shake well to dissolved the precipitate.
5. Remove either the whole contents, or a part of them ( 50 100 ml ) in conical flask for
titration. Prevent any bubbling to avoid further mixing of oxygen.
6. Titrate the contents, within one hour of the precipitate against sodium thiosulphate
solution using starch as an indicator. At the end point, initial dark blue colour change to
colourless.
CALCULATION:
When whole contents have been titrated:
Burette reading 8000 N of Na2S2O3
DO, mg/l =
ml of sample taken

40

## Burette reading 8000 N of Na2S2O3

DO, mg/l =
V1 - V
V2 (

)
V1

Where,
V1 = volume of sample bottle after placing the stopper.
V2 = volume of the part content titrated.
V = volume of MnSO4 and KI.

## DETERMINATION OF KJELDAHL NITROGEN (Org- N + ammonia- N)

41

PRINCIPLE:
The digestion of the sample with H2SO 4 and potassium sulphate, converts all the organic
nitrogen and ammonia in to ammonium sulphate. However, most of other forms remain
unaffected. NaCl is added to prevent the partial reduction of nitrate to ammonia which converts
the NO3 into NOCl. The nitrogen in the form of ammonia sulphate can be determined by
distillation (as ammonia) at higher pH
REAGENTS:
a) Sulphuric acid H2SO4 conc. (sp. Gr. 1.84)
b) Copper sulphate solution 10%:
Dissolved 10 g copper sulphate in 100 ml of distilled water.
c) NaCl solution 10%:
Dissolved 10 g of NaCl in 100 ml of distilled water.
d) Potassium sulphate K2SO4, Solid
e) Sodium hydroxide, 10 N:
Dissolved 400 g of NaOH in distilled water to make 1 liter of solution.
f) Sodium hydroxide, 5 N:
Dissolved 200 g of NaOH in distilled water to make 1 liter of solution.
g) Hydrochloric acid, 0.01 N:
Dilute 12 N concentrated HCl (sp. gr. 1.18) to 12 times (8.34 100 ml) to prepare 1.0 N HCl.
Dilute it further to make 0.1 N HCl (100 1000 ml).Dilute 0.1 N HCl to 10 times (100 1000
ml).
h) Boric acid + Mixed indicator
a) Prepare 4% solution of H3 BO3 by dissolving 4 g boric acid in 100 ml warm distilled water.

42

b) Mix alcoholic solutions of bromocresol green (0.5 %) and methyl red ( 0.1%) in 2:1 ratio. Add
5 ml of mixed indicator in 100 ml of boric acid. If necessary ( only when the colour becomes
blue), adjust the pH with 0.01 N HCL until colour just turns faint pink to brown.
i) Phenolphthalein indicator:
Dissolved 0.5 g of phenolphthalein in 50 ml of 95% ethanol and add 50 ml of distilled water.
Add 0.05 N CO2 free NaOH solution drop wise, until the solution turns faintly pink.

PROCEDURE:
Digestion:
1. Take 40 ml of sample in a 100 ml Kjedahl flask.
2. Add 4 ml H2SO4 , 10 drops of Cuso4 solution (0.3ml), 6.0 g of solid potassium sulphate and
1ml of 10 % Nacl solution.
3. Heat the flask on a heater to avoid loss through foaming.
4. After the water boils off, the sample will turn dark due to decomposition of organic matter by
H2SO4. As the digestion proceeds, he colour of the sample turn pale green. Continue the heating
for additional 30 minutes.
5. Cool the flask and make up the volume to 100ml. Sometimes a cake is formed on dooling of
the digest. This cake can easily by dissolved by warming it gently with water. If the same digest
is to be utilised for determination of total phosphorous, neutralize it with 5N Naoh using
phenolphthalein as an indicator. At the end point the colour changes to faint pink, After the
neutralisation, make up the volume to 100 ml with distilled water.
Distillation:
6. Take 25 ml of the digest and perform the distillation according to the ammonia method. Exept
that 10 nml of 10 N NaOH is added instead of borax buffer.
7. Run a separate blank with distilled water using same amount of the chemicals.
8. Titrate the distillate ( in boric acid + mixed indicator) with 0.01 N HCL until the colour
changes from blue to brown or faint pink.

43

CALCULATION:
Kjeldahl N- mg/l = a b 0.01 100014 D / ml sample distilled
Where,
a = ml of HCL used with sample.
b= ml of HCL used with blank.
D = dilution factor (2.5). the original volume ( 40 ml) of sample has been made to
100 ml after digestion.
ORGANIC NITROGEN
Organic nitrogen can be determined by subtracting the concentration of ammonia from the
Kjeldahl nitrogen values.
Organic N = Kjeldahl-N NH3N

44

## DETERMINATION OF NITRATES BY BRUSINE METHOD

PRINCIPLE:
The reaction between nitrate and brucine produces a yellow color that can be used for the
colorimetric estimation of nitrate. The intensity of colour is measured at 410 nm. The reaction
rate between brucine and nitrate ion is affected significantly by the amount of heat generated
during the test. Heat control in the procedure is achieved by reagent addition sequence and
incubation of the reaction mixture for a precise interval of time at a known temperature. The
method is recommended only for the concentration range of 0.1 to 2.0 mg/l of NO 3-N, because
above this range the sensitivity of the method is poor. Ideal range for this method is from 0.1 to
1 mg/l of NO3-N else suitably dilute the samples.
INTERFERENCE:
All strong oxidising or reducing agents interfere. The presence of oxidising agents may be
determined by the addition of orthotolidine reagent, as the measurement of residual chlorine.
The interference by residual chlorine may be eliminated by the addition of sodium
arsenite, provided that the residual chlorine does not exceed 5 mg/l. A slight excess of
sodium arsenite will not affect the determination. Ferrous and ferric iron and quadvalent
manganese give slight positive interferences, but in concentrations less than 1 mg/l these are
negligible. The interference due to nitrite up to 0.5 mg/l NO 2-N is eliminated by the use of
sulfanilic acid. Chloride interference is masked by the addition of excess NaCl.
High concentrations of organic matter such as in un-diluted raw waste water usually will
interfere.
APPARATUS:
1.

2.5 cm.

2.

45

REAGENTS:
1.

## Stock nitrate solution:

Dissolve 721.8 mg anhydrous potassium nitrate, KNO 3 and dilute to 1 litre with
distilled water (1 ml = 0.1 mg N).

2.

## Standard nitrate solution:

Dilute 10 ml of stock solution to one litre with distilled water. Prepare immediately
before using.

3.

## Sodium arsenite solution:

Dissolve 5 gm NaAsO2 and dilute to one litre with distilled water (Toxic, take care to
avoid ingestion).

4.

## Brucine-sulfanilic acid solution:

Dissolve 1.0 g brucine sulfate and 0.1 g sulfanilic acid in approximately 70 ml hot
distilled water. Add 3 ml conc. HCl, cool and make up to 100 ml. This solution is
stable for several months. The pink color that develops slowly does not affect its
usefulness (Brucine is toxic-take care to avoid ingestion.)

5.

## Sulfuric acid solution:

Carefully add 500 ml conc. sulfuric acid to 125 ml distilled water. Cool to room
temperature before using and keep tightly stoppered to prevent absorption of
atmospheric moisture.

6.

## Sodium chloride solution:

Dissolve 300 g NaCl and dilute to 1000 ml with distilled water.

PROCEDURE:
1

## Preparation of nitrate standards:

Prepare nitrate standards in the range 0.1 to 1 mg/l by diluting 1, 2, 4, 7, 10 ml std.
nitrate solution to 10 ml with distilled water.

2.

Pre-treatment of sample:

46

If the sample contains residual chlorine, remove by adding 1 drop (0.05 ml) sodium
arsenite solution for each 0.10 mg chlorine and mix. Add 1 drop in excess to a 50 ml
portion.
3.

Color development:
Set up the required number of tubes in the wire rack, spacing them so that each
tube is surrounded by empty spaces. Include a reaction tube for a reagent blank and
reaction tubes for as many standards as desired. to each tube add 10 ml sample or a
portion diluted to 10 ml so that the sample volume taken for analysis contains between
0.1 and 8 ug NO3-N. Place the rack in a cool water bath and add 2 ml NaCl solution.
Mix thoroughly by hand and add 10 ml sulfuric acid solution. In no case use a
"vortex" mixer, since this type of

mixing

## produces inconsistent results in the

analysis. Mix again thoroughly by swirling and allow to cool. At this point, in any
turbidity or color is present or if optically unmatched colorimeter tubes, dry the
tubes and read a sample blank value against the reagent blank tube at 410 nm. Replace
the rack of tubes in the cool water bath and 0.5 ml brucine-sulfanilic acid reagent.
Swirl the tubes to mix thoroughly and then place the rack of tubes in a well stirred
boiling water bath that maintains a temp. of not less than 95oC. After exactly 20
min. remove the samples and immerse in a cold water bath. When thermal equilibrium
is reached at around room temperature, dry the tubes with tissue and

read the

standards and samples against the reagent blank at 410 nm in spectrophotometer. Check
the technique and the onsistancy of

## standards with each batch of samples.

Prepare a curve from the absorbance values of the standard minus the blank run together
with the samples. Correct the absorbance readings of the samples by subtracting their
sample blank values from their final absorbance values. Read the NO3-N directly from
the standard curve.

47

CALCULATION:
g NO3-N
mg/l Nitrate N =
ml sample
mg/l NO3 = mg/l Nitrate N x 4.43

48

DETERMINATION OF NITRITE
PRINCIPLE:
Nitrite forms a diazonium salt with sulphanilic acid in acid medium (2.0 2.5 pH), which
combines with a-naphthlylamine hydrochloride to form a pinkish dye. The colour so produce
obyes Beers law and can be determined colorimetrically.
REAGENT:
a. Disodium ethylene diamine tetra acetic acid (Na EDTA) solution
Dissolved 500 mg of disodium salt of EDTA in distilled water to make 100 ml solution.
b. Sulphanilic acid solution
Dissolved 600 mg of sulphanilic acid in 70 ml of hot distilled water and add 20 ml of
concentration HCL after cooling and dilute to 100ml.
c. a-naphthylamine hydrochloride solution
Dissolved 600 mg of a-naphthylamine hydrochloride in distilled water to which 1 ml
concentrated HCL has been add. Dilut the contents to 10ml and place in a cool place. If the
precipitate occurs after few day, reagent can be use further by filtering the solution.
d. Sodium acetate solution
Dissolved 1.232 g of anhydrous CH3COONa or 27.2 g of CH3COONa.3H2O in distilled
water to prepare 100 ml solution.

## e. Standard nitrite solution (1mg/NO2-N)

Dissolved 1.232 g NaNO2 in diluted to 1 litre (250mg/l NO 2-N). Dilute this solution 250
times (4

## 1000 ml) to prepare standard solution having 1 mg/l NO2-N.

49

PROCEDURE:
1. Take 50 ml of colourless filtered sample not having more than 1.0 mg/l NO2 N in a
conical flask. The colour can be removed by activated charcoal in case of coloured
sample.
2. Add 1 ml each EDTA , sulphanilic acid, a-naphthylaimine Hydrochloride and sodium
acetate solution in sequence.
3. A wine red colour will appear in the presence of nitrites. Take the reading at520nm.
4. Compare the absorbance with the standard curve to calculated the nitrite content.
5. Prepare the standard curve between 0.0 to 1.0 mg NO2- N/1 at the interval of 0.1
employing the same procedure as for the sample.

50

## DETERMINATION OF PHOSPHOROUS (PHOSPHATES PO4-3)

IS-3025 PART 31 (1988)
STANNOUS CHLORIDE METHOD
PRINCIPLE:
Ammonium molybdate reacts with ions of phosphorous in acidic medium to form a Heteropoly
acid i.e. Molybdo-phosphoric acid, which is reduced to an intensely colored complex
molybdenum blue by SnCl2. This is very reliable method and has minimum interference. The
minimum detectable concentration by this method is 3 micro gm per litre of phosphorous.
APPARATUS:
1.

2.

REAGENTS:
1.

## Phenolphthalein Indicator solution:

Dissolve 500 mg phenolphthalein in 100 ml of 50 % Ethyl alcohol.

2.

## Strong acid solution:

Slowly add 300 ml conc. sulfuric acid to about 600 ml distilled water. When cool, add 4
ml conc. nitric acid and dilute to 1000 ml.

3.

## Ammonium Molybdate Reagent - I:

Dissolve 25 g of ammonium molybdate (NH 4)Mo7O24.4H2O in 175 ml of distilled water.
Continuously add 280 ml conc. sulphuric acid to 400 ml distilled water in a separate
beaker, cool it and add ammonium molybdate solution to the acid solution and make the
volume 1000 ml with distilled water.

4.

## Stannous Chloride solution - I:

Dissolve 2.5 gm of fresh stannous chloride (SnCl 2. 2H2O) in 100 ml glycerol. Heat it in
water bath and stir with a glass rod to hasten dissolution. The solution is stable and
requires no preservation or special storage.

51

5.

## Standard Phosphate solution:

Dissolve 219.5 mg of dry anhydrous potassium di-hydrogen phosphate in 1000 ml
distilled water. Its 1 ml = 50 ug of orthophosphate phosphorous.

6.

PROCEDURE:
1.

## Preliminary sample treatment:

a)

If coloured, decolourise the sample by shaking about 200 ml sample with 250
mg activated carbon in an Erlenmeyer flask for 5 minutes. Filter the solution
through Whatman No.42 or equivalent to remove carbon.

b)

To a 100 ml sample (containing not more than 0.2 mg P) free from color and
turbidity, add 0.05 ml (1 drop) phenolphthalein indicator. If the sample turns pink,
add strong acid solution drop-wise to discharge the color. If acid requirement is
more than 0.25 ml (5 drops), reject and take a smaller sample and dilute to 100 ml
with distilled water after first discharging the pink color with acid.

2.

Color development:
Add, with thorough mixing after each addition, 4.0 ml Ammonium Molybdate Reagent -I
and 0.5 ml (10 drops) of Stannous Chloride reagent -I. The rate of color development
and the intensity of color depends on the temperature of the final solution, each 1 oC
increase producing about 1 % increase in color. Hence, samples, standards, and reagents
should be within 2 oC of one another and at a temperature between 20 and 30 oC.

3.

Color measurement:
After 10 minutes but before 12 minutes, allowing the same specific interval for all
determinations, measure the color photometrically at 690 nm and compare with a
calibration curve, using a distilled water blank. Prepare at least one standard with each
set of samples.

52

EXTRACTION:
REAGENTS FOR EXTRACTION:
1

Benzene-isobutanol solvent:
Mix equal volumes of benzene and isobutyl alcohol (Caution: The solvent is highly
flammable)

2.

## Ammonium Molybdate Reagent - II:

Dissolve 40.1 g of ammonium molybdate (NH 4)Mo7O24.4H2O in 500 ml of distilled
water. Slowly add 396 ml molybdate reagent I. Cool and dilute to 1000 ml.

3.

## Alcoholic sulfuric acid solution:

Cautiously add 20 ml conc. sulfuric acid to 980 ml methyl alcohol with continuous
mixing.

4.

## Dilute Stannous Chloride Reagent -II:

Mix 8 ml stannous chloride reagent I with 50 ml glycerol. This reagent is stable for at
least 6 months.

## PROCEDURE FOR EXTRACTION:

When increased sensitivity is desired or interferences must be overcome, extract the phosphate
as follows:
a)

Pipette a suitable portion into a 100 ml graduated extraction cylinder and dilute, if
necessary, to 40 ml with distilled water.

b)

## Add 50 ml benzene-isobutanol solvent and 15 ml Ammonium Molybdate Reagent - II.

c)

Close container at once and shake vigorously for exactly 15 seconds (If poly-phosphate is
present, any delay will increase the amount of it that will be included in the
orthophosphate value).

d)

Remove the stopper and using a pipette and a safety aspirator, withdraw 25 ml of
separated organic layer. Transfer to a 50 ml volumetric flask, add 15 to 16 ml alcoholic
sulfuric acid solution, swirl and add 0.5 ml (10 drops) dilute Stannous Chloride ReagentII, swirl, and dilute to the mark with alcoholic sulfuric acid. Mix thoroughly. After 10
min. but before 30 min. read against the blank at 625 nm. Prepare the blank by carrying

53

40 ml distilled water through the same procedure used for the sample. Read the
phosphate concentration from a calibration curve prepared by taking known phosphate
standards through the same procedural steps used for the samples.
CALIBRATION CURVE:
Using the Standard Phosphate solution having concentration 50 ug/ml of orthophosphate
phosphorous and preparing different concentration standards and following the same procedure
as per the preliminary treatment, colour development and colour measurement (absorbance) at
690 nm , plot a standard curve.
CALCULATIONS:
Calculate the results from the direct or the extraction procedures by the following equation:
mg of Phosphorous from standard curve
Phosphorous mg/l = ------------------------------------ml sample

54

1000

## DETERMINATION OF SULPHATES (SO4-2) IS-3025 PART 24 (1986)

GRAVIMETRIC METHOD
Sulfate is precipitated in acid medium as barium sulfate by theaddition of barium chloride.The
precipitation is carried out near the boiling temperature, and after a period of digestion the
precipitate is filtered, washed with water until free of chloride, ignited or weighed as BaSO4.
APPARATUS:
1.

Muffle furnace

2.

Dessicator

3.

4.

5.

Glass wares

REAGENTS:
1.

## Barium chloride solution (10%):

Dissolve 100 gm of BaCl2.2H2O in 1 litre distilled water.

## Silver nitrate - nitric acid reagent:

Dissolve 8.5 gm of AgNO3 and 0.5 ml of conc. nitric acid in 500 ml distilled water.

4.

## Methyl red indicator solution:

Dissolve 100 mg of methyl red sodium salt in 100 ml distilled water.

PROCEDURE:
1.

Take about 150 ml of sample and make it just acidic with HCl.

2.

Heat the solution to boiling and while stirring gently add warm barium chloride solution
slowly until precipitation appears to be complete.

55

3.

Then add about 2 ml in excess. If precipitation is small add about 4 ml of barium chloride
solution.

4.

## Digest the precipitate at 80-90oC for not less than 2 hrs.

5.

Filter the solution through whatmann filter paper. Wash the precipitate with small portion
of warm distilled water until the washing are free form chloride as indicated by testing
with silver nitrate solution.

6.

Dry the filter paper and precipitate and ignite at 750 oC for at least 30 minutes in weighed
crucible. Cool in dessicator and weigh.

CALCULATIONS:
1000 mol.
SO4 mg/lit

mg Residue x

wt of SO4
x

ml of sample
OR
mg BaSO4 x 411.5
SO4 mg/l =
ml of sample

56

mol. wt of BaSO4

DETERMINATION OF BROMIDE
PHENOL RED COLORIMETRIC METHOD
PRINCIPLE:
When sample contain bromide (Br-) is treated with diluted solution of chloroamine T in the
presence phenol red occur readily. If the reaction is buffered to pH 4.5 to4.7, the colour of
brominated compound will range from riddish to violet, depending on the bromide
concentration. Thus a sharp differentiation can be made among veriou concentration of
bromide. The concentration of chloroamine T and timing of the reaction before
dechlorination are critical.
APPRATUS:
Spectrophotometer
Acid washed glassware:
wash all glassware with 1 + 6HNO 3 rinse with distilled water to remove all trace of adsorbed
bromide.
REAGENTS:
a. Acid buffer solution
Dissolve 90 g Nacl and 68g sodium acetate trihydrate, NaC2H3O2.3H2O in distilled water.
Add 30ml con. (glacial) acetic acid and make up to 1 L. The pH should be 4.6 to 4.7.

## b. Phenol red indicator solution

Dissolved 21 mg phenolsulfonephthalein sodium salt and diluted 100 ml with distilled water.
c. Chloroamine T solution
Dissolved 500 mg chloramines T, sodium p- toluenesulfonchloroamide, and dilute to 100 ml
with distilled water. Store in dark bottle and refrigerate.

57

d. Sodium thiosulfate 2 M
Dissolved 49.6 g Na2S2O3.5H2O OR Na2S2O3 and dilute to 100 ml with distilled water.
e. Stock bromide solution
Dissolved 744.6 mg anhydrous KBr in distilled water and make up 1000 ml: 1.00ml =
5.00gBr-.
f. Standard bromide water solution
Diluted 10.00 ml stock bromide solution to 1000 ml with distilled water: 1.00 ml = 5.00 g
Br-.
PROCEDURE:
a. Preparation of bromide water standards:
Prepare at least six standard, 0 , 0.20, 0.40, 0.60, 0.80, and 1.00 mg Br - / L by diluting 0.0,
2.00, 4.00, 6.00, 8.00, and 10.00 ml standard bromide solution to 50.00 ml with distilled
water
b. Treatment of sample:
Add 2 ml buffer solution, 2 ml phenol red solution, and 0.5 ml chloramine T to 50.0 ml
sample or two separate sample dilution such that final bromide concentration in the range of
0.1 to 1.0 mg Br-/L. Mix thoroughly immediately after each addition Exactly 20 min. after
adding, with mixing, 0.5 ml Na2S2O3 solution Compare visually in nessler tubes against
bromide tandard prepared simultaneously, or preferably read in photometer at 590 nm against
areagent blank. Determine the bromide value from calibration cruve of mg Br -/ L (55ml final
volum) against absorbance. A 2.54 cm light path yield an absorbance value of
approximately 0.36 for 1 mg Br - / L.
CALCULATION:
mg Br - / L = mg Br- / L (from calibration curve) Dilution factor (if any)
Results on base on 50 ml final volume for samples and standard.

58

## DETERMINATION RESIDUAL CHLORINE

PRINCIPLE:
Chlorine is primarily added to the water for destroying the harmful micro-organism. Presence of
excess chlorine intensifies the test and odours of many other compounds such as phenol etc. It
may also many aquatic organism in combination with ammonia.
Chlorine is a strong oxidizing agent and liberates iodine is equivalent to the amount of chlorine
and can be titrate against sodium thiosulphate using starch as an indicator.
REAGENT:
a. Acetic acid, concentrated (glacial)
b. potassium iodide (KI crystals)
c. Sodium thiosulphate, 0.025 N
Dissolved 24.82 g of Na2S2O3.5H20 in boiled distilled water and make up the volume to 1
litre. Add 0.4 g of borax or a pallet of NaOH as stabilizer. This is 0.1 N stock solution Dilute
it to 4 times with boiled distilled water to prepare 0.025 N solution ( 250 100ml ).Keep in a
boron glass stoppered bottle.
d. Starch solution
Dissolved 1 g of starch in 100 ml of warm ( 80o C 90o C ) distilled water and filter
PROCEDURE:
1. Take 100 ml of sample in an Erlenmeyer flask and add 5 ml acetic acid. The pH after
addition of acetic acid should be between 3 and 4.
2. Add approximately 1 g of KI crystal and mix thoroughly with a stirring rod for about 15
minutes keeping it away from the direct sunlight.
3. Add a few drops of starch indicator and titrate against 0.025 N sodium thiosulphate until
the contents turn colourless from blue

59

CALCULATION:
(ml N) of titrant 1000 35.5
Residual chlorine, mg/l =
ml of sample

60

## DETERMINATION OF CARBON DIOXIDE

PRINCIPLE:
Free CO2 can be determine by titrating the sample using strong alkali ( such as carbonate free
NaOH ) to pH 8.3. At this pH all the free CO2 is converted into bicarbonate.
REAGENTS:
a. Sodium hydroxide, 0.05 N
Prepare 1.0 N NaOH by dissolving 40 g NaOH in CO 2 free distilled water (boiled) to make 1
litre of solution. Dilute 50 ml of 1.0 N NaOH to 1 litre. Standardize it with H 2SO4, HCL or
oxalic acid.
b. Phenolphthalein indicator
Dissolve 0.5 g of phenolphthalein in 50 ml 95% ethanol and add 50ml of distilled water. Add
0.05 N CO2 free NaOH solution dropwise, until the solution turns faintly pink.
PROCEDURE:
1. Take 100 ml of sample in a conical flask and add a few drops of phenolphthalein
indicator.
2. If the colour turn pink, free CO2 is absent. If the sample remain colourless titrate it against
0.05 N NaOH. At the end point pink colour appears.
CALCULATION:
( ml N ) of NaOH 1000 44
Free CO2, mg/l =
ml sample

61

DETERMINATION OF CYANIDE
COLORIMETRIC METHOD
PRINCIPLE:
CN - in the alkaline distillate from preliminary treatment is converted to CNCl by reaction with
chloramines Tat pH < 8 without hydrolyzing to CNO - .1 (Caution CNCl is a toxic gas; avoid
inhalation.) After the reaction is complete, CNCl forms a red- blue dye on addition of a pyridine
barbituric acid reagent . If the dye is kept in an aqueous solution, the absorbance is read at 578
nm. To obtain colour of comparable intensity, have the same salt content in sample and
standards.
INTERFERENCE:
All known interferences are eliminated or reduce to a minimum by distillation.
REAGENTS:
a. Chloramine-T solution
Dissolved 1.0 g white, water-soluble powder in 100ml water. Prepare weekly and store in
refrigerator.
b. Stock cyanide solution:
Dissolved approximately 1.6 g NaOH and 2.51 g KCN in 1 litre distilled water (CAUTION KCN is highly toxic; avoid contact or inhalation) Standardize against standard silver nitrate
(AgNO3) using KCN solution. Check titer weekly because the solution gradually loses
strength;1 ml = 1 mg CN-.
c. Standard cyanid solution
Base on the concentration determine for the KCN stock solution calculate volume requried
to prepar 1 L of 10 g CN-/ ml solution. Dilute with the NaOH dilution solution. Dilute 10 ml
of the 10 g CN -/ml solution to 100ml with the NaOH solution 1.0 ml = 1.0 g CN -/ml
prepare fresh daily and keep in a glass-stoppered bottle.

62

## d. Pyridine-barbituric acid reagent:

Place 15 g barbituric acid in a 250-ml volumetric flask and add just enough water to wash
sides of flask and wet barbituric acid acid. Add 75 ml pyridine and mix. Add15 ml conc
hydrochloric acid (HCl), mix, and cool to room temperature. Dilute to mark with water and
mix. This reagent is stable for up one month; discard if a precipitate develops.
e. Sodium dihydrogen phosphate, 1 M
Dissolved 138 g NaH2PO4. H2O in 1 L distilled water. Refrigerate.
f. Sodium hydroxide dilution solution
Dissolved 1.6 g NaOH in 1 L distilled water.
PROCEDURE:
a. Preparation of calibration curve
Prepare a blank of NaOH dilution solution. From the standard KCN solution prepare a series
of standards containing from 0.2 to 6 g CN- in 20ml solution using the NaOH dilution
solution for all dilution. Plot absorbance of standards against CN- concentration.
Recheck the calibration curve periodically and each time a new reagent prepare.
On the basis of the first calibration curve, prepare additional standards containing less than 6
g CN- to determine the limits measurable with the Photometer being used.
b. Colour development
Adjust photometer to zero absorbance each time using a blank consisting of the NaOH
dilution solution and all reagent. Take a portion of absorption liquid obtained in method c,
such that the CN- concentration fall in the measurable range, and dilute to 20ml with NaOH
dilution solution. Place in a 50 ml volumetric flask. Add 4 ml Phosphate buffer solution and
and mix thoroughly. Add 2.0 ml Chloramine T solution and swirl to mix. Immediately and
5ml pyridine-barbituric acid solution and swirl gently. Dilute to mark with water; mix well
by inversion.
Measure absorbance with the photometer at 578 nm after 8 min but within 15 min form the
time of adding the pyridine-barbituricacid reagent. Even with the specified time of 8 to 15
min there is a slight change in absorbance. To minimize this, standardize time for all

63

readings. Using the calibration curve and the formula in below, determine CN- concentration
in original sample.
CALCULATION:
A B
CN- , mg/L =
C D
Where:
A = g CN- read form calibration curve (50 ml final volume)
B = Total volume of absorbing solution from the distillation, ml.
C = Volume of original sample used in sample distillation, ml. and
D = Volume of absorbing solution used in colorimetric test, ml.
PRECISION:
The analysis of mixed cyanide solution containing sodium zinc, copper, and silver cyanides in
tap water gave a precision within the designated range as follows:
ST = 0.115 X + 0.031
Where:
ST = overall precision and
X = CN- concentration, mg/L.

64

## TOTAL CYANIDE AFTER DISTILLATION

1.

GENERAL:
Hydrogen cyanide (HCN) is liberated from an acidified sample by distillation and
purging with air. The HCN gas is collected by passing it through an NaOH scrubbing
solution. Cyanide

concentration

in the scrubbing

solution

is determined by

2.

APPARATUS:

## Apparatus is shown in Figure 4500 CN:1 (APHA) and includes:

a)

Boiling flask, one litre, with inlet tube and provision for water-cooled condenser.

b)

Gas absorber, with gas dispersion tube equipped with medium porosity fritted
outlet.

c)

d)

## Ground glass ST joints, TFE sleeved or with an appropriate lubricant for

the boiling flask and condenser.

3.

REAGENTS:
a)

Sodium hydroxide solution: Dissolve 40 g NaOH in water and dilute to one litre.

b)

Magnesium chloride reagent: Dissolve 510 g MgCl 2. 6H2O in water and dilute to
one litre.

c)

d)

e)

## Sulfamic acid. NH2SO3H.

65

4.

PROCEDURE:
a)

Add 500 ml sample, containing not more than 10 mg CN/l (diluted if necessary
with distilled water) to the boiling flask. If a higher CN content is anticipated, use
the spot test (4500-CN.K) to approximate the required dilution. Add 10 ml
NaOH solution to the gas scrubber and dilute, if necessary, with distilled water to
obtain an adequate liquid depth in the absorber. Do not use more than 225 ml
total volume of absorber solution. When S' generation from the distilling flask is
anticipated add 50 or more mg powdered PbCO3 to the absorber solution to
precipitate S2. Connect the train, consisting of boiling flask, air inlet, flask,
condenser, gas washer, suction flask trap, and aspirator. Adjust suction so that
approximately one air bubble/second enters the boiling flask. This air rate will
carry HCN gas from flask to absorber and usually will prevent a reverse flow of
HCN through the air inlet. If this air rate does not prevent sample backup in the
delivery tube, increase air-flow rate to two air bubbles per second. Observe air
purge rate in the absorber where the liquid level should be raised not more than
6.5 to 10 mm during purging. Maintain air flow throughout the reaction.

b)

Add 50 ml H2SO4 (1+1) through the air inlet tube and wash down with distilled
water and let air mix flask contents for 3 min.

c)

Add 20 mL MgCl2 reagent through air inlet and wash down with stream of water.
A precipitate that may form redissolves on heating.

d)

Heat with rapid boiling, but do not flood condenser inlet or permit vapors to rise
more than halfway into condenser. Adequate refluxing is indicated by a reflux
rate of 40 to 50 drops/min from the condenser lip. Reflux for at least 1 hour.
Discontinue heating but continue air flow. Cool for 15 minutes and drain gas
washer with distilled water, add rinse water to drained liquid, and dilute to 250 ml
in a volumetric flask.

e)

## Determine cyanide content by the titration method (D) if cyanide concentration

exceeds 1 mg/l and by the colorimetric method (E) if the cyanide concentration is
less than 1 mg/l.

66

DETERMINATION OF CYANIDES
COLORIMETRIC METHOD
1. PRINCIPLE:
CN in the alkaline distillate from preliminary treatment is converted to CNCl by
reaction with chloramine-T at pH < 8 without hydrolyzing to CNO (CAUTION-CNCI
is a toxic gas: avoid inhalation.) After the reaction is complete, CNCI forms a red-blue
dye on addition of a pyridine barbituric acid reagent. If the dye is kept in an aqueous
solution, the absorbance is read at 578 nm. To obtain colors of comparable intensity, have
the same salt content in sample and standards. All known interferences are eliminated or
reduced to a minimum by distillation.
2. APPARATUS:
Colorimetric equipment: One of the following is required:
a) Spectrophotometer, for use at 578 nm, providing a light path of 10 mm or longer.
b) Filter photometer, for use at 578 nm, providing a light path of 10 mm and equipped
with a red filter having maximum transmittance at 570 to 580 nm.
3. REAGENTS:
a)

## Chloramine: T solution: Dissolve 1 g white, water-soluble powder in 100 ml

water. Prepare weekly and store in refrigerator.

b) Stock cyanide solution : Dissolve approximately 1.6 g NaOH and 2.51 g KCN
(highly toxic; avoid contact or inhalation) in one litre of distilled water. Take 25
mL of KCN solution, add one ml of potassium dichromate solution (50 g in 1 litre).
Standardize against Standard Silver Nitrate (AgNO3) 0.0141 N, titrant and find
out the normality of KCN solution. Check titer weekly because the solution gradually
loses strength; 1 ml contains 1 mg CN.
c) Standard Cyanide Solution : Based on the concentration determined for the KCN
stock solution in 3 (b) above, calculate volume required (shall be approximately 10
mL) to prepare 1 L of a 10 ug CN/mL solution (use dilute NaOH solution for any
dilutions). Dilute 10 ml of 10 ug CN/ml solution to 100 ml with the NaOH

67

dilution solution which corresponds to 1 mL = 1 ug CN. Prepare fresh daily and keep
in a glass stoppered bottle. (CAUTION - Toxic ; take care to avoid ingestion.)

15

## g barbituric acid in a 250 ml

volumetric flask and add just enough water to wash side of flask and wet
barbituric acid. Add 75 mL pyridine and mix. Add 15 mL conc hydrochloric acid
(HCl), mix and cool to room temperature. Dilute to mark with water and mix. This
reagent is stable for up to one month; discard if a precipitate develops.
e) Sodium dihydrogen phosphate: 1 M: Dissolve 138g NaH2PO4 H2O in 1 L distilled
water. Refrigerate.
f) Sodium hydroxide dilution solution : Dissolve 1.6 g NaOH in 1 L distilled water.
4. PROCEDURE:
a) Preparation of calibration curve : Prepare a blank of NaOH dilution solution. From the
standard KCN solution prepare a series of standards containing from 0.2 to 6 ug CN in
20 mL solution using the NaOH dilution solution for all dilutions. Treat standards
in accordance with (b) below. Plot absorbance of standards against CN concentration
(micrograms).

Recheck calibration curve periodically and each time a new reagent is prepared. On the
basis of the first calibration curve, prepare additional standards containing less
than 0.2 and more than 6 ug CN to determine the limits measurable with the
photometer being used.

b) Color development: Adjust photometer to zero absorbance each time using a blank
consisting of the NaOH dilution solution and all reagents. Take a portion of absorption
liquid obtained in distillation process such that the CN concentration falls in the
measurable range, and dilute to 20 mL with NaOH dilution solution. Place in a 50-mL
volumetric flask. Add 4 mL phosphate buffer

68

and mix

## thoroughly. Add 2.0 mL

chloramine-T solution and swirl to mix. Immediately add 5 mL pyridine barbituric acid
solution and swirl gently. Dilute to mark with water; mix well by inversion.

Measure absorbance with the photometer at 578 nm after 8 min but within 15 min from
the time of adding the pyridine

the specified

## time of 8 to 15 min there is a slight change in absorbance. To minimize this, standardize

time for all readings. Using the calibration curve and formula below determine CN
concentration in original sample.
5. CALCULATION:
CN, mg/L

(AxB)/ (CxD)

Where

## ml absorbing solution used in colorimetric test.

69

DETERMINATION OF IODINE
LEUCO CRYSTAL VIOLET METHOD
PRINCIPLE:
Mercuric chloride added to aqueous elemental iodine solution causes essentially complete
hydolysis of iodine and the stoichiometric production hypoiodus acid. The compound 4,4,4
methylidynetris (N,N- dimetylaniline), also known by the common name of leuco crystal violet,
react instantaneously with the hypoiodous acid to form crystal violet dye solution is produce in
pH range of 3.5 to 4.0 and measured the at a wavelength of 592 nm. The absorbance follows
Beers Law over a wide range of iodine concentrations and the develop color is stable for several
hours.
In presence of certain excess oxidations such as free chlorine or chloroamine, the iodine residual
will exit exclusively

in form

## of hypoiodous acid. The leuco crystal violet is relatively

insensitive
to the combined forms of chlorine while any free chlorine by reaction with an ammonium salt
incorporated in the test reagent. All hypoiodous acid is determine and, when exprexx as an
equivalent elemental I2 concentration , will yield a weight concentration value twice that found in
an elemental I2 solution of the same weight concentration.
INTERFERENCE:
Oxidizing form of manganes interfere by oxidizing the indicator to crystal violet dye and yield
apparent high iodine concentration.
Iodine and chloride ion concentration above 50 mg/l and 200 mg/l respectively, interfere by
inhibiting full color production . Dilute sample eliminate this interference.
Combine chlorine residual normally do not interfere provided that the test is completed within in
5 min after adding the indicator solution. Eliminate interference from free chlorine by adding an
ammonium salt buffer to form combine chlorine.

70

## MINIMUM DETECTABLE CONCENTRATION

10 g I asI2/L
REAGENTS:
a. Iodinedemandfree water
Prepare a 1-m ion exchange column of 2.5 to 5 cm diam, containing strongly acid cation
strong basic anion exchange resins. If a commercial analytical- grade mixed bed resin is
used, verify that compound that react with iodine are removed. Pass distilled water at aslow
rate through the risin bed and collect in clean container that will protect the treated water
form unduse exposure to the atmosphere. Prepare all stock Iodine and solution with iodine
demand free water. Prepare all stock Iodine and solution with iodine demand free water.
b. Stock Iodine solution
Prepare the saturated iodine solution by dissolving 20 g elemental iodine in 300 ml. water
Let stand several hours. Decent iodine solution and dilute 170 ml to 2000 ml.
Prepare a working solution of g I as I2 / ml by appropriate dilution of the standardized
stock
solution.
c. Citric buffer solution, pH 3.8
1. Citric acid
Dissolved 192.2 g C6H8O7, or 210.2 g C6H8O7. H2O and dilute to 1 L with water.
2. Ammonium hydroxide, 2 N
Add 131 ml conc. NH4OH to about 700 ml water and dilute 1L

store in a

polyethylene bottle
3. Final buffer solution
Slowly add, with mixing, 350 ml 2 N NH4OH solution to 670 ml citric acid. Add 80 g
ammonium di-hydrogen phosphate (NH4 H2PO4)

## d. Leuco crystal violet indicator

71

Measure 200 ml water and 3.2 ml conc. Sulphuric acid (H 2SO4) into brown glass container
of at list 1-L capacity. Introduce a magnetic stirring bar and mix at moderate speed. Add 1.5 g
4, 4 ,4 methylidynetris (N,N- dimethylaniline)* and with small amount of water wash
down any reagent adhering to neck or side of container. Mix until dissolved.
To 800 ml water , add 2.5 g mercuric chloride (HgCl 2) and stir to dissolve. With mixing, add
HgCl2 solution to leuco crystal violet solution. For maximum stability , adding , if necessary,
conc. H2SO4 drop wise. Store in brown glass bottle away from direct sunlight. Discard after 6
months, Do not use rubber stopper.
e. Sodium thiosulphate solution
Dissolve 5.0 g Na2S2O3 . H2O in water and dilute to 1L.
PROCEDURE:
a. Preparation of temporary iodine standards:
Prepare standards in the range of 0.1 to 6.0 mg I as I 2/L by adding 1 to 60 ml working solution
to

## 100 ml glass stoppered volumetric flasks in increment of 1 ml or larger. Adjust these

volumes if the measured iodine concentration of working solution varies by 5% 0r more from
10 g I as I2 /mL.
Measure 50.0 mL of each diluted iodine working solution into a 100-mL glass- stoppered
volumetric flask. Add 1.0mL leuco crystal violet indicator and swirl to develop color. Dilute to
100mL and mix.
b.photometric calibration :
Transfer colored temporary standards of known iodine concentrations to cells of 1 cm light
path and read absorbance in a photometer or spectrophotometer at a wavelength of 592 nm
against a distilled water reference. Plot absorbance values against iodine concentrations to
construct a curve that follows Beers law.
c. Color development of iodine sample :
measure 50.0mL sample into a 100-mL volumetric flask and treat as described for preparation
of temporary iodine standards, match test sample visually with temporary standards or read
absorbance photometrically and refer to standard calibration curve for the iodine equivalent.

72

## d. Samples containing >6.0 mg I as I2/L:

Place approximately 25 mL water in a 100mL volumetric flask. Add 1.0 mL citric buffer
solution and a measured volume of 25 mL or less of sample. Mix and let stand for at least 30
s. Add 1.0 ml leuco crystal violet indicator, mix, and dilute to mark. Match visually with
standards or read absorbance photometrically and compare with calibration curve from which
the initial iodine is obtained by applying the dilution factor. Select one of the following
sample to remain within optimum iodine range:

Iodine mg/ L

6.0 12.0
12.0 30
30 60

25.0
10.0
5.0

## f. Samples containing chlorine and iodine

For sample containing free or combined chlorine and iodine, follow procedure given in c &
d above but read absorbance within 5 min after adding leuco crystal violet indicator.
g. Compensation for turbidity and colour:
Compensate for natural colour or turbidity by adding 5 ml Na 2S2O3 solution to a 50 ml
sample.

Add reagent to sample as described previously and use as blank to set zero

absorbance on the photometer. Measure all sample in relection to this blank and, from
calibration curve, determine concentration of iodine.

73

DETERMINATION OF SULFITE
IODOMETRIC METHOD
PRINCIPLE:
An acidify sample containing sulfite (SO32 -) is titrate with a standardized potassium iodide
iodate titrant. Free iodine, liberate by the iodide-iodate reagent react with SO 32 -. The tration
end point is signalled by the blue colour resulting from the frist excess of iodine reacting with
a starch indicator.
INTERFERENCE:
The presence of other oxidizable material, such as sulphide, thiosulfate, and Fe2+
ions, can cause apparently high result for sulfite. Some metal ions such as Cu 2+, may catalyze
the oxidation of SO32 to SO4+ when the sample is exposed to air, thus leading to low result.
NO2- will react with SO32- in the acidic reaction medium and lead to low the sulfite results
unless sulfamic acid is added to destroy nitrite. Addition of EDTA as a complexing agent at
the time of sample collection inhibits Cu2+ catalysis and promots oxidation of ferrous to ferric
iron before analysis. Sulfide and thiosulfate ions normaly would be expected only in samples
containing certain industrial discharge, but must be accounted for if present. Sulfide may be
removed by adding about 0.5 g zinc acetate and analyzing the supernatant of the settled
sample. How ever thiosulfate may have to be determined by an independent method (e.g., the
formaldehyde/iodometric method1), and then the sulphide determine by difference.
MINIMUM DETECTEBLE CONCENTRATION:
2mg SO32-/ l
REAGENTS:
a. Sulfuric acid: H2SO4, 1+1
b. Standard potassium iodide-iodate titrant,0.0125 M:
Dissolved 0.4458 g primary grade anhydrous KIO 3 (dried for 4 h at 120oC), 4.35 g KI, and
310 mg sodium bicarbonate (NaHCO3) in distilled water to dilute to 1000mL; 1.00mL= 500
g SO32 -.

74

## c. Sulfamic acid, NH2SO3H, crystalline.

d. EDTA reagent:
Dissolved 2.5 g disodium EDTA in 100 ml distilled water.
e. Starch indicator:
To 5 g starch (potato, arrowroot, or soluble) in a mortar, add a little cold distilled water and
grind to a paste. Add mixture to 1 L boiling distilled water, stir, and let settle overnight. Use
clear supernatant. Preserve by adding either 1.3 g salicylic acid, 4 g ZnCl 2 or a combination
of 4 g sodium propionate and 2 g sodium azide to 1 L starch solution.
PROCEDURE:
a. sample collection:
Collect a fresh sample taking care to minimize contact with air. Fix cooled sample (< 50 oC)
Immediately by adding 1 ml EDTA solution/100 ml sample. Cool hot sample to 50 oC or below.
Do not filter.
b.Titration:
Add 1 ml H2SO4 and 0.1 g NH2SO3H crystals to a 250-ml Erlenmeyer flask or other suitable
titration vessel. Accurately measure 50 to 100 ml EDTA- stabilized sample in to flask, keeping
pipet tip below liquid surface. Add 1 ml starch indicator solution .Titrate immediately with
standard KI-KIO3 titrant, while swirling flask, until a faint permanent blue color develop.
Analyze a reagent blank using distilled water instead of sample.
CALCULATION:
( A-B ) M 40000
2-

MgSO3 / L =
mL sample
Where:
A= ml titrant for sample,
B= ml titrant for blank,
M= molarity of KI-KIO3

75

DETERMINATION OF SULFIDE
IODOMETRIC METHOD

REAGENT:
a. Hydrochloric acid: HCl, 6N.
b.Standard iodine solution, 0.0250 N:
Dissolved 20 to 25 g KI in a little water and add 3.2 g iodine. After iodine has dissolved, dilute
to 1000 ml and standardize agains 0.0250 N Na2S2O3 using starch as indicator.
c. Standard sodium thiosulfate solution,0.0250 N:
Dissolve 6.205 g Na2S2O3. 5H2O in distilled water. Add 1.5 ml 6N NaOH or 4 g solid NaOH and
dilute to 1000 ml standize with bi-iodite solution.
d. Starch:
To prepare aqueous solution solution, dissolved 2 g laboratory-gard soluble starch and 0.2 g
salicylic acid, as a preservative, in 100 ml hot distilled water.
PROCEDURE:
a. Measure from a buret into a 500 ml flask an amount of iodine solution estimated to be an
excess over the amount of sulfide present. Add distilled water, if necessary, to bring volume to
about 20 ml. Add 2 ml 6 N HCl. Pipete 200 ml sample into flask, discharging sample under
solution surface. If iodine colour disappears, add more iodine so that color remains. Back titrate
with Na2S2O3 solution, adding a few drop of starch solution as end point is approached, and
continuing until blue color disappears.
b. If sulphide was precipitate with Zinc and ZnS filtered out, return filter with precipitate to
original bottle and add about 100 ml water. Add iodine solution and HCl and titrate as in above.

76

CALCULATION:
One milliliter 0.0250N iodine solution react with 0.4 mg S2- :]
[ (A B) (C D) ] 16000
2-

Mg S /L =
ml sample
Where:
A = ml Iodine solution,
B = normality of iodine solution,
C = ml Na2S2O3 solution, and
D = normality of Na2S2O3 solution.

77

## DETERMINATION OF CHEMICAL OXYGEN DEMAND (C.O.D.)

PRINCIPLE
Most type of organic matter is destroyed by a boiling mixture of chromic and sulfuric acids. A
sample is refluxed with known amounts of potassium dichromate and sulfuric acid and the
excess dichromate is titrated with ferrous ammonium sulfate. The amount of oxidizable organic
matter, measured as oxygen equivalent, is proportional to the potassium dichromate consumed.
APPARATUS
Reflux apparatus consisting of round bottom 300 ml. flasks and Liebig condensers.
REAGENTS
1.

## Concentrated H2SO4 with Ag2SO4:

Add approx. 10 g of Ag2SO4 to one litre of conc.H2SO4.

2.

Ferroin indicator:
Dissolve 1.485 g of 1,10 - phenanthroline (monohydrate) together with 0.695 g of
ferrous sulphate in 100 ml distilled water.

3.

## Standard K2Cr2O7 (0.25 N):

Dissolve 12.259 g of potassium dichromate, primary standard grade, previously dried at
103oC for 2 hrs., in distilled water and make the volume to one litre with distilled water.

4.

## Standard Ferrous Ammonium Sulfate (0.1 N) (FAS):

Dissolve 39.0 g of ammonium ferrous sulfate in distilled water. To that add 20 ml of
conc. sulphuric acid and make the volume to one litre with distilled water. Standardize
against the standard potassium dichromate.

5.

6.

78

PROCEDURE:
1.

following table.

Sample Size

0.25N

Standard

with Ag2SO4

Fe

Dichromate

## (NH4)2- after Reflux

(SO4)2

ml

ml

nk

ml

10

15

0.2

0.05

70

20

10

30

0.4

0.10

140

30

15

45

0.6

0.15

210

40

20

60

0.6

0.20

280

50

25

75

1.0

0.25

350

2.

Attach the flask to condenser and reflux the mixture for two hrs. Cool it. Then wash
down the condenser with little distilled water. Remove the flask and cool.

3.

Dilute the mixture to about 140 ml with distilled water and titrate excess of dichromate
with standard ferrous ammonium sulfate using ferroin as indicator. The colour change
is sharp, changing from blue-green to wine red.

CALCULATION:

79

(B-S) x 8000 x N
COD in mg/litre

=
ml of sample taken

where,
B

## ml of FAS used for Sample

Normality of FAS

SIGNIFICANCE:
COD test is extensively used for analysis of industrial waste. It is particularly valuable to
determine and control efficiency of sewage systems. Results may be obtained in relatively short
time.

## DETERMINATION BIOLOGICAL OXYGEN DEMAND

80

PRINCIPLE:
Biochemical oxygen demand is a measure of the degradable organic material present in a water
sample and is defined as the amount of oxygen required by the micro-organisms in stabilising
biologically degradable organic matter under aerobic conditions. The method consists of filling
with sample, to overflowing, an airtight bottle of the specified size and incubating it at the
specified temperature 270C for 3 days. 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 immediately after the dilution is made, all oxygen uptake, including
that occurring during the first 15 minutes, is included in the BOD measurement.

APPARATUS :
Incubation bottles, 250 to 300-ml special BOD bottles.
Air incubator, thermostatically controlled AT 27 + 10C
REAGENT :
Phosphate buffer solution:
Dissolve 8.5 g of Dihydrogen Potassium

## phosphate KH 2PO4, 21.75 g of

Dipotassium

Hydrogen phosphate K2HPO4, 33.4 g of Disodium Hydrogen phosphate Na 2HPO4 .7H2O and
1.7 g of ammonium chloride NH 4Cl in distilled water and dilute to 1 L. The pH should be
7.2 Magnesium sulfate solution: Dissolve 22.5 g MgSO 4.7H2O in distilled water

and

dilute to 1 L.
Calcium chloride solution:
Dissolve 27.5 g anhydrous CaCl2 in distilled water and dilute to 1 L.
Ferric chloride solution :
Dissolve 0.25 g FeCl3.6H2O in

distilled

water

and

dilute to 1 L.

## Manganous sulfate solution :

Dissolve 480 gms MnSO4.4 H2O /400GMS MnSO4.2H2O/364gm MnSO4.H2O in 1000ml of
boiled distilled water and filter.MnSO4 solution should not give a color with starch when added
to an acidified KI solution.
Alkali- iodide azide solution.:

81

Dissolve 500gms NaOH or 700GMS KOH and 135 GM NAI or 150 GMS KI in 1000 ml
distilled water, Dissolve 10 gms sodium azide in 40 ml distilled water, mix both this reagent
should not give a color with starch solution when diluted and acidified.
Starch aqueous solution 2 % :
Dissolve 2 gms laboratory grade soluble starch and 0.2 gm of salicylic acid as a preservative in
100ml of hot distilled water.
Sodium thiosulfate solution 0.1 N :
Dissolve 24.82 gms of NA2 S2O3 .5H2O in 1000 ml distilled water. Add 1.5 ML 6N NaOH or
1.6 gms solid NaOH pellets or 0.4 gms borax. Standardise the same.
PROCEDURE:
Preparation of dilution water:
Place desired volume of water in a suitable round bottom flask and add 1 ml each of Phosphate
buffer, MgSO4, Cacl2 and FeCl3 solutions/L of water. Seed dilution water and mix thoroughly
with compressed air, Store dilution water
Seeding material:
Take 1 gm of vacuum dried biological sludge of aeration tank in 250 ml distilled water, shake
vigorously so that mixing is through.
The sample containing either acidity or alkalinity should be neutralised. Use either dilute
hydrochloric acid or sodium hydroxide.
To check the quality of the dilution water to be used and the effectiveness of the seeding material
determine the BOD of a standard solution of 150-mg glucose and 150 mg of glutamic acid to be
diluted to 1 L with distilled water. The above standard solution should show a BOD of 198 mg/l
with a standard deviation of 35 mg/L
Add 6.25 ml/L of previously prepared seeding material solution. Bubble for minimum 1 hour.
Take required volume of sample in a 500-ml beaker. Make the volume to 350 ml and add this
into previously numbered BOD bottles. Fill one bottle with dilution water only for blank
reading.
Stopper the bottles and keep in incubator for 3 days at 27 0C.After 3 days take out BOD Bottles
from incubator add 2 ml of MnSO4 and 2 Ml of alkali iodide -azide solution. Shake well and
keep for settling. When precipitate settles down add 1 to 2 ml of conc. H 2SO4. Shake well. Take
out 100ml from this bottle and titrate against 0.01 N sodium thiosulfate solution using starch as

82

indicator at the end point initial dark blue colour changes to colourless . Note down the burette
reading and calculate BOD as below.
CALCULATION :
BOD = (DO of Blank - DO of Sample) Dilution Factor
Dilution Factor = Total Diluted Volume(350ml) + Volume of Sample
Sample Volume
When only a part of the contents has been titrated i.e. 100 ml
BOD mg/L = B.R X N of titrant X 8000
V2 (V1-V)
V1
Where
V1 = Volume of sample bottle after placing the stopper.
V2 = Volume of the part of the contents titrated
V = Volume of MnSO4 and KI added.

83

## DETERMINATION OF OIL AND GREASE

PRINCIPLE:
This method of measurement is applicable to the determination of relatively non-volatile
hydrocarbons, vegetable oils, animal fats, waxes, soaps, greases and related matter.
APPARATUS:
1.

Separating funnel

2.

Evaporating dish

3.

Oven

REAGENTS:
1.

3.

Sodium sulfate

2.

4.

Sodium chloride

PROCEDURE:
a)

b)

## Acidify with few drops of conc. HCl to pH 2.

c)

Shake well and transfer to the separating funnel. Add 25 ml of petroleum ether. If
emulsion is formed, add few NaCl crystals, as required to break the emulsion.

d)

e)

f)

g)

## Drain the lower layer.

h)

Collect the upper clear layer, by filtering through a funnel containing solvent-moistened
filter paper and Na2SO4 in a preweighed evaporating dish.

j)

## Repeat solvent treatment to aqueous phase earlier separated.

k)

Wash the filter paper with additional 10 to 20 ml petroleum ether. Pour the remaining
extraction also in the evaporating dish.

84

l)

Dry it in oven at 70oC. After drying, take final weight. Find out increase in weight in
mg.

m)

Run one blank by evaporating the same quantity of solvent used for extraction.

CALCULATIONS:
Difference in weight in mg x 1000
Oil and Grease mg/lit =
ml of sample
=

Where,
X

## Weight of the residue of blank

85

DETERMINATION OF PHENOLS
CHLOROFORM EXTRACTION METHOD
PRINCIPLE:
The steam distillable phenols react with 4-aminoantipyrine at a pH of 10.0 + 0.2 in the presence
of potassium ferricyanide to form a colored antipyrine dye. This dye is extracted from aqueous
solution with chloroform and the absorbance is measured at 460 nm. The concentration of
phenolic compounds is expressed as micro-gram/l of phenol. All interferences are eliminated or
reduced to a minimum if the sample has been preserved, stored, and distilled in accordance
with the foregoing instructions.
APPARATUS:
1.

Photometric equipment:
Spectrophotometer, for use at 460 nm.

2.

Separatory funnels.

3.

pH meter.

REAGENTS:
1.

## Stock phenol solution:

Dissolve One gram phenol in freshly boiled and cooled distilled water and dilute to
one litre.
Standardization :
a)

## To 100 ml distilled water in a 500 ml glass stoppered conical flask, add 50 ml

stock phenol solution and 10 ml 0.1 N bromate-bromide solution. Immediately
add 5 ml conc. HCl and swirl the stoppered flask gently. If the brown color of
free bromine does not persist, add 10 ml portions of bromate-bromide solution

86

until the color does persist. Keep the flask stoppered and let stand for 10
minutes. Then add approximately 1.0 g KI.

## Usually four 10 ml portions of bromate-bromide solution are required if the

stock phenol solution contains 1,000 mg/l phenol.

b)

Prepare a blank in exactly the same manner, using distilled water and 10 ml
0.1 N bromate-bromide solution. Titrate the blank and sample with the 0.025 N
sodium thiosulfate titrant, using starch as the indicator.

c)

## Calculate the conc. of the phenol solution as follows:

mg/l phenol = 7.842 (A x B) - C
Where, A = ml sodium thiosulfate for blank
B = ml bromate-bromide solution used for sample divided by 10, and
C = ml sodium thiosulfate used for sample.

2.

## Intermediate phenol solution:

Dilute 10 ml stock phenol solution to 1 lit. in freshly distilled water; (1 ml = 10
micro-gram phenol). Prepare a fresh solution on each day of use.

3.

## Standard phenol solution:

Dilute 50 ml intermediate phenol solution to 500 ml with freshly boiled and cooled
distilled water; (1 ml = 1 micro-gram phenol). Prepare this solution within 2 hrs. of use.

87

4.

## Bromate-bromide solution, (0.1 N):

Dissolve 2.784 g anhydrous KBrO3, in distilled water and add 10 g KBr crystals, dissolve
and dilute to one litre.

5.

6.

## Standard sodium thiosulfate, titrant, (0.025 N):

Stock Sodium thiosulphate (0.1 N):
Dissolve 24.82 gm of sodium thiosulphate in one litre distilled water. Store the
solution with 5 ml CHCl3 or by one gram of NaOH.
Std. Sodium thiosulfate (0.025 N):
Take 250 ml of stock solution and dilute to one litre with distilled water or by dissolving
6.205 g of sod. thiosulfate in one litre distilled water.

7.

(1 %) Starch indicator:
Dissolve 5 g of starch soluble powder in little amt. of distilled water. Digest this solution
by heating till the solution gets clear. Store the solution with 1.25 g of salicylic acid per
litre or by few drops of toluene.

8.

## Ammonium chloride solution:

Dissolve 50 gm NH4Cl in D/w and dilute to 1 lit.

9.

## Ammonium hydroxide, NH4OH, conc.:

10.

4-Aminoantipyrine solution:
Dissolve 2 g, 4-aminoantipyrine in distilled water and dilute to 100 ml. Prepare a
fresh solution on each day of use.

88

11.

## Potassium ferricyanide solution:

Dissolve 8 g K3Fe(CN)6 in distilled water and dilute to 100 ml. Filter if necessary.
Prepare fresh each week of use.

12.

Chloroform, CHCl3:

13.

14.

15.

## Copper sulfate solution (10 %):

Dissolve 100 g CuSO4.5H2O in distilled water and dilute to one litre.

16.

## Phosphoric acid solution, (1+9):

Dilute 10 ml 85% H3PO4 to 100 ml with distilled water.

17.

## Methyl orange indicator:

Dissolve 0.5 g methyl orange in one litre distilled water.

18.

## Special reagents for turbid distillates:

Sulfuric acid, 1 N, Sodium chloride, Chloroform or ethyl ether and sodium hydroxide,
2.5 N (Dilute 41.7 ml 6 N NaOH to 100 ml distilled water or dissolve 10 gm NaOH to
100 ml distilled water.

PROCEDURE:
Distillation:
1.

Place 500 ml sample in the distillation flask (if the sample is known to contain
enough phenols, smaller aliquots may be taken and volume of distillate may be
reduced accordingly).

89

2.

Add 5 ml copper sulfate solution (10%) and bring the pH below 4 using H 3PO4 (1+9).
(omit these additions if the sample is already preserved).

3.

Start distillation and collect 450 ml distillate. Cool the flask and add 50 ml distilled
water and continue the distillation until total volume of the distillate is 500 ml.

(If distillate is turbid, acidify with 1+9 H 3PO4 to pH below 4, add 5 ml CuSO4 solution
(10%) and distill as described above. If the second distillate is also turbid, acidify 500
ml of original sample using 1 N. Sulfuric acid and methyl orange indicator and add
150 g NaCl. Extract with 40 ml chloroform. Extract further with 25 ml chloroform for
four times. Combine all the chloroform extracts in a second separating funnel and
extract with 2.5 N NaOH solution (100 g NaOH /l) first with 4 ml and then with 3 ml
volumes

two times.

## Combine the alkaline extracts, heat on a water

bath until

chloroform is removed. Cool and dilute to 500 ml and proceed as described above
starting from step No. 2).

Notes:
1.

Appropriate smaller volumes of reagents may be used in cases where smaller volumes of
samples are taken for distillation.

2.

## Diethyl ether is recommended in place of chloroform as it eliminates emulsion

problem and use of NaCl. Chloroform is preferred because of the hazards in handling
ether.

90

DETERMINATION OF PHENOLS
4-AMINO ANTIPYRINE COLORIMETRIC METHOD
(This method is applicable to most of the phenolic compounds excepting para cresol and
similar para substituted phenols)
Principle:
Phenols react with 4-amino antipyrine at a pH of 10.0 +/- 0.2

in presence of potassium

## ferricyanide to form a red antipyrine dye.

Interferences:
All the interferences are eliminated or reduced to a minimum level by preliminary treatment
and distillation.
Procedure:
1.

Into a series of separating funnels, place 0.0, 2.0, 5.0, 10.0, 20.0, .50.0 ml.of phenol
working solution and dilute each to 500 ml. with D/w.

2.

Place 500 ml distillate in 1 lit. separating funnel. (Lower aliquots may also be use if
phenol concentration is more, but dilute to 500 ml with D/w.)

3.

To the blank , standard and distillate, add 10 ml NH 4Cl solution and adjust pH to 10.0
+/- 0.2 by using conc. NH4OH solution.

4.

## Add 3 ml of 4-amino antipyrine solution. Mix immediately and add 3 ml potassium

ferricyanide and again mix immediately.

5.

Allow to stand for 3 min. and extract with 25 ml chloroform if 5 cm. cell is used with 50
ml if 10 cm cell is used.

6.

Pass through fritted-glass funnel containing 5 gm layer of sodium sulfate and collect
the dried extract in 50 ml Nessler tube.

7.

Measure the optical density of sample and standards at 460 nm setting blank at 100 %
transmittance and prepare a calibration curve. Find out the micro-gm equivalent of

91

phenol from the curve. Visual comparison can also be made. Express the result as mg
phenolic substances as phenol (C6H5OH) per litre of sample.
CALCULATION:
Micro-gm phenol

## micro-gm phenol in sample, from calibration curve.

ml original sample.

92

## DETERMINATION OF pH OF SOIL/SLUDGE/SEDIMENT AND SOLID WASTE

PRINCIPLE:
pH is a measure of hydrogen ion concentration. It is defined as the negative logarithm of H + ion
concentration or more precisely hydrogen ion activity of a sodium and expressed by equation. It
can be measured electrometrically using pH meter.
pH= -log [H+]
APPARAUS:
PH meter, Beaker100 ml.
REAGENT:
Standard PH buffers, PH 4.0, 7.0, & 9.2.
PROCEDURE:
Take about 10g of powdered and dried (at room condition around 250C for 24 hours) sample in
beaker.
Add 50 ml of distilled water and stir for one hour (1:5 ratio, 1 part sample and 5 part of distilled
water).
Calibrate the ph meter using standard pH buffer of pH 4.0, 7.0, & 9.2.
Measured the pH of the solid suspension using standard pH meter following the procedure.
Record the pH value (1:5) in 2 decimal plant.
Note:
The conductivity of the distilled water to be used for the preparation of standard buffer solution
and KCl solution should be less than 2.0 S/cm.
Discard the buffer if there is any mold growth or concentration.
EXPRESSION OF RESULTS:
Express the result as pH (1:5) in 2 decimal units.

93

## DETERMINATION OF ELECTRICAL CONDUCTIVITY

PRINCIPLE:
Electrical conductivity (EC) of the sample saturation indicates ionisable constituents of the
solution and is measured using a conductivity meter. This method is applicable to solid samples
like soil, solid waste, sewage sludge , municipal waste, Compost.
APPRATUS:
Conductivity meter, Beaker 100 ml.
REAGENT:
Standard KCl (0.01M) :
Dissolved 0.7456 g dry KCl in 1 liter of distilled water. This solution has an electrical
conductivity of 1412 S/cm. at 250C.
PROCEDURE:
Take 10 g of air dried sample in a beaker, add 50 ml of distilled water and stir at one hour (1:5
ratio).
Calibrate the conductivity meter using the standard 0.01 KCl solution.
Immerse the conductivity cell in the solution and measure conductivity as per the procedure
given in the instruction Manual.
CALCULATION:
Report the conductivity in S/cm (1:5 ratio)
EXPRESSION OF RESULTS:
Express the results as conductivity (1:5) in decimal units.
Reference:
Jackson, M.L. (1967): Soil chemical analysis. Prentice-Hill of India Pvt. Ltd New Delhi.

94

## DETERMINATION OF TOTAL KJELDAHL NITROGEN

PRINCIPLE:
Total kjeldahl nitrogen (TKN) is the sum of ammonia nitrogen and organic nitrogen present in a
sample. It does not include nitrite nitrogen and nitrate nitrogen. In presence of sulphuric acid,
potassium sulphate and cupric sulphate (catalysts), the nitrogen of organic matter as well as free
ammonia is converted in to ammonium sulphate on digestion at 360 410 0C. An excess of alkali
is then added to liberate ammonia and distilled. The liberated ammonia is absorbed in boric acid
solution (mixed with indicator) and TKN is determined titrimetrically with standard sulphuric
acid. This method is applicable to solid samples like soil, solid waste, sewage sludge, municipal
solid waste, compost.
APPARATUS:
Kjeldahl tube or flask, 500ml capacity
Heating device with temperature range of 360-410oC
Fume hood or scrubber unit
Kjeldahl Distillation unit
REAGENTS
a)

b)

## Potassium Sulphate Copper Sulphate mixture (10:1 ratio):

Prepare a mixture 10 gm of Potassium sulphate ( K 2SO4 ) and 1gm of Copper Sulphate
(CuSO4.5H2O). Alternatively use a kjeldahl tablet.

c)

## Phenolphthalein indicator solution:

dissolve 0.5g of Phenolphthalein in 50 ml ethyl or Isopropyl alcohol and add 50ml.
distilled water.

d)

95

## Dissolve 40 gm. NaOH in 100 ml.distilled water.

e) Mixed indicator solution:
dissolve 0.2 g methyl red in 100 ml of 95% ethyl or isopropyl alcohol and 0.1 g
methylene

## f) Boric acid + mixed indicator solution:

Dissolve 20g boric acid (H 3BO3) in ammonia free distilled water , add 10 ml of mixed
indicator solution and dilute to 1 litre.
g) Standard Sulphuric acid solution (0.02N):
Dilute 20ml of 1N H 2SO4 to 1000ml with distilled water. Standardise it against 0.02N
Sodium carbonate solution (1.06g of Na2CO3/ liter) using methyl red indicator (0.2%
Solution in 95% ethyl or isopropyl alcohol). A faint orange colour will appear at the end
point.
PROCEDURE:
Digestion:
Take 2.0g or suitable quantity of oven dried (105 oC) sample thoroughly ground and sieved
through 0.2 mm sieve in a Kjeldahl digestion tube or flask.
Add 10g of Potassium sulphate- Copper sulphate mixture (10:1 ratio) or kjeldahl tablet into the
tube and 35 ml of conc. H2SO4.
Heat initially at low temperature for first 10 to 30 min. until frothing stops and then raise the
temperature gradually to 360-410oC. Continue the digestion until the contents become light
yellow colour.
Distillation:
cool the digested sample and add 50ml of distilled water. Mix thoroughly, let it stand for few
minutes and transfer the content in 1 litre distillation flask. Carry out 4-5 washings with 50ml of
distilled water and transfer the content of every washing into the same distillation flask making
the final volume of about 300ml. the solution is made alkaline (pH>11) with 40% sodium
hydroxide solution using phenolphthalein indicator. Immediately attach the distillation flask or

96

tube to the distillation unit. Start distillation after immersing the tip of the condenser in 50ml
boric acid solution (with mixed indicator) in a conical flask. Collect about 150ml of the distillate.
Titration:
titrate the boric acid solution against the standardised H 2SO4 (0.02N). The end point is the
appearance of purple colour. Carry out blank titration similarly using distilled water as blank
starting from the digestion step to final titration.
Note:
In case if laboratory has automated or semiautomatic TKN assembly, the same may be used as
per procedure given in operation manual.
CALCULATION:

% TKN = (S B) X N X 1.4
Wt.

Where,
S = ml of standard H2SO4 acid used for sample
B = ml of standard H2SO4 acid used for blank
N = Normality of standard H2SO4 acid
Wt. = weight of the sample in gm.
EXPRESSION OF RESULTS:
Express the results as TKN % on in 2 decimal units on oven dry wt. basis.
REFERENCE:
Jackson, M. L. (1967): Soil chemical analysis. Prentice- Hill of India Pvt. Ltd.

97

## DETERMINATION OF TOTAL PHOSPHOROUS

PRINCIPLE:
The organic phosphorous is converted into inorganic form after digestion with conc. Nitric acid
Phosphorous as phosphate reacts with ammonium molybdate to form phosphomolybdic acid,
which in presence of stannous ions gives a blue colour complex , which can be determined
spectrophotometricallly at 690 nm.
APPPARATUS:
a)

b)

Hot plate

c)

d)

## Spectrophotometer for use at 690 nm.

REAGENTS:
a) Ammonium molybdate solution:
Dissolve 25 g (NH 4)6MO7O24.4H2O in 175 ml distilled water. Cautiously add 280ml
concentrated H2SO4 to 400 ml distilled water, cool add molybdate solution and dilute to 1
L.
b) Stannous chloride solution:
Dissolve 2.5 g fresh Sncl 2.2H2O in 100ml glycerol, heat and stir in water bath until
dissolved. Prepare freshly every time.
c)

## Nitric Acid (HNO3) conc. Sp. Gr. 1.40

d) Triacid mixture:
Mix 10ml of nitric acid (HNO 3), 1ml of Sulphuric acid H2SO4) and 4ml of 60%
Perchloric acid HClO3) in a conical flask.

98

e)

## Stock phosphate standard solution (100 mg of P/L):

Dissolve 0.439 g anhydrous Potassium hydrogen phosphate (KH 2PO4) in 1000ml of
distilled water.

## f) Working standard phosphate solution (1mg of P/L):

Dilute 10ml of stock Phosphate standard solution to 1000ml.
PROCEDURE:
Take 1.0 g or suitable quantity of oven dried (105 oC) sample thoroughly ground and sieved
through 0.2 mm sieve in a conical flask and add 5 ml of conc. HNO3 (5 ml per gm of sample).
Swirl the flask to moisten the entire mass of the sample and place it on a hot plate at a
temperature of 180-200oC in a fume cupboard and allow to boil to dryness.
Cool the flask and add 5 ml (5ml per gram of sample) of Triacid mixture (HNO 3-H2SO4-HClO4)
to the sample, digest at 180-200oC till the sample appears whitish or light yellow in colour.
Add 20 ml distilled water and 2-3 drops of phenolphthalein indicator . Neutralize the solution by
adding 1N NaOH drop by drop as until a faint pink colour appears.
Filter the whole content through whatman No. 42 filter paper and make up the volume to 100 ml
with distilled water in a volumetric flask. This accounts the total volume of digested solution.
Pipette out 50 ml of solution in a Nesslers tube and add 1ml of ammonium molybdate solution ,
followed by 5 drops of freshly prepared stannous chloride solution.
A blue colour will appear, wait for 10 minutes and record absorbance at 690 nm before 12 min.
using a spectrophotometer. Carry out a blank determination using distilled water.

Determine the concentration from the standard calibration curve (preferably 0.0, 0.1,0.2,0.4,0.5
mg of P/L) for phosphate prepared from appropriate dilutions of the working standard
phosphate solution.

99

CALCULATION:
Total Phosphorous as P mg/gm =

_AxV__
1000 x Wt.

Where,
A = conc. of phosphorous as P in mg/l obtained from calibration graph
Wt. = Weight of sample taken for digestion
V = Total vol. of digested solution

The result can also be expressed as% P2O5 of oven dry sold sample
A V
Total Phosphorous as % P2O5 =
1000 Wt
Where,
A = conc. Of phosphorous as P in mg/l obtained from the calibration graph
Wt. = weight of the sample taken for digestion
V = Total vol. of the digested solution
Note:
The factor 4.58/10 is applied to convert the mg/gm of P into % P2O5
EXPRESSION OF RESULTS:
Express the result as % P2O5 in 2 decimal units
REFERENCE:
Jackson, M.L. (1967): Soil Chemical Analysis. Prentice- Hill of India Pvt. Ltd, New Delhi.

100

## PREPARATION SOIL EXTRACT

REAGENTS:
Ethyl alcohol, 40%:
Mix 600 ml of distilled water with 400 ml of absolute or 95% alcohol.
Absolute alcohol:
Ammonium acetate solution:
Dilute 57 ml of glacial acetic acid to 800 ml with distilled water with distilled water and then
neutralize to pH 7.0 with concentrated NH4OH. Make up the final volume to 1 litre.
PROCEDURE:
Take 50 g of air dried soil in 500 ml beaker and add about 100 ml of 40% alcohol. Shake well
and keep for 15 min.
Filter the suspension through Whatman filter paper No. 50 filter paper using Buchner funnel and
vacuum pump.
Wash the soil 4-5 time with 50 ml portions of 40% alcohol. Perform the final washing with 50 ml
absolute alcohol to dry the soil.
Remove the filter paper and scrap the soil in 250 ml beaker. Wash the Buchner funnel and the
filter with 100 ml ammonium acetate solution for removing any adhered portion of soil.
Stir the suspension and keep over night.
Filter the supernatant and finally the soil, with additional ammonium acetate through Whatman
filter paper No. 42 using Buchner funnel and vacuum pump Leach the soil 4-5 time more with
portion ammonium acetate and make up the final volume of the filtrate 500 ml in a volumetric
flask.

101

DETERMINATION OF CHLORIDES
PRINCIPLE:
Most of the chlorides in the soil are soluble in the water and determined directly in soil solution.
The most common method is titrimetric, involving direct titration of the soil solution withAgNO3
using K2Cr4 as an indicator.
APPARATUS:
Mechanical or magnetic stirrer, vacuum pump, Buchner funnel, side arm conical flask, Whatman
filter paper No. 50 etc.
REAGENTS:
Silver nitrate, 0.02 N:
Dissolved 3.400 g of dried AgNO3 (A.R.) in distilled water to make 1 liter of solution and keep
in a dark bottle.
Potassium chromate,5%:
Dissolved 5 g of K2CrO4 in 100 ml distilled water.
PROCEDDURE:
Prepare 1:5 soil suspension by adding 100 ml of distilled water 20 g of soil. Stir mechanically for
about one hour at regular interval.
Filter the suspension through Whatman No. 50 filter paper using Buchner funnel and vacuum
pump.
Take 50 ml of sample in an Erlenmeyer flask and add 2 ml of K2CrO4 solution.
Titrate the contents against 0.02 N AgNO3 until a persistent red ting appears.

102

CALCULATION:

( ml N ) of AgNO3 35.5
% OF Chlorides =
ml of soil solution 2

103

REAGENTS:
1.

## Ammonium Oxalate Extractant:

Take 58 ml of Glacial Acetic Acid in 500 ml

## measuring cylinder & make up the

volume to 400 ml. Take 70 ml Ammonium Hydroxide (NH 4 OH) and make up the
volume upto 400 ml. Mix above two solutions and adjust pH to 7.0, either by adding
Acetic acid or ammonium hydroxide as required. Make up the volume to 1000 ml.
2.

Murexide indicator:

3.

## Sodium Hydroxide - 1N:

Dissolve 40 g Sodium Hydroxide in some distilled water in a 1000 ml beaker. After
dissolving it completely, transfer the contents into one litre volumetric flask and make
up to exactly 1000 ml.

4.

EDTA 0.01 M:
Weigh accurately 3.723 gm of disodium ethylene diamine tetra acetate dihydrate and
dissolve in distilled water. Dilute to 1000 ml with water. One ml of this = 0.4008 mg of
Calcium.

PROCEDURE:
1.

2.

3.

4.

5.

## Take 20 ml filtrate; add 100 mg Murexide indicator & 5 ml of 1 N Sodium Hydroxide

solution.

6.

Titrate against 0.01 N EDTA solution, with purple colour appearance end point and
note volume of EDTA titrant.

104

CALCULATIONS:
0.4008
Calcium %

100

Vt

Vt

105

Where,

## ESTIMATION OF MAGNESIUM CONTENT IN A SOIL SAMPLE

REAGENTS:
1.

EDTA 0.01 N:
Weigh accurately 3.723 gm of disodium ethylene diamine tetra acetate dihydrate and
dissolve in distilled water. Dilute to 1000 ml with water.

2.

Ammonia buffer:
Dissolve 6.75 g of NH4Cl in 57 ml of Ammonium Hydroxide and dilute to 100 ml with
distilled water.

3.

## Erichrome Black T indicator:

Mix 0.5 g of Erichrome Black T with 100 g of Sodium Chloride and grind it to get a fine
powder.

PROCEDURE:
1.

## Take 20 ml of soil extract in a conical flask and add 10 ml of distilled water, 2 ml of

ammonia buffer and 100 mg Erichrome black T indicator. Shake well to mix all
contents.

2.

Titrate with 0.01 M EDTA solution until solution changes from red to blue. Note the
volume of EDTA used.

3.

CALCULATIONS:

0.4008
Magnesium % =

100

Vt

106

Where,

Vt

## = Weight of soil sample used for extraction, g

107

DETERMINATION OF AMMONIA
PRINCIPLE:
Ammonia ions present in the exchangeable complex of the soil can be extracted by leahing with
sodium chloride in acid medium. Ammonia in the extract is then determined by any of the
method described for water analysis.
REAGENT:
A. Sodium chloride solution
Dissolve 100g NaCl in distilled water to prepare 1 litre solution. Adjust the pH to 2.5 with dilute
HCl.
B. Standard ammonia solution
Dissolve 3.819 g of anhydrous NH4Cl in distilled water to prepare 1 litre of solution. This
solution contains 1000mg/l NH3-N, Dilute this solution 100 times (101000ml) to prepare the
solution containing 10 mg/l NH3-N.
C. sulphuric acid, 0.04 N
Add 2ml of (1+1) H2SO4 (sp. Gr. 1.84) to 1 litre of distilled water.
D. Borax buffer
Add 4 g of Na2B4O7.10H2O to 100ml of distilled water. Heat to dissolve the crystals.
E. Nesslers reagent
a) Dissolve 25 g of HgI2 and 20 g of KI in 500 ml of distilled water.
b) Dissolve 100 g of NaOH in 500ml of distilled water. Store these two solutions in brown glass
air tight stoppered bottles. Mix (1+1) just before use.

PROCEDURE:
1. Take 100 g freshly collected soil in a 500ml conical flask and add 200 ml of acidified NaCl
solution. Keep the flask for about 30 min. with intermittent thorough shaking.

108

2. Filter the suspension through Whatman No. 42 filter paper using Buchner funnel and vacuum
pump. Rinse the conical flask with about 50 ml NaCl solution to remove the residual soil and
transfer the rinsings to the buchner funnel. Leach the soil with 200ml additional NaCl solution.
Make up the final volume of the leachate to 500ml with NaCl solution in a volumetric flask.
3.

Take 50 ml of sample in the distillation assembly through tap a and add 1 ml of borax

buffer solution.
4.

put 2.5 ml of 0.4 N H 2SO4 in a 100ml Erlenmeyer flask and place below the condenser so

## that the tip of outlet dips in the acid.

5.

Keep the boiling fask (C) on the heater to pass the steam into the sample through chamber
( D ).

6.

Ammonia will be distilled off and collects in the sulphuric acid as (NH4)2 SO4. Continue

## the distillation until nearly 40 ml of distillate is collected.

7. Remove the flask having distillate. Cool the boiling flask so that all the waste contents will
be sucked into the chamber ( D ).
8. Remove the waste contents through tap B.
9. Run a blank with distilled water using same quantity of the chemicals.
10. make up the volume of distillate to 50 ml and add 1ml of Nesslers reagent .A brown colour
will develop.
11. Measure the absorbance at 425 nm.
12. Prepare a standard curve between 0.05 to 2.0 mg/l of NH3-N by diluting the standard
NH3-N solution.
13. Find out the moisture content of the soil separately by oven drying method.

109

CALCULATION:
a. Colorimetric Method:
%ammonia-N = NH3-N mg/l soil extract x V
10000 X S
Where,
M = % Moisture content of soil
V = volume of total extract prepared (ml )
S =Weight of soil taken (g)

110

100
100-M

## TOTAL ALKALINITY, SOLUBLE CARBONATES AND BICARBONATES

PRINCIPLE:
Like water, total alkalinity, soluble carbonate and bicarbonates can be determine by the direct
titration of the soil solution with strong acid (HCl or H2SO4) using methyl and phenolphthalein
indicators.
REAGENTS:
A. Hydrochloric acid, 0.1 N:
Dilute 12 N concentrated HCL (sp. Gr. 1.18) to 12 times (8.34 100 ml) to prepare 1.0 N HCl.
Diluted it further to make 0.1 N HCl (100 1000 ml). Standardize it again sodium carbonate
solution.
B. Methyl orange indicator, 0.05%:
Dissolved 0.5 g of methyl orange in 100 ml distilled water.
C. Phenolphthalein indicator:
Dissolved 0.5 g of phenolphthalein in 50 ml of 95% of ethanol and add 50 ml of distilled water.
Add 0.05 N CO2 free NaOH solution dropwise, until the solution turns faintly pink.
D. Sodium carbonate, 0.1 N:
Dissolved 5.300 g of Na2 CO3, previously dried at 250oC for about 4 hours in distilled water to
prepare 1 liter of solution.
PROCEDURE:
1. Prepare 1:5 soil solution as described in determination of soil chloride.
2. Take 100 ml of Erlenmeyer flask and add 2 drops of phenolphthalein indicator.
3. If the solution remain colourless, PA=O, and total alkalinity is determined as described in step
v.
4. If the colour changes to pink after addition of phenolphthalein, titrate it with 0.1 N HCl until
the colour disappears at end point This is phenolphthalein alkalinity (PA).

111

5. Now add 2-3 drops of methyl orange to the same sample and continue the titration further,
until the yellow colour change to pink at end point. This is total alkalinity (TA).
CALCULATION:
(ml N) of HCl 500
a. Total alkalinity, meq/100 g =
ml of soil sample
V1 N of HCl 1000 60
b. Carbonate % =

= V1 0.03
ml of soil solution 2000
(V2 V1) N of HCl 1000 61

c. Bicarbonate % =

## = (V2 V1) 0.0305

ml of soil solution 2000

Where,
V1 = Volume of HCl used for phenolphthalein end point.
V2 = Additional volume of HCl used from phenolphthalein end point to methyl orange end
point.
To converted in to values in mg/100 g for carbohydrate, multiply the results in % with 1000.

112