1

DISSOLVED OXYGEN ( DO)

Method:- Iodometric Method (Winkler’s Method)

Reagents:-

1.Manganous Sulfate, MnSO4

Dissolve 91.0g of MnSO4.H2O in d/w. Filter and dilute to 250 ml.

2.Alkali-Iodide-Azide reagent
Dissolve 175g of KOH and 37.5 g of KI in freshly boiled and cooled d/w.
Disolve 2.5 g of Sodium Azide (NaN3) in 10 ml of d/w .Mix the two solutions
and make upto 250ml.

3.Conc. Sulfuric Acid(H2SO4)

4.Starch Indicator
Make a smooth paste of 0.5 g soluble starch in cold water and pour this into 100
ml of boiling d/w with constant stirring. Boil for one minute and allow to cool
before use. Use clear supernatant. Preserve with 0.2g of salicylic acid or by
adding a few drops of toluene.

5.Sodium Thiosulfate Stock Solution (Na2S2O3.5H2O), 0.125N

Dissolve 31.5 g of Sodium Thiosulfate pentahydrate, Na2S2O3.5H2O in boiled and
cooled distilled water and make the volume upto 1L.Add 5 ml chloroform or 1g
of NaOH as preservative.

6.Standard Potassium Iodate (KIO3), 0.0125N

Dissolve 0.446 g of Potassium Iodate, KIO 3, previously dried at about 120˚C and
desiccated in d/w and dilute to 1 L. The solution is stable for a long period if
stored in dark glass stoppered bottle.

7.Standard Sodium Thiosulfate Titrant ( Na2S2O3.5H2O), 0.0125N

Dilute 100 ml of Sodium Thiosulfate Stock Solution to 1L with d/w. Standardize
this diluted solution against the Standard Potassium Iodate (KIO 3), 0.0125N
before use.
 2

Principle:

The Iodometric test (Winkler’s method ) is precise and reliable titrimetric for DO
analysis. The test is based on the addition of divalent manganese solution, followed by strong
alkali, to the sample in a BOD glass-stoppered bottle. DO present , rapidly oxidize an
equivalent amount of the dispersed divalent manganous hydroxide of higher oxidation state
(brownish orange in colour).This brownish-orange colour indicates presence of oxygen. In the
presence of iodide and on subsequent acidification, higher manganese hydroxide revert to the
divalent state, and liberate iodine equivalent to the original dissolved oxygen content of the
sample. The iodine liberated is titrated with a standard solution of sodium thiosulphate using
starch as indicator.

Standardization:-

Standadize Sodium Thiosulfate Solution( 0.0125N Na 2S2O3.5H2O) with 0.0125 N

Potassium Iodate (KIO3)
Pipette 10 ml of 0.0125 N Potassium iodate solution into a conical flask containing
about 100 ml distilled water.
Add 2 ml of Conc. Sulfuric Acid followed by about 2g (one spatula) of Potassium
iodide (KI) solid. Titrate immediately against 0.0125 N Sodium Thiosulfate using starch as
indicator.

Procedure:-

1.Fill the BOD bottle with water sample upto the mouth and stopper it.

2.Add 2ml of Manganous Sulfate followed by 2 ml of Alkali Iodide Azide reagent ,

dipping the pipette little below the surface.

3.Stopper carefully and thoroughly mix the contents by inverting the bottle at least 15
times.

4.Allow the yellow precipitate to settle completely leaving a clear supernant liquid.

5.Add 2ml Conc. Sulfuric Acid immediately after removing the stopper by the side of
the bottle. Restopper and mix by gentle inversion until all the precipitates dissolve.

6.Pipette 101.3 ml of the above solution into 250 ml Conical flask (1.3ml extra to
account for reagents added)

7.Immediately titrate with Standard Sodium Thiosulfate Titrant (Na 2S2O3.5H2O) to a

pale yellow straw colour. Add 2 ml starch solution and continue the titration till the
first disappearance of the blue colour.
 3

Calculations:-

1.Standadization of Sodium Thiosulfate (Hypo)

N1V1 = N2V2

Where, N1 is Normality of Hypo

V1 is vol. of Hypo
N2 is Normality of Pot. Iodate
V2 is Vol. of Pot. Iodate

N1 = 10 × 0.0125
V1

2.Concentration of D.O in µg/l

D.O = Normality of Hypo × Eq.wt. of O2 × 1000 × titrant (Vol.of Hypo)

Vol. of sample taken for titration

= Normality of Hypo × 8 × 1000 × Vol. of titrant (Hypo)

100

Precautions:-

1.Manganous solution should not give colour with starch when added to an acidified
solution of Potassium Iodide.
2.Alkali Iodide Azide reagent should not give colour when diluted and acidified.
3.while sampling, remove bubbles entrapped in the bottles by tapping the neck of the
bottle with the stopper
4.Determine DO at site, if not possible, fix at the site by adding Manganous Sulfate and
Alkali Iodine Azide and titrate in the laboratory (add acid in the laboratory)
5.Do not use soap or synthetic detergents for washing the DO bottles.

Result:

Report the value to the nearest of first decimal i.e. 0.1 mg/l
 4

CHLORIDE

Method: Argentometric titration

Reagents:

1. Pot. Chromate (K2CrO4) Indicator Solution (5 %).

Dissolve 25g K2CrO4 in d/w.Let stand for 12 hrs. Filter if required and dilute to
500 with d/w.

2. Standard Silver Nitrate (AgNO3), (0.0141N)

Dissolve 2.395g AgNO3 in d/w and dilute to 1L.

3. Standard Sodium chloride (NaCl), (0.0282N)

Dissolve 1.648 g of NaCl (dried at 140ºC) and dilute to 1 L.

4. Sodium hydroxide (NaOH), 1 N

Dissolve 20 g NaOH in d/w and dilute to 500 ml.

5. Sulphuric Acid (H2SO4), IN

Prepare 1:36 Conc. H2SO4.

Principle:

Chlorine as Chloride(Cl-) is determined in neutral or slightly alkaline solution by

titration with Silver Nitrate (AgNO3) , using Potassium Chromate as an indicator.
Silver Chloride is quantitatively precipitated before red Silver Chromate is
formed.
Ag+ + Cl- = AgCl (white ppt.)
2Ag + CrO4 = AgCrO4 (Red ppt.)
 5
Procedure
1. Take 50 ml of Sample, pH adjusted (7.0 – 8.0) in Erlenmeyer flask and add 0.5
ml K2CrO4. as indicator.
2. Titrate with standard AgNO3 solution with constant stirring until it turns yellow
to brick red which persists for few seconds.
3. Standardize AgNO3 against 0.0282 N NaCl solution.
Pipette 10 ml NaCl (0.0282N). Add 0.5 ml Pot. Chromate (indicator). Titrate
against AgNO3 (0.0141N) with constant stirring until it turns yellow to brick red
end point.
Calculations:
1. Standardization:

N1V1 = N2V2
Where N1 is Normality of AgNO3
V1 is Vol. of AgNO3 consumed
N2 is Normality of NaCl
V2 is vol. of NaCl consumed

Therefore, N1 = 10 X 0.0282 (N)

V1

2. Conc. Of Chloride in Mg / l

= Normality of AgNO3 X At. Wt. of Chloride X 1000 X Vol of AgNO3 consumed

Vol. of Sample (ml)

= Normality of AgNO3 X 35.45 X 1000 X Vol of AgNO3 consumed

50

Precaution :
1.The vol. of a sample should be selected in such a way that it should not contain less
than 20mg/l chloride. On the other hand, it may be increased if the chloride content is too low
and may be reduced , if the content is more than 100 mg/l.
2. Use Chloride free distilled water or deionized H2O for preparation of chemicals and
dilution.
3.Titration should be carried out on a opaque white base (white porcelain dish maybe)

Result: Round off the results to the nearest Whole Number.

 6

TOTAL HARDNESS as CaCO3

Method:- Complexometric titration solution (Ammonia–ammonium chloride buffer)

Reagents:-
1. Ammonium Buffer Solution (Ammonia – ammonium chloride buffer)
Dissolve 16.9g of Ammonium Chloride (NH4Cl) in 143ml of Conc. Ammonia
Solution (NH4OH of Sp. Gr.0.90) or if Sp.gr. is 0.91, take 165ml. Add 1.25g of
Magnesium salt of EDTA and dilute to 250 ml with d/w. If Magnesium Salt of
EDTA is not available, dissolve 1.179g di-Sodium salt of EDTA(AR grade) and
780 mg MgSO4.7H2O or 644 mg of MgCl2.6H2O in 50 ml d/w.
Add this solution to NH4Cl + NH4OH solution and dilute to 250 ml.

2. Standard EDTA (Ethylene – Diamine Tetraacetic – Acid), 0.01 M (0.02N)

Dissolve 3.723g di Sodium Salt of EDTA, AR grade disodium ethylene diamine
tetra acetate dihydrate, also called (ethylene dinitrilo) – tetra acetic acid disodium
salt EDTA (Na2 C10 H14 O8 N2. 2H2O) in d/w and make upto 1L.
Store it preferably in polythene bottle or in a pyrex bottle.

3. a)Erichrome Black– T (EBT) Indicator – Sodium salt of 1 (1-hydroxy,2-

napthylazo) 6-nitro-2napthol -4 sulphonate is a dye having 203 no. in the color
index. EBT is used in powder form with sodium chloride, NaCl. Mix 0.5 g EBT
wth 100 g of NaCl, grind together in mortar to obtain homogenous mixture of 40-
50 mesh size. Use about 0.2 to 0.4 g per 100 ml sample for each titration.
b)Calmagite – 1-(1-hydroxy-4 methyl,-2 phenylazo) 2 napthol -4 sulphonic acid
is a dye which produces same colour change as EBT when titrated for hardness
estimation. It has a more sharp end poinjt. Dissolve 0.1 g calmagite I in 100 ml
d/w. use 1 ml indicator per sample to be titrated

General:

Hardness of water is the traditional measure of the capacity of water to react with
soap. More hard the water, more the soap required to produce lather. Hardness is not a specific
constituent but is variable and complex mixture of cations and anions. It is mainly due to
Calcium and Magnesium ions, however, some other polyvalent ions such as aluminium, iron,
manganese, strontium and zinc also contribute to hardness. In natural hardness, it is defined as
source of the calcium and magnesium ions expressed as calcium carbonate. The degree of
hardness of drinking water has been classified in terms of the equivalent CaCO 3 concentration
as follows:
Soft 0 - 60 mg/l
Medium 60 – 120 mg/l
Hard 120 – 180 mg/l
Very Hard > 180 mg/l
 7

Principle:

EDTA ( Ethylene diamine tetra acetic acid ) and its sodium salt forms a chelate
complex when added to a solution of certain metal cations. AT pH 10+ 0.1, EDTA preferably
forms complex with calcium and magnesium. If, a small quantity of dye such as Erichrome
Black T (EBT) or calmagite is added to an aqueous solution containing calcium and
magnesium at pH 10 + 0.1,the solution becomes wine red because the metals form an unstable
complex with dyes. If, EDTA is added as titrant, the calcium and magnesium will be chelated
with EDTA to form EDTA metal complex, which is more stable. When all the magnesium and
calcium has been complexed, the solution turns blue as the indicator will be free from metal-
indicator unstable complex marking the end point of titration. Magnesium ion must be present
in solution to yield a satisfactory end point, To ensure it, a small amount of
compleximetrically neutral magnesium salt of EDTA is added to the buffer, this automatically
introduces sufficient magnesium and obviates the need to blank correction. The sharpness of
the end point increases with increasing pH. However, the pH cannot be increased indefinitely
because of hr danger of precipitating calcium carbonate (CaCO3) or magnesium hydroxide
[Mg(OH)2]. So, pH at 10+ 0.10 is a satisfactory compromise.

Procedure: -

1. Take 50 ml of Sample in a conical flask.

2. Add 0.5 ml of Ammonia buffer (pH 10 ± 01)
3. Add 1 drop of EBT indicator.
4. Titrate with 0.01 M EDTA solution slowly until a reddish tinge appears and add
the last few drops within 3-5 seconds till the end point of blue colour.
The whole titration procedures should be completed within 5 minutes after
addition of buffer.
 8

Calculations:-

Conc. of Total Hardness as CaCO3 in mg/l.

= Volume of EDTA consumed for sample X Molarity of EDTA X Mol. Wt. of CaCO3 X1000
Vol. of sample taken for titration.

= Volume of EDTA X 0.01 X 100 X 1000

50

Precaution:

1. Carry out the titration within 5 minutes after the addition buffer to minimize the tendency
toward CaCO3 precipitation.

2. Select a suitable sample volume that requires less than 15 ml titrant (EDTA) and complete
titration within 5 minutes of buffer addition.
 9

CALCIUM

Method:- Complexometric (EDTA titritometric method)

Reagents:-

1. Standard EDTA Solution (0.01M)

Dissolve 3.723 g AR grade Ethylene diamine tetra acetic acid (EDTA) di-
sodium salt, Na2C10H12O8N2.2H2O in d/w. and dilute to 1 L. Standardize it against
standard calcium solution. Standard EDTA titration 0.0100 (M) is equivalent to
400.8 µg calcium/ml.

2. Sodium hydroxide, NaOH (IN)

Dissolve 20 g NaOH in d/w and dilute to 500 ml.

3. Indicator –
a)Erichrome Blue Black RC (Blue end point): Mix 200 mg dye with 100 g
NaCl and grind it to 40 – 50 mesh, 0.2 tp 0.4 g of the mixture is required
for each titration. During the course of titration , the colour changes from
red through purple blkuish purple to a pure blue without any trace of
reddish or purple tinge.pH of the sample maybe raised to 14 by adding 8
N NaOH to get a sharper colour change.

b)Murexide (Amm. Purpurate) (Pink to purple): Prepare by mixing 200 mg

dye in 100 g solide sodium chloride, NaCl .Grind it to 40 to 50 mesh.
This indicator is unstable in alkaline.

c)Calcon Indicator (Calcium Indicator) light pink to Blue end point

Principle:

When EDTA is added to water containing both calcium and magnesium, it

combines first with the calcium when the pH of the test solution is sufficiently high (12 to 13),
magnesium gets precipitated as magnesium hydroxide, Mg(OH) 2. Therefore, at pH of 12 to
13, calcium can be directly estimated by titration with EDTA using a suitable indicator, which
can only combine with calcium (Murexide or Erichrome blue black R)
 10

Procedure: -

1. Take 50 ml of Sample in a conical flask.

2. Add 0.5 ml of Sod. Hydroxide (NaOH) buffer.
3. Add 0.1 – 0.2g indicator.
4. Titrate with standard 0.01M EDTA slowly with continuous stirring to proper
end point.

Complete titration within 5 minutes.

Calculations:-

= Volume of EDTA consumed X Molarity of EDTA X At. Wt. of Calcium X 1000

Vol. of sample taken

= Volume of EDTA consumed X 0.01 X 40 X 1000

50

Precaution:

1.Titrate immesiately after adding indicator because it is unstable under alkaline

conditions.
2. For recognizing end point, use a colour comparison blank prepared under
identical conditions.
 11

MAGNESIUM

Method:- Calculation method

Principle:-
While measuring total hardness, calcium and magnesium ions react with EDTA
to form soluble complexes and completion of reaction is indicated by the colour
change of a suitable indicator. By subtracting the value of calcium hardness from
total hardness, magnesium hardness can be measured. Magnesium as Mg ++ can
be calculated by multiplying magnesium hardness with 0.243.

Calculations:-

= Morality of EDTA X (Total Hardness Reading – Cal. Reading) X At. Wt. of Mg. X 1000
Vol. of sample taken

= 0.01 X (Total Hardness Titrant R. – Cal R.) X 24 X 1000

50
 12
ALKALINITY

Method: Visual titration

Reagents:-

1. Standard H2SO4 , 0.02 N

Dilute 28ml of Conc. H2SO4 to 1L with d/w to make 1N H2SO4.
Further dilute 100 ml of 1N H2SO4 to make 0.1 N H2SO4.
Dilute 200 ml of 0.1N H2SO4 to 1L with d/w to make 0.02 N H2SO4.

2. Standard Sod. Carbonate Sol., 0.05 N

Dry approximately 5 gm. of Na2 CO3 (AR Grade) at 250º C for 4 hrs. and then
cool in a desiccators for 20 mins. Weight approx. 2.65 gm and dissolve this Na 2
CO3 in 1 L of d/w.

Prepare fresh solution every week.

3. Phenolphthalein Indicator Solution (pH 8.3) –

Dissolve 1 gm phenolphthalein in 100 ml 95 % ethyl alcohol or isopropyl
alcohol and 100 ml d/w.

4. Methyl Orange Indicator(pH-4.5)

Dissolve 0.5 g of methyl-orange powder in d/w and dilute to 1 L with d/w.

General:
The alkalinity of water is a quantitative measure of the basic constituents of water
and is defined as the capacity of the water to neutralize a strong acid at a
designated pH. The alkalinity of natural waters is mainly due to the presence of
salts of carbonates, bi-carbonates and hydroxide of calcium, magnesium, sodium
and potassium. The borates, silicates and phosphates also contribute to some
extent. Alkalinity value help in deciding chemical doses in water and wastewater
treatment processes particularly in coagulation, softening and operational control
of anaerobic digestion and suitability of water for irrigation purposes.
Principle:
Alkalinity of water sample can be estimated by titration with standard acid using
phenolphthalein indicator (pH-8.5) or methyl orange indicator (pH-4.5 ) or with ph
meter (potentiometer method). Alkalinity is reported in terms of CaCO 3 in mg/l. In
the absence of above indicators, metacresol purple (pH-8.3) and bromocresol green
(pH-4.5) can also be used for titration,Methyl Orange indicator will indicare total
alkalinity ( complete neutralization of OH CO3HCO3
 13
Procedure: -

1. Take 50 ml of Sample in a conical flask.

2. Add few drops of phenolphthalein indicator if pink colour appears .titrate with
0.02N of H2SO4 to the colorless end point (pH-8.3). Note down the vol. of acid
consumed.
3. Add few drops of Methyl Orange Indicator to the above soln., if yellow colour
appears, continue titration with acid will till pink end point (pH-4.5). Note down the
vol. of acid consumed.

Standardizations of H2SO4 (0.02N):-

Take 10 ml of Na2CO3 soln. in a conical flask. Add few drops of Methyl Orange indicator
and titrate against 0.02N H2SO4. End point – Reddish Orange.

Calculations:-

1. Standardization of H2SO4
N1V1 = N2V2
N1 = N2V2
V1
Where, N1 is Normality of H2SO4
V1 is Vol. of H2SO4 consumed
N2 is No. of Na2CO3
V2 is vol. of Na2CO3
Therefore, N1 = 0.05 X 10
V1

2. Phenolphthalein Alkalinity

TO BE CONTINUED

3. Total Alkalinity as CaCO3, Mg / l

= R X N1 X 50 X 1000 .
Vol. of Sample taken for titration

Where, R is acid consumed.

N1 is Normality of H2SO4
50 is Mol. Wt. of CaCO3.
 14
SULPHATE, SO4 2-

Method: Turbidimetric Method

Principle: Sulphate (SO42-) ions are precipitated in a hydrochloric medium with Barium
Chloride (BaCl2) so as to form an uniform suspensions of BaSO 4. The absorbance
of the BaSO4 suspensions is measured by a photometer and the SO 42- concentration
is determined by comparison of the reading with the standard curve.

Reagents:

1.Conditioning reagent: Mix 50 ml. of glycerol with a solution containing 30 ml

Conc. HCl, 300 ml of d/w, 100 ml of 95% ethyl alcohol and 74 g of NaCl.
2. Barium Chloride : crystals, 20 – 30 mesh
3. Stock Sulphate Solution : Dissolve 1.4798 g. of previously dried, anhydrous
Na2SO4 in d/w and dilute to 1000 ml.
1ml = 1mg SO4
4. Working Standard Sulphate Solution: Dilute 25 ml of stock solution to 250 ml
with d/w
1ml = 0.1mg or 100 ppm, 1mg/ml = 1000 ppm
1mg/l = 1 ppm

Procedure:

A. Calibration curve preparation:

1.Take 5, 10, 15, 20 and 25ml.of Standard Sulphate solution and dilute to 50 ml.
with d/w. to make standard solns. of 10, 20, 30, 40 and 50 ppm respectively.
2. Take 50ml Sulphate free d/w for blank reagent.
3. Add to the above 1 & 2
a) 5 ml conditioning reagent and mix well using magnetic stirrer .
b) While stirring, add about 0.5g (half a spatula) of BaCl 2 crystals and continue to
stir exactly one minute.
4. Immediately after 1 min, measure absorbance reading at 420 nm for the above
solutions carrying out the reagent blank using Sulphate free d/w.
5.Prepare Calibration curve for Sulphate concentration v/s. absorbance.

B. Sample Measurement:
 15

1.Take 50 ml. of sample, filtered with filter paper, GF-41.

2. Add 5 ml conditioning reagent and mix well using magnetic stirrer. While stirring,
add about 0.5g (half a spatula) of BaCl 2 crystals and continue to stir exactly one
minute.
3. Immediately after 1 min, measure absorbance reading at 420 nm and not down.

Calculations:

Conc. of SO4 in mg/l = R× mf

Where, R = absorbance reading of sample

mf = absorbance
Conc.
 16
PHOSPHATE, PO4 3-

Method :Colorimetric Method (Stannous Chloride Reduction Method)

Principle: Molybdophosphoric acid is formed and reduced by stannous chloride to intensely

coloured molybdenum blue. The method is applicable for lower level upto 7 µg
P/L by use of increased path length.

Reagents:
1.Ammonium molybdate reagent: Dissolve 25g reagent (NH4 6 MO7
O24.4H2O) in 175 ml d/w .Carefully add 280 ml con, Sulphuric acid to 400 ml
d/w , cool and add Molybdate solution and dilute to 1 L.
2.Stannous chloride reagent: Dissolve 2.5 g fresh SnCl2.2H2O in 100 ml
glycerol . Heat and stir in waterbath until dissolved.
3.Stock phosphate Standard Soln. (100mg/I - P): Dissolve 0.439 g. anhydrous
KH2PO4 in d/w and dilute to 1 L.
4.Working Standard Phosphate solution: Dilute 10 ml. of stock solution to 100
ml with d/w to make 10 ppm. Dilute 10 ml of 10 ppm soln to 100 ml with d/w to
make 1 ppm soln.
Procedures:

A. Calibration curve preparation:

1. Take 5, 10, 15, 20 and 25ml.of working Phosphate solution (1 ppm) and dilute
to 50 ml. with d/w. to make standard solns. of 0.1, 0.2, 0.3, 0.4, 0.5 ppm
respectively.
2. Take 50 ml d/w for blank reagent.
3. Add to the above 1 & 2 with thourough mixing after each addition, the
following:
a. 1ml of Ammonium molybdate reagent
b. 4 drops of Stannous chloride reagent.
4. After 10 mins, but before 12 mins, measure absorbance for the standard solns.
and at 690 nm and prepare calibration curve

B. Sample Measurement:

1. Take 50 ml. of sample and add 1ml of Ammonium molybdate reagent and 4
drops of Stannous chloride.
2. After 10 mins, but before 12 mins, measure the absorbance using d/w as
reagent blank and dote down.
 17

Calculation:-
Conc. of PO43- in mg/l = R × mf

Where, R = absorbance reading of sample

mf = absorbance
Conc.

Precautions: Clean all the glasswares with hot HCl and rinsed well with d/w. Preferably
reserve the glassware only for phosphate determination. Avoid use of phosphate containing
detergents for cleaning of glasswares. All reagents should be prepared by phosphate free
distilled water.
 18
Nitrogen Ammonia (N-NH3)

Method: Colorimetric method

Reagents:
1. Rochella salt: Dissolve 50g Potassium/Sodium tarterate in 100 ml of d/w
2. Nessler’s reagent: (A)Dissolve 100g of HgI and 70g of KI in small quantity of
d/w and cool .(B) Dissolve ………g of NaOH in 500 ml of d/w and cool. Mix
soln. B very slowly to soln. A with stirring.
3. Stock Ammonia Standard Solution: Dissolve 3.819 g of NH4Cl in d/w and
dilute to 1 L to make 1000mg/l – N soln.
4. Working standard Ammonia Solution:
Dilute 10ml of Stock soln to 100ml to make 100ppm soln. Again dilute 10ml of
100ppm soln. to 100ml to make 10ppm soln.

Procedures:

A.Calibration curve preparation:

1.Take 3, 6, 9, 12 and 15ml.of working Ammonia solution (10 ppm) and dilute to
50ml. with d/w. to make standard solns. of 0.6, 1.2, 1.8, 2.4, 3.0 ppm
respectively.
2. Take 50 ml d/w for blank reagent.
3.Add to the above 1 & 2 , 1ml of Nessler’s reagent and 1ml of Rochella’s salt.
4. Measure absorbance at 415 nm and prepare calibration curve.

B.Sample measurement:
1. Take 50ml of sample (if NH3 is more concentrated, it may be diluted so that
absorbance comes within the range, for this, take 10ml of sample and make
upto 50ml ie., five times dilution )
2. Add 1ml of Nessler’s reagent and 1ml of Rochella’s salt.
3. Measure absorbance at 415 nm using d/w as reagent blank and note down .
Calculation:

Conc. of N-NH3 in mg/l = R × mf × D (Dilution factor/no of times of dilution)

Where, R = absorbance reading of sample

mf = absorbance
Conc.
 19

Nitrogen Nitrate (N-NO3)

Method: Ultra violet Spectrophotometric Method

Principle: Measurement of UV absorption at 220 nm enables rapid determination of NO 3-

because dissolved organic matter also may absorb 220nm and NO3- does not absorb
at 275nm and measurement made at 275nm may be used to correct the NO 3- value.
The nitrate calibration curve follows Beer’s law upto 11 mg/l NO3- N.

Reagents:
1. Nitrate free water: Use double distilled, deionised water of high purity to
prepare all solutions and dilutions.
2. Hydrochloric acid: 1:1 HCl.
3. Preservative, CHCL3
4. Stock Nitrate standard solution: Dissolve 7.220 g of previously dried KNO3 in
1 L of d/w to make 1000mg/l-N ).
5. Intermediate Nitrate Solution: Dilute 10 ml of stock soln. to 100ml with d/w to
make 100 ppm .Again, dilute 10ml of 100 ppm soln. to 100 ml with d/w to
make 10 ppm soln. Preserve with 2ml CHCL3/l. This solution is stable for 6
months.

Procedures:
A.Calibration curve preparation:

1.Take 2.5, 5 , 7.5, 10, 12.5 ml of Nitrate solution (10 ppm) and dilute to 50 ml.
with d/w. to make standard solns. of 0.5, 1.0, 1.5, 2.0, 2.5 ppm respectively.
2.To the above, add 1ml of 1:1 HCl and mix thoroughly.
3.Read absorbance or transmittance against double distilled water set at zero
absorbance or 100% transmittance. Use a wavelength of 220 nm to obtain NO 3-
reading and a wavelength of 275 nm to determine interference due to dissolved
organic matter.
4.Prepare a calibration curve (subtract absorbance reading at 275 nm from that at
220nm) by plotting absorbance due to NO3- against No 3—N concentration of
standards.

B.Sample measurement:

1.Take 50 ml of sample in Nessler’s tube and add 1ml of 1:1 HCl and mix well.
2.Read absorbance at 220nm and 275 nm usinf d/w as blank reagent. Subtract the
absorbance reading at 275 nm from that at 220nm and note down as sample
absorbance reading
 20

Calculation:

Conc. of N-NO3 in mg/l = R × mf × D (Dilution factor/no of times of dilution)

Where, R = absorbance reading of sample

mf = absorbance
Conc.
 21
Nitrogen Nitrite (N-NO2-)

Method: Colorimetric Method (Diazotization method)

Principle: Nitrite (NO2-) is determined through formation of reddish purple azodye produced
at pH 2 to 2.5 by coupling di-azotized Sulphanilamide with N-(1-naphthyl)-
ethylenediamine dihydrochloride.The applicable range of the method for
Spectrophotometric measurements is 10 to 1000ug NO 2-N/L.The photometric
measurements can be made in the range 5 to 50 ug N/2 The colour system obeys
Beer’s law upto 180ug N/L with a 1 cm. light path. Higher NO2- concentratio9ns
can be determined by diluting the sample. The colour measurement is done at
543nm.

Reagents:
1.Nitrite free water.
2.Sulphanilamide reagent: Dissolve 5g od Sulphanilamide in a mixture of 50 ml
conc. HCl and 300 ml d/w and make upto 500 ml.
3.NEDA reagent: Dissolve 0.5 g of NEDA hydrochloride in 500 ml of d/w.
4.Stock Nitrite solution: Dissolve 0.4928 g of NaNO 2 in d/w and dilute to 1L with
d/w to make 100mg/l-N soln.
5.Intermediate Nitrite solution: Dilute 10 ml of stock soln. to 100ml to make 10ppm
soln. Again, dilute 10 ml of 10 ppm soln. to 100 ml with d/w to make 1 ppm
standard.
Procedures:
A.Calibration curve preparation:

1.Take 1, 2, 3, 4, 5 ml of Nitrate solution (1 ppm) and dilute to 50 ml. with d/w. to

make standard solns. of 0.02, 0.04, 0.06, 0.08, 0.10 ppm respectively.
2.Take 50ml d/w for blank reagent
3.To the above, add 1ml of Sulphalinamide reagent and 1 ml of NEDA reagent
and mix thoroughly.
4. After 10 mins, read absorbance at wavelength of 540 nm.
5.Prepare a calibration curve by plotting absorbance against concentration of
standards.

B.Sample measurement:

1.Take 50 ml of sample in Nessler’s tube and add 1ml of Sulphalinamide reagent

and 1 ml of NEDA reagent and mix thoroughly.
2.Read absorbance at 540nm.
 22
Calculation:

Conc. of N-NO2 in mg/l = R × mf × D (Dilution factor/no of times of dilution)

Where, R = absorbance reading of sample

mf = absorbance
Conc.
 23
BORON

Method: Colorimetric method

Principle: In the presence of Boron, a solution of carmine or carminic acid in concentrated

Sulphuric acid changes from a light red to a bluish red or bluish, depending on the
concentration of Boron present.

Reagents:
1.Carbine reagent: Dissolve 0.92 g reagent in 1000 ml of conc. H2SO4
2. Dilute HCl: 1:11
3. Conc. H2SO4 , analytical grade.
4. Stock Boron standard: Dissolve 571.6 mg of anhydrous Boric Acid in 1000 ml of
d/w to make 100 mg/l.
5. Working standard Boron Solution: Dilute 10 ml of the stock soln. to 100 ml of
d/w to make 10 ppm

Procedures:

A.Calibration curve preparation:

1.Take 5, 10, 15, 20, 25 ml of Boron solution (10 ppm) and dilute to 50 ml. with
d/w. to make standard solns. of 1, 2, 3, 4, and 5 ppm respectively.
2.Take 50ml d/w for blank reagent
3.To the above, add 0.5 ml HCl and then 10 ml of Conc. H2SO4 and let cool to
room temperature. Add 10 ml of Carbine indictor and mix well.
4.After 45 to 60 mins, read absorbance at wavelength of 580 nm.
5.Prepare a calibration curve by plotting absorbance against concentration of
standards.

B.Sample measurement:

1.Take 50 ml of sample and add 0.5 ml HCl and then 10 ml of Conc. H2SO4 and
let cool to room temperature. Add 10 ml of Carbine indictor and mix well.
2. After 45 to 60 mins, read absorbance at wavelength of 580 nm using blank
(d/w) as reference.
Calculation:

Conc. of Boron in mg/l = R × mf × D (Dilution factor/no of times of dilution)

Where, R = absorbance reading of sample

mf = absorbance
Conc.
 24
Total Suspended Solids (TSS)

Method: Gravimetric Method

Principle: The basic principle involved is gravimetric. A well mixed sample is filtered
through a wet glass fibre filter and the residue is dried to a constant weight at
103–105°C. The increase in weight of the filter paper represents the Total
Suspended Solids.

Sample Preservation : Preserve samples at 4°C. The determination of the TSS shall be
carried out as early as possible .Holding time, 2 to 9 days, if preserved at 4°C.

Apparatus:
1. Millipore Filtration assembly with suction flask and pump.
2. Whatman filter paper – GF/C grade ( No. approx. 40 / 41)
3. Drying oven , for operation at 103 – 105 °C
4. Desiccator provided with a desiccant , preferably silica or CaCl2 fused.
5. Analytical balance capable of weighing of 0.1 mg.
6. Forceps.
7. Petri dishes

Procedure:
1. Assemble the filtration assembly and place the dried and pre-weighed filter
paper on the holder.
2. Wet the filter paper with small volume of distilled water.
3. Take 100 ml. of homogenous sample.
4. Apply suction and transfer the sample to the filltration.
5. Wash with three successive 10 ml volume of distilled water.
6. Continue suction for about 3 mins after filtration is complete.
7. Remove the filter paper using forceps and transfer to the Petri dish.
8. Dry for at least 1 h at 103-105°C. Prolong drying, if necessary.
9. Cool in a desiccator and weigh.
10.Repeat the cycle of drying, cooling and weighing until a constant weight is
obtained.
Calculation:
Total Suspended solids, mg/L = (A-B) × 106
V
Where, A = weight of the filter paper & residue in g
B = weight of the filter paper in g
V= Volume of sample in ml.

Expression of result:Express the result as ‘Total Suspended Solids’ in mg/L. Round off to
the nearest whole number.
 25

Total Dissolved Solids (TDS)

Method: Gravimetric Method

Principle: The basic principle involved is gravimetric. A well mixed sample is filtered
through standard glass fibre filter and known quantity of the filtrate is evaporated
to dryness on steam bath then further in an oven at 180°C using porcelain dish .
The increase in weight represents the Total Dissolved solids.

Sample Preservation: Chemical preservation is not practical. The samples should be

analysed within 7 days of collection.

Apparatus: 1. Millipore Filtration assembly with suction flask and pump)

2 Whatman filter paper – GF/C grade ( No. approx. 40 / 41)
3. Drying oven , for operation at 180°C
4. Evaporating dishes of 100 ml capacity of porcelain or high –silica glass.
5. Desiccator provided with a desiccant , preferably silica or CaCl2 fused.
6. Analytical balance capable of weighing of 0.1 mg.

Procedure:
1. Dry the well cleaned dish in an hot air oven at 103 – 105 °C for 1 hour.
2. Cool, desiccate and take the initial weight of the dish.
3. Repeat the above step 2. until a constant weight is obtained.
4. Transfer a known volume(100 ml) of filtered homogenous sample into the
dish.
5. Evaporate to dryness on a steam bath.
6. Dry the evaporated sample in an hot air oven at 180°C for 1 hour.
7. Cool in a desiccator and weigh.
8. Repeat the steps 6 & 7 until a constant weight is obtained.

Calculation:
Total Dissolved solids, mg/L = (A-B) × 106
V
Where, A = weight of the dish residue in g
B = weight of the dish in g
V= Volume of sample in ml.

Expression of result:

Express the result as ‘Total Dissolved Solids’ in mg/L. Round off to the nearest whole
number.
 26

MIZORAM POLLUTION CONTROL BOARD

WATER ANALYSIS METHODS

Sl.No. Parameter Page No.

1. Dissolved Oxygen (D.O)……………………………1

2. Chloride……………………………………………..4

3. Total hardness as CaCO3…………………………...6

4. Calcium……………………………………………...7

5. Magnesium…………………………………………..8

6. Alkalinity……………………………………………9

7. Sulphate…………………………………………….11

8. Phosphate…………………………………………..13

9. Nitrogen-Ammonia………………………………...15

10. Nitrogen Nitrate (N-NO3)………………………….16

11. Nitrogen Nitrite (N-NO2-)………………………….18

12. Boron………………………………………………..20

13. Total Dissolved Solids……………………………...21

14. Total Suspended Solids…………………………….22

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