CHAPTER 2

MATERIALS & METHODOLOGY

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 MATERIALS AND METHODOLOGY

Physico chemical analysis is the prime consideration to assess the quality of water
for its best usage say for drinking, bathing, fishing, industrial processing and so on, while
for waste water either domestic or industrial to know the pollution strength and its effect
on the ecology.

River water analysis often necessitates examination of water samples from

different points and under varying conditions to find out the extent of pollution and
natural purification that takes place in the water

2.1 WATER SAMPLES AND THEIR ANALYSIS

Water samples from the surface of all the 3 stations were collected at an interval
of about 30 days in polythene cans at 10.00 AM from May, 2009 to April, 2011. Separate
samples were collected for the estimation of dissolved oxygen taking all the precautions
and fixed in the field by adding the reagents. All the samples were kept in an icebox and
transported to the laboratory. After returning to the laboratory they were analysed for
various physico-chemical parameters by following the standard methods.

2.2 WATER TEMPERATURE

Measurement of temperature is an important parameter required to get an idea of
self purification of rivers. Water temperature is also important parameter for aquatic life.
It is the important factor, for calculating the solubility of oxygen and carbon dioxide,
bicarbonate and carbonate equilibrium. The temperature of drinking water has an
influence on its taste.

2.2.1 APPARATUS
A thermometer having a quick response, with 0.1° C division, checked against a
precision thermometer.

16
2.2.2 PROCEDURE
Immerse the thermometer directly in the water body for a period of time sufficient
to permit constant reading. If it is not possible to take reading directly, then collect water
in a sampling bottle, nearly one litre, and measure the temperature by dipping the
thermometer in the sample. But while collecting the sample, it must not be exposed to
heat or direct solar radiation.
Record temperature in Celsius scale to the nearest 0.1° C

2.3 pH
For most practical purposes the pH of aqueous can be taken as negative logarithm
of hydrogen ion activity. pH values from 0 to 7 are diminishingly acidic, 7 to 14
increasingly alkaline and 7 is neutral.

The pH of natural water usually lies in the range of 4.4 to 8.5. Its value is
governed largely by the carbon dioxide/bicarbonate/carbonate equilibrium. It may be
affected by humic substances by changes in the carbonate equilibrium due to the
bioactivity of plants and in some cases by hydrolysable salts. The effect of pH on the
chemical and biological of plants and in some cases by hydrolysable salts. The effect of
pH on the chemical and biological properties of liquid makes its determination very
important. It is used in several calculations in analytical work and its adjustment is
necessary for some analytical procedures.

The determination of pH conventional chemical means is not practicable and the

equilibrium which is involved depends to some extent on temperature. The precise
accepted scale of pH must therefore be based on an agreed primary standard. The
colorimetric indicator methods can be used only if approximately pH values are required.

The pH determination is usually done by electrometric method which is the most

accurate method and free of interference.

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2.4 CARBONATES AND BICARBONATES
Organic matter present in water or wastewater can be determined as total organic
carbon with the help of special instrument. Tolerable range is from 1 to 150 mg/L. The
sample has to be homogeneous which can be injected by a micro syringe.

2.4.1 PRINCIPLE
Homogenised or diluted sample when injected into a heated packed tube in
presence of oxygen, the water vapourises and organic matter is oxidised to CO2. CO2 is
measured with a non-dispersive type IR analyser. Total carbon estimation followed by
inorganic carbon estimation gives organic carbon.

2.4.2 INTERFERENCES
Carbonates & Bicarbonates

2.4.3 APPARATUS
1. Total carbon analyser
2. Hypodermic syringe 0-500 pi capacity
3. Waring blender
4. Magnetic stirrer

2.4.4 REAGENTS
As listed in total organic carbon (Demand Analysis)

2.4.5 PROCEDURE
1. Follow manufacturer’s instructions.
2. Vary the sample size from 20 to 100 pi.
3. If the sample is turbidor colloidal or oily homogenise it in the blender
4. To free from inorganic carbon reduce its pH to pass CO2 free N2 gas.
5. Take sample from this by a capillary syringe and inject into the analyser.
6. Measure the peak height and repeat with three such estimations to obtain an
average.

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7. Take standard solutions from 10-100 jj.1 with a difference of 10. Total final volume
should be 500 ml.
8. Inject sample and record peak heights for this.
9. Run a blank
10. Plot carbon concentration versus corrected peak height in mm on a rectangular
coordinate paper.
11. Read the sample value from the curve.

2.4.6 CALCULATIONS
„ , , • Peak height for standard
Corrected peak in mm =-----------5------------------
Peak height for blank

2.5 CHLORIDE
Chloride anion is generally present in natural water. The presence of chloride in
natural waters can be attributed to dissolution of salt deposits, discharges of effluent from
chemical industries, oil well operations, sewage discharges, irrigation, drianage,
contamination from refuge leachates, and seawater intrusion in coastal areas. Each of
these sources may result in local contamination of both surface water and ground water.

Three methods are suggested for the estimation of chloride i) Involving titration
against standard mercuric nitrate solution, ii) An argentometric method, and iii) A
potentiometric method. The mercurimetric method is recommended when an accurate
determination of chloride is required, particularly at low concentrations. The
potentiometric method is suitable only when the sample is coloured or turbid;
argentometric method is the simplest one and can easily be carried out.

2.5.1 PRINCIPLE
Chloride is determined in a natural or slightly alkaline solution by titration with
standard silver nitrate, using potassium chromate as an indicator. Silver chloride is
quantitatively precipitated before red silver chromate is formed.

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2.5.2 INTERFERENCES
Bromide, iodide and cyanide are measured as equivalent of chloride ions. If the
sample contains sufficient thiosulphate, thiocyanate cyanide, sulphite and sulphide to
interfere seriously with the determination, they may be oxidized to non-interfering
substances as follows.

Measure a suitable quantity of sample into a conical flask and dilute to 150 ml
with water. Add 25 ml H2O2 (3%) and boil for 15 minutes, then add further 10 ml H2O2
and boil for 5 minutes. Repeat the same until the solution is thiocyanate free.

If the sample is too coloured or turbid to allow the end point to be readily
detected, this interference may be reduced by the following treatment with suspension of
aluminium hydroxide.

Add 3 ml Aluminium hydroxide suspension to the measured quantity of sample.

Stir thoroughly, set aside for a few minutes and filter. Wash the precipitate with distilled
water. Collect the washings with the filtrate and continue as described under procedure.

2.5.3 REAGENTS
1. Potassium Chromate indicator: Dissolve 50 g K2C1O4 in distilled water. Add
AgN03 till red precipitate is formed. Allow to stand for 12 hours. Filter and dilute
to 1000 ml.
2. Silver nitrate 0.0141 N: Dissolve 2.395 g AgN03 and dilute to 1000 ml.
Standardize against Nacl, 0.0141 N. 1 ml of 0.0141 N AgNOs = 0.5 mg, Cl
3. Sodium Chloride 0.014 N: Dissolve 824.1 mg Nacl (dired at 140 °C) and dilute to
1000 ml. 1 ml = 0.5 mg, cl.
4. Special reagent to remove colour and turbidity: Dissolve 125 g. ALK (S04)2.
12H20 or ALNH4(S04)2. 12H20 and dilute to 100 ml. Warm to 60 °C and adds 55
ml Cone. NH4OH slowly. Allow to stand for one hour. Solution should be free
from Cl.

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2.5.4 PROCEDURE
1. Take 100 ml sample and adjust the pH between 7.0 and 8.0.
2. Take 50 ml well mixed sample adjusted to pH 7.0 and add 1.0 ml K2Cr04
3. Titrate with standard AgN03 solution till AgCr04
4. Standardize AgN03 against std. Nacl
5. For better accuracy titrate distilled (50 ml) in the same way to establish reagent
blank.
6. Calculate as follows

(A -B)xNx35.45x1000
Chloride mg/1 -
ml sample

Where A= ml AgN03 required for sample

B = ml AgN03 required for blank
N = Normality of AgN03 used

2.6 DISSOLVED OXYGEN

The Winkler Method with Azide Modification

2.6.1 PRINCIPLE
Oxygen present in sample rapidly oxidizes the dispersed divalent maganous
hydroxide to its higher valency which precipitates as a brown hydrated oxide after
addition of NaOH and KI. Upon acidification, manganese reverts to divalent state and
liberates iodine from KI equivalent to the original dissolved content. The liberated iodine
against Na2S203 (N/80) using starch and an indicator.

2.6.2 INTERFERENCES
Ferrous ion, ferric ion, nitrite, microbial mass and high suspended solids constitute
the main sources of interferences. Modifications in the estimation procedure to reduce
these interferences are described in the procedure.

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2.6.3 APPARATUS
1. BOD bottles capacity 300 ml.
2. Sampling device for collection of samples.

2.6.4 REAGENTS
1. Manganese sulphate: Dissolve 480 g tetrahydrate manganous sulphate and dilute
to 1000 ml. Filter if necessary. This solution should not give colour with starch
when added to an acidified solution of K3.
2. Alkali iodide-azide reagent: Dissolve 500 g NaOH and 150 g KI or 135 g dilute to
1000 ml. Add 10 g NaN3 dissolved in 40 ml distilled water. This solution should not
give colour with starch solution when diluted and acidified.
3. Sulphuric Acid: H2SO4, concentration 1 ml equivalent to about 3 ml alkali-iodide-
azide reagent.
4. Starch indicator: Prepare paste or solution of 2.0 g L.R grade soluble starch
powder and 0.2 g salicylic acid as preservative in distilled water. Pour this solution
in 100 ml boiling water. Allow to boil for few minutes, cool and then use.
5. Stock sodium thiosulphate 0.1 N: Dissolve 24.82 g Na2S2C>3 5H2O in boiled
distilled water and dilute to 1000 ml. Preserve by adding 5 ml chloroform per litre.
6. Standard sodium thiosulphate 0.025 N: Dilute 250 ml stock Na2S2C>3 solution to
1000 ml with freshly boiled and "cooled distilled water. Preserve by adding 5 ml
chloroform per litre. (This solution will have to be standardized against standard of
dichromate solution for each set of titrations).

2.6.5 PROCEDURE
1. Collect sample in a BOD bottle using DO sampler.
2. Add 2 ml MnS04 followed by 2 ml of alkali-iodide-azide reagent. The tip of the
pipette should be below the liquid level while adding these reagents. Stopper
immediately.
3. Mix well by inverting the bottle 2-3 times and allow the precipitate to settle leaving
150 ml clear supernatant.

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4. At this stage, add 2 ml concentration H2SO4. Mix well till precipitate goes into
solution.
5. Take 203 ml in a conical flask and titrate against Na2S2C>3 using starch as an
indicator. When 2 ml MnS04 followed by 2 ml alkali-iodide-azide reagent is added
to the samples as in (2) above 4.0 ml of original sample is lost. This 203 ml taken
for titration will correspond to 200 ml of original sample.
200 x 300/(200-4) = 203 ml

2.6.6 CALCULATIONS
1 ml of 0.025 N Na2S203 = 0.2 mg of 02

T_<_. (0.2x1000) ml of thiosulphate

D.O. in mg/L = -----------------------------------------
.200

2.7 OXIDIZABLE ORGANIC MATTER

100 ml of water sample was used with adequate KMn04 and 1:4 H2SO4 and
incubated at 40 Centigrade for 4 hours. Afterwards all the samples were cooled to room
temperature, and then 2 ml of 5% potassium iodide solution was added and titrated
against N/40 Na2 S2O3 by using 1% starch solution as indicator.

2.8 FREE AMMONIA

2.8.1 PRINCIPLE
Ammonia produces a yellow coloured compound when reacted with alkaline
Nessler reagent, provided the sample is clarified properly. Pre-treatment with ZnS04 and
NaOH precipitates Ca, Fe, Mg and sulfide and removes turbidity and apparent colour.
Addition of EDTA (before Nessler reagent) or Rochelle salt solution prevents
precipitation of residual Ca and Mg in the presence of alkaline Nessler reagent.

2.8.2 INTERFERENCES
Colour, turbidity, Ca, Mg, salts and Fe in the sample constitute the prime sources of
interferences.

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2.8.3 APPARATUS
1. Spectrophotometer having a range of 300 to 700 nm.
2. Nessler tubes or 100 ml capacity volumetric flasks.

2.8.4 REAGENTS
1. Zinc sulphate: Dissolve 10 g ZnSC>4.7H20 in distilled water and dilute to 100 ml.
2. Sodium hydroxide: Dissolve 24 g NaOH and dilute to 100 ml (6N).
3. EDTA reagent: Dissolve 50 g EDTA in 60 ml water containing 10 g NaOH. Cool
and dilute to 100 ml.
4. Rochelle salt solution: Dissolve 50 g potassium sodium tartrate in 100 ml.
Remove ammonia by boiling of30 ml solution, cool and dilute to 100 ml.
5. Nessler reagent: Mix well 100 g HgU and 70 g KL Dissolve in small quantity of
water. Add this mixture to a cooled solution of 160 g NaOH in 500 ml: water.
Dilute to 1000 ml. Keep overnight. Store supernatant in coloured bottle.
6. Standard ammonium solution: Dissolve 3.819 g NH4CI dried at 100°C in
distilled water and dilute to 1000 ml. Dilute 10 ml of the solution to 1000 ml.
1 ml = 10 pg N or 12.2 pg NH3.

2.8.5 PROCEDURE
1. Take 100 ml of sample. Add 1 ml ZnS04 solution and 0.4 or 0.5 ml NaOH to
obtain a pH of 10.5. Allow to settle and filter the supernatant though 42 No.
Whatman filter paper.
2. Take a suitable aliquot of sample and dilute to 50 ml.
3. Add 3 drops of Rochelle salt solution or 1 drop of EDTA, mix well.
4. Add 3 ml Nessler reagent if EDTA is used or 1 ml if Rochelle salt solution is used.
Make up to 100 ml.
5. Mix well and read % transmission after 10 min at 410 nm, using a blank prepared in
the same way by taking distilled water instead of sample.
6. Prepare a calibration curve using suitable aliquots of standard solution in the range
of 5 to 120 pg/100 ml for reference following the same procedure as 1 to 5 but
using the standard solution in place of sample.

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2.9 ALBUMINOID AMMONIA
Distillation method as above but after expelling free ammonia. After expelling free
ammonia from the sample, alkaline potassium permanganate was added, distilled and the
liberated ammonia was collected in N/20 H2SO4 containing the mixed indicator. This was
titrated against N/20 NaOH.

2.10 NITRATES
Measurement of the ultraviolet absorption at 220 nm enables rapid determination of
nitrate. The nitrate calibration curve follows Beer’s law up to 11 mg/L N. Because
dissolved organic matter may also absorb at 220 nm and nitrate does not absorb at 275
nm a second measurement can be made at 275 nm to correct the nitrate value. The extent
of this empirical correction is related to the nature and concentration of the organic matter
and may vary from one water to another. Filtration of the sample is intended to remove
possible interference from suspended particles.

Acidification with 1 N hydrochloric acid is designed to prevent interference from

hydroxide or carbonate concentrations up to 1,000 mg/L as CaCC>3. Chloride has no
effect on the determination. Minimum detectable concentration is 40 pg/L N03-N.

2.10.1 INTERFERENCES
Dissolved organic matter, nitrite hexavalent chromium, and surfactants interfere.
The latter three substances may be compensated for by the preparation of individual
correction curves.

Organic matter can cause a positive but variable interference, the degree depending
on the nature and concentration of the organic material.

2.10.2 APPARATUS
1. Spectrophotometer, for use at 220 nm and 275 nm with matched silica cells of 1 cm
or longer light path.
2. Filter: One of the following is required.
i) Membrane filter: 0.45 pm membrane filter, and appropriate filter assembly.

25
 ii) Paper: Acid-washed, ashless hard-finish filter paper sufficiently retentive for
fine precipitates.
3. Nessler tubes, 50 ml, short form.

2.10.3 REAGENTS
1. Redistilled water: Use redistilled water for the preparation of all solutions and
dilutions.
2. Stock nitrate solution: Prepare as described in PDA method
1.00 ml = 100 pg N = 443 pg NO” 3
3. Standard.nitrate solution: Dilute 100.0 ml with stock nitrate solution tolOOO ml
with distilled water:
1.0 ml = 10.0 pg NO" 3 N = 44.3 pg NO" 3-
4. Hydrochloric acid solution: HC1, l.N.
5. Aluminum hydroxide suspension: Prepare as directed in PDA Method.

2.10.4 PROCEDURE
a. Colour removal: If the sample has a high colour or is known to contain organic
interference add 4 ml AUCOHh suspension/100 ml sample in an erlenmeyer flask.
Swirl to mix and let settle for 5 min. Filter through a 0.45 pm membrane filter
previously washed with about 200 ml distilled water.
b. Treatment of sample: To 50 ml clear sample, filtered if necessary, or to 50 ml
sample filtered after colour removal, add 1 ml IN, HC1 and mix thoroughly.
c. Preparation of standard curve: Prepare nitrate calibration standards in the range
j

0 to 350 pg N by diluting to 50 ml the following volumes of the standard nitrate

solution: 0,1.00, 2.00, 4.00, 7.00...35.0 ml. Treat the nitrate standards in the same
manner as the samples.
d. Spectrophotometric measurement: Read the absorbance or transmittance against
redistilled water set at zero absorbance or 100% transmittance. Use a wavelength
of 220 nm to obtain the nitrate reading and, if necessary, a wavelength of 275 nm to
obtain interference due to dissolved organic matter.

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2.10.5 CALCULATION
For correction for dissolved organic matter, subtract 2 times the reading at 275 nm
from the reading at 220 nm to obtain the absorbance due to nitrate. Convert this
absorbance value into equivalent nitrate by reading the nitrate value from a standard
calibration curve obtained at 220 nm.

Calculate as follows:
xt- xt ,T Net mg nitrate N
Nitrate N mg/L =-------- -------------
ml of sample

NO3 mg/L = Nitrate N mg/L x 4.43

2.11 NITRITES
2.11.1 PRINCIPLE
Nitrite (NO2 ) is determined through formation of a reddish purpleazodye produced
at pH 2.0 2.5 by compiling diazotized sulphanilic acid with N-(l-Naphthy)
ethylenediamine dihydrochloride (NED - dihydrochloride). The method is suitable for
determination of NO'2 down to 1 pi NO-2-N/L. The colour system obeys Beer’s law up
to 180 pg N/L with 1 cm light path at 543 nm.

Sample can be preserved by addition of HgCL for a short period, necessary. Acid
preservation is not used in any case.

2.11.2 APPARATUS
1. Colorimeter or spectrophotometer that can be operated at 543 nm.
2. Nessler tubes or 100 ml capacity volumetric flask.

2.11.3 REAGENTS
1. Sulphacilamide reagent: Dissolve 5 g sulphanilamide in a mixture of 50 ml
concentration HC1 and about 300 ml water. Dilute to 500 ml. with water. The
solution is stable for many months.

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2. NED-dibydrochloride solution: Dissolve 500 mg N- (1-naphthyl)-
ethylenediamine dihydrochloride in 500 ml water. Store in a dark bottle. Replace
monthly or immediately when it develops a strong brown colour.
3. Stock nitrite solution: Dissolved .2320 sodium nitrite and dilute to 1000 ml;
1 ml = 250 pg N. Standardize N02 as follows:

Take 50 ml 0.05 N KMn04 add 5 ml concentration H2SO4 and 50 ml stock nitrite

solution well below surface by pipette. Warm to 70 °C-80 °C; decolourize KMn04 using
0. 05.N sodium oxalate. Calculate NO2 by the equation:

A_ [(B.C) - (D.E)] x 7
F

Where A = mg N02-H./ml in stock solution B = Total ml of KMnC>4 used,

C = Normality of KMnQt, D = Total ml Standard oxalate added.
E = Normality of sodium oxalate and F = ml. of stock NaNC>2 used.
1725 mg of NaNC>2 or 350 mg
N consumes 1 ml of 0.05 KMn04. Preserve with 1 ml chloroform.
Standard nitrite solution: Dilute appropriate aliquot of stock to 1000 ml so as to get
lml = 0.5 pg N in the solution.

2.11.4 PROCEDURE
1. If sample contains suspended solids, filter through a 0.45 pm porediam membrane
filter.
2. To 50 ml clear sample neutralized to pH 7, or to a portion diluted to 50 ml, add 1 ml
sulphanilamide solution. Let reagent react of 2 to 8 min.
3. Add 1.0 ml NED dihydrochloride solution and mix immediately. Measure
absorbance after 10 min but before 2 hours ata 543 nm.
4. Prepare blank in the same way substituting water for the sample.
5. Prepare calibration curve by pipetting suitable volume of standard nitrite solution in
the range 0.25 pg NO-2-N/L using 1 cm light path for reference.

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6. Run parallel check frequently against nitrite standards preferable in the
concentration range of sample. Redetermine complete calibration curve after
preparing new reagents.

2.12 PHOSPHATES
2.12.1 PRINCIPLE
In acidic condition, orthophosphate reacts with ammonium molybdite to form
molybdophosphoric acid. It is further reduced to molybdenum blue by adding reducing
agent such as stannous chloride. The intensity of the blue coloured complex is measured
which is directly proportional to the concentration of phosphate present in the sample.

2.12.2 INTERFERENCES
Silica, arsenic in concentration of 100 mg/L and NO2 Fe. Alum; S2O3 in large
concentration interfere with the test. For NO~2 interference sulfamic acid is added to the
sample prior to addition of Ammonium molybdate.

2.12.3 APPARATUS
1. Colorimeter for use at 690 nm providing 1 cm light path.
2. Nessler tubes, cap. 100 ml.

2.12.4 REAGENTS
1. Stock phosphate solution: Dissolve 0.7111 g anhydrous KH2PO4 and dilute
to1000ml.
1 ml = 0.5 mg P04
2. Phosphate working solution: Dilute 100 ml stock solution to 1000 ml.
1 ml = 0.05 mg PO4
3. Ammonium molybdate solution: Dissolve 31.4 g in about 200 ml distilled water.
Add carefully 252 ml concentration H2SO4 to 400 ml distilled water. Cool and add
3.4 ml concentration HNO3. To this solution add molybdate solution and dilute to
100 ml.

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4. Strong acid reagent: Add 300 ml concentration H2SO4 to 600 ml distilled water.
Add 4 ml concentration HNO3 cool dilute to 1000 ml.
5. Sodium hydroxide: (3N) Dissolve 12.0 g NaOH and dilute to 100 ml.
6. Phenolphthalein Indicator: Prepare as described in alkalinity measurement.
7. 1-Amino 2-Naphthol 4-SuIphonic acid (ANSA): Weigh out seperately 0.75 g
ANSA 42 g Na2SC>3 and 70 g Na2S205. Grind ANSA with small portion of
Na2S205 in mortor. Prepare solution of remaining salts in small quantity of water.
Dissolve the above mixture in this solution. Dilute to 1000 ml filter through
whatman filter paper No.42.
OR
Stannous chloride: Dissolve 2.5 gm fresh SnCl2.2H20 in 100 ml glycerol. Heat on
water bath to ensure complete dissolution.

Preparation of calibration graph:

1. In to a series of 100 ml nessler tubes pipette appropriate amounts of phosphate

working solution to cover the range of 5-30 mg/L or 0.32 mg/L when ANSA/SnC^

is used as a reducing agent.

2. Add 2 ml ammonium molybdate and mix well.

3. Add 2 ml or 0.5 ml stannous chloride and dilute to 100 ml.

4. Prepare blank using distilled water in the same way.

5. Measure the intensity of blue coloured complex at 690 nm and 1 cm light path

between 10 and 12 minutes after the development of the colour.

6. Prepare a graph relating net absorbance to mb phosphate.

2.12.5 PROCEDURE
1. Orthophosphates:
Take suitable volume of the sample in a nessler tube and continue according to the
procedure described for the preparation of calibration curve. From graph read the
concentration of phosphate in the volume of sample taken. Calculate in mg/L.

30
<

2.13. SILICATES
Silicates cause a problem in use of water for boilers due to scale formation. They
also stimulate growth of organisms which, in turn, deteriorate water quality. The
existence Of silica is always associated with hard water.

Silica in the form of alumino silicates or activated silica is used in water softening
and coagulation processes.

2.13.1 PRINCIPLE
Ammonium molybdate at a pH 1.2 reacts with silica to produce molybdosilic acid.
The yellow colour developed corresponds to reactive silica present in the sample. This
molybdosilicic acid is reduced to heteropoly blue by amino naphthol sulphonic acid
which increases sensitivity of the method. \

2.13.2 INTERFERENCES
Phosphate, tannin, iron in large cone., colour, turbidity and sulphide are potential
sources of interference. Interference caused by PO4 and tannin can be eliminated by
addition of oxalic acid. The interference due to colour and turbidity can be compensated
by using an appropriate blank.

2.13.3 APPARATUS
1. Spectrophotometer for use at 410 & 690 nm providing 1 cm light path.
2. Nessler tubes, cap. 100 ml.

2.13.4 REAGENTS
1. Hydrochloric Acid : HC1: 1 + 1
2. Ammonium molybdate reagent: Dissolve 10 g amm. Molybdate in distilled
water, with stirring and warming and dilute to 100 ml. Adjust pH to 7-8 with
NH4OH or NaOH and store in polyethene bottle.
3. Oxalic acid solution: Dissolve 10 g H2C2O4. 2H2O and dilute to 100 ml. Store in
polyethene bottle.

31
4. Stock silica solution: Dissolve 4.73 g NaiSiOs. 9H2O in recently boiled cooled
distilled water and dilute to 900 ml. Estimate silica concentration. Gravimetrically
and dilute accordingly to. have 1000 mg/L Si02. Stored in a tightly stoppered
plastic bottle.
5. Reducing reagent: Dissolve 500 mg 1-amino, 2-naphthol, 4-sulphonic acid and lg
Na2 SO3 in 50 ml water. Add this to a solution of 30 g NaHSC>3 in 150 ml water.
Filter through whatman paper no. 42 and store in a plastic bottle.

2.13.5 PROCEDURE
1. Take suitable volume of filtered sample in Nessler tube. If sample is turbid or
colored prepare approximate blank as follows: To the identical aliquot add 1 ml 1 +
1 HC1 followed by 1.5 ml oxalic acid. Do not add ammonium molybdate or
reducing agent. Dilute to 100 ml. Use, for setting zero of the instrument.
2. Add 1 ml 1 + 1 HC1 followed by 2 ml ammonium molybdate. Allow to stand for 5-
10 min.
3. Add 1.5 ml oxalic acid to the samples and mix well.
4. Add 2 ml reducing reagent and dilute to 100 ml. Measure the colour at 690 nm
after 10 min. using 1 cm path.
5. In case of clear sample prepare blank substituting sample by distilled water and
processing through all above steps from 1 to 4.
6. Prepare a standard curve using standard silica solution covering 0.0 to 240 pg SiC>2
7. Calculate the concentration from standard graph and express as Si02 mg/L.
8. Eliminating step-4, direct measure of the yellow colour developed, can be made at
410 nm.

2.14 TOTAL HARDNESS

2.14.1 PRINCIPLE
In alkaline condition, EDTA reacts with Ca and Mg to form a soluble chelated
complex. Ca and Mg ions develop wine red colour with eriochrome black ‘T’ under
alkaline condition. When EDTA is added as a titrant, Ca and Mg divalent ions get
complexed resulting in a sharp change from wine red to blue which indicates end-point of

32
the titration. The pH for this titration has to be maintained at 10.0+0.1. At a higher pH
about 12.0 Mg++ ion precipitates and only Ca++ ion remains in solution. At this pH
Murexide indicator forms a change from pink to purple which indicates end point of the
reaction.

2.14.2 INTERFERENCES
Metal ions do interfere but can be overcome by addition of inhibitors.

2.14.3 REAGENTS
1. Buffer Solution: Dissolve 16.9 g NH4OH. Add 1.25 g magnesium salt of EDTA to
obtain sharp change in colour of indicator and dilute to 250 ml. If magnesium salt
of EDTA is unavailable, dissolve 1.179 gm. disodium salt of EDTA (AR grade) and
7S0 mg MgSC>4.7H20 or 644 mg MgCl2.6H20 in 50 ml; distilled water. Add to
above solution of NH4CI in NH4OH and dilute to 250 ml.
2. Inhibitor: Dissolve 4.5 g hydroxyl amine hydrochloride in 100 ml 95% ethyl
alcohol or isopropyl alcohol.
3. Erichrome balck T indicator: Mix 0.5 g dye with 100 g NaCl to prepare dry powder.
4. Murexide Indicator: Prepare a ground mixture of 200 mg of murexide with 100 g
of solid NaCl.
5. Sodium hydroxide 2N: Dissolve 80 g NaOH and dilute to 1000 ml.
6. Standard EDTA solution 0.01M: Dissolve 3.723 g EDTA sodium salt and dilute to
1000 ml. Standardize against standard Ca solution, 1 ml = 1 mg CaCC^.
7. Weigh accurately 1.0 g AR grade CaC03 and transfer to 250 ml conical flask. Place
a funnel in the neck of a flask and add 1+1 HCL till CaC03 dissolves completely.
Add 200 ml distilled water and boil for 20-30 min. to expel CO2. Cool and add few
drops of methyl red indicator. Add NH4OH 3N drop wise till intermediate orange
colour develops. Dilute to 1000 ml to obtain 1 ml = 1 mg CaCC>3.

33
2.14.4 PROCEDURE
Total Hardness
1. Take 25 or 50 ml well mixed sample in porcelain dish or conical flask.
2. Add 1-2 ml buffer solution followed by 1 ml inhibitor.
3. Add a pinch of Eriochrome black T and titrate with standard EDTA (0.01M) till
wine red colour changes to blue. Note down the volume of EDTA required. (A).
4. Run a reagent blank. Note the volume of EDTA (B).
5. Calculate volume of EDTA required by sample, C= (A-B) from volume of EDTA
required in steps 3 & 4.
6. Calculations as follows

t t i u a r- n CxDx 1000
Total Hardness as CaCo3 --------------------
ml sample

Where C = Volume of EDTA required by sample

D = mg CaCC>3 equivalent to 1.0 ml EDTA tltrant.

2.15 CALCIUM AND MAGNESIUM

Calcium can be estimated by atomic absorption spectrophotometer, flame
photometer, permanganate or EDTA titrimetric method and can also be calculated from
calcium hardness data. The multiplying factor is 0.4 when hardness is expressed as
CaC03. Magnesium can be estimated by atomic absorption spectrophotometric method
and gravimetric method after removing calcium salts. However it is permissible in
almost all cases to calculate the magnesium content from the difference between the total
hardness in the same units. The appropriate factor to be used to convert the difference to
magnesium is 0.244 if both total and Calcium hardness are expressed as CaC03.

Most common method applicable for routine analysis, because of its simplicity
and rapidity is EDTA titrimetric method, which is discussed earlier in hardness.

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2.16 SODIUM AND POTASSIUM
The estimation of sodium and potassium is based on the emission spectroscopy,
which deals with the excitation of electrons from ground state to higher energy state and
coming back to its original state with the emission of light.

2.16.1 PRINCIPLE
The sample solution is sucked by an atomsier under controlled conditions. The
radiation from the flame enters a dispearing device in order to isolate the desired region
of the spectrum. The intensity of isolated radiation can be measured by a phototube. After
carefully calibrating the photometer with solution of known composition and
concentration, it is possible to co-relate the intensity of a given spectral line of the
unkown with the amount of an element present that emits the particular radiation.

2.16.2 EQUIPMENT
A flame photometer, with flame accessories.

2.16.3 REAGENTS
1. Deionised distilled water
2. Stock sodium solution: Dissolve 2.542 g dried at 140°C sodium chloride in 1000
ml distilled water. 1 ml =1 mg Na
3. Working sodium solution: Dilute 10.0 ml of the stock solution to 1 litre. 1 ml =
0.10 mg Na
4. Stock Potassium solution: Dissolve 1.907 g dried 110°C KCL in one litre distilled
water 1 ml = 1 mg K
5. Working potassium solution: Dilute 10 ml of the stock solution to 1 litre. 1 ml =
0.01 mg K

2.16.4 PROCEDURE

1. Follow the instructions given by the manufacturer.

2. Start the electrical supply and switch on the air supply. Stabilize the air. The needle
should be steady at the mark.

35
3. Switch on the gas and maintain the gas fuel mixture so that the blue flame is seen
through the viewing window.
4. Aspirate distilled water and adjust the galvanometer reading to zero.
5. Calibrate the instrument by aspirating the standard and adjusting the galvanometric
reading to desire mark.
6. Aspirate distilled water to bring the reading zero mark.
7. Aspirate sample and note down the galvanometric reading.
8. Put off the fuel supply first followed by air and then main switch.

2.17 TOTAL SOLIDS

Residue left after the evaporation and subsequent drying in oven at specific
temperature 103-105°C of a known volume of sample are total solids. Total solids include
"Total suspended solids" (TSS) and "Total dissolved solids" (TDS). Whereas loss in
weight on ignition of the same sample at 500°C, 50°C, in which organic matter is
converted to CO2 and H2O while at controlled temperature to prevent decomposition and
volatilisation of inorganic matter as much as consistent with complete oxidation of
organic matter, are volatile solids.

2.17.1 APPARATUS AND EQUIPMENT

a. Electrically heated temperature controlled oven
b. Monopan balance
c. Evaporating dish (200 mL)
d. Pipettes
e. Measuring cylinder (100 mL)

2.17.2 CALIBRATION AND STANDARDIZATION

The oven thermometer and balance need to be properly calibrated regularly.

2.17.3 PROCEDURE
a. Take a known volume of a well-mixed sample in a tarred dish ignited to constant
weight (Wl)

36
 b. Evaporate the sample to dryness at 103-105°C for 24 hrs.
c. Cool in desiccator, weigh and record the reading (W2)
/

d. Ignite the dish for 15-20 minutes in a muffle furnace maintained at 550 + 50°C.
e. Cool the dish partially in air until most of heat has been dissipated, and then
transfer to a desiccator for final cooling in a dry atmosphere and record final
weight (W3).
f. The concentration is to be calculated in percent by weight.

2.17.4 CALCULATION
The total and the volatile solids are expressed as:

(W2-W1) xlOOO
Total solids, mg/L
ml sample

Where, Wl, W2 are recorded in mg.

2.17.5 TOTAL DISSOLVED SOLIDS

The filterable residue is the material that passes through a standard glass filter
disk and remains after evaporation and drying at 180°C.

2.17.6 APPARATUS AND EQUIPMENT

Evaporatory dish (porcelain) - 100/200 ml
Drying oven - equipped with thermostatic control capable
of maintaining the temperature within 2°C range.
Desiccator - provided with desiccants
Analytical balance - 200 g capacity capable of weighing to 0.1 mg
Filter holder - Gooch crucible adapter or membrane filter funnel

Gooch crucible with glass fibre filter disk or membrane filters

Suction flask - 500 ml capacity

37
2.17.7 PROCEDURE
Filter the well-mixed sample under vacuum through membrane filter or Gooch
Crucible. Transfer 100 ml or more, depending upon the concentration of dissolved solids,
in a weighed evaporating dish.

Evaporate to dryness on steam bath. Dry the evaporated sample for at least 1 hour
in an oven at 180 + 2°C. Cool in a desiccator and weigh. Repeat the drying until constant
weight is obtained or weight loss is less than 0.5 mg

2.17.8 CALCULATION

mg/1 total filterable residue at 180° C = B)xl000

C
Where: A = weight of dried residue + dish
B = weight of dish
C = ml of filtrate used

2.17.9 SUSPENDED SOLIDS

Suspended solids were calculated by deducting the value of dissolved solids from
the total solids.

2.18 IRON (TOTAL)

2.18.1 PRINCIPLE
The ferric form of iron is reduced to ferrous form by boiling with hydrochloric
acid and hydroxylamine hydrochloride. Later phenanthroline is added at pH between 3.2
and 3.3 to form soluble chelated complex of orange red colour with iron. Three molecules
of 1, 10 phenanthroline are required to form a complex iron with each Fe++. The colour
obeys Beer’s law and the intensity of colour is independent of pH from 3 to 9. Total
dissolved and suspended iron can be measured with unfiltered and filtered samples (prior
to acidification or preservation). For study of ferrous ferric equilibrium (and also to get
an idea of redox potential) it is sometimes desirable to determine the ferrous content of
the water sample. For such determination add 10 ml of 1% 1, 10-phenonthroline or

38
bathophenonthroline and 1ml glacial acetic acid to the sample bottle before sample
collection for iron. It will become complex soluble ferrous iron before it is oxidized to
ferric state. The addition of nitrilotriacetic acid to the system further helps to stablise the
ferrous ferric system.

2.18.2 INTERFERENCES
Strong oxidizing agents such as CN~, NO" 2 , polyphosphates, Cr, Zn in
concentration. 10 times the Fe concentration, Co and Cu if 5 mg/L, Ni if 2 mg/L, colour
and organic matter constitute sources of interference in the development of colour.

Boiling with HC1 and addition of hydrochloride remove interferences due to CN“,
NO2, PO4, and other oxidizing reagents. The metal ions get complexed with
phenanthroline.

2.18.3 APPARATUS
1. Colorimeter with an operating range of400-700 nm.
2. Nessler tubes

2.18.4 REAGENTS
1. Hydrochloric Acid: HC1 concentration containing less than 0.00005% iron.
2. Hydroxylamine HC1 solution: Dissolve 10 g NH2OH. HC1 and dilute to 100 ml.
3. Ammonium acetate buffer: Dissolve 250 g NH4C2H3O2 in 150 ml distilled water.
Add 700 ml concentration glacial acetic acid. Final volume will be slightly more
than 1000 ml.
4. Phenanthroline solution: Dissolve 100 mg 1,10 - phenanthroline monohydrate in
100 ml distilled water warm slightly or add 2 drops concentration HC1 if necessary.
1 ml of this solution can chelate 100 mg iron.
5. Stock iron solution: Add 20 ml concentration H2SO4 to 50 ml distilled water and
dissolved 404 g Fe (NFLi)2 (SC>4)2 6H2O . Add drop-wise 0.1N KMnQt till faint
pink colour persists. Dilute to 1000 ml. 1 ml = 200 pg Fe.

39
6. Standard iron solution: Dilute 50 ml stock Fe solution to 1000 ml. Prepare this
solution freshly. 1 ml = 10 pg Fe.

2.18.5 PROCEDURE
1. Take suitable aliquot about 50 ml (having 2 mg/L Fe) of well mixed sample in 125
ml conical flask.
2. Add 2 ml concentration, HC1 followed by 1 ml hydroxylamine HC1 solution.
3. Add 2-3 glass beads and boil for 20-25 minutes to ensure dissolution of Fe.
4. Cool to room temperature and transfer to nessler tubes.
5. Add 10 ml Acetate buffer and 2 ml 1, 10- phenanthroline solution.
6. Dilute to 100 ml and mix well.
7. Prepare blank by substituting the sample by distilled water.
8. For soluble iron determination, take known volume of filtered sample, acidify by
adding 2 ml concentration HC1 per 100 ml of sample add 1 ml hydroxylamine HC1
solution and treat from step 5 onwards for colour development.
9. Prepare calibration curve taking standard iron solution in the same way in the range
1000-4000 pg/L with 1 cm light path.
10. Measure the developed colour after 10 min. at 510 nm.
11. Calculate the concentration of total or soluble Fe present in the sample from
calibration curve and express as mg/L.

40