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. Evaporating Dishes.b. Drying oven.c. Analytical balance.d. Muffle furnace for the operation at 550 + 50O C.e. 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

1

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) × 1000Mg volatile solid/L =

Sample volume, mL

(B – C) × 1000Mg 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, andC = Weight of dish or filter, mg

2

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 non-

homogeneous 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.

3

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) × 1000Mg total solids/ L =

Sample volume, mL

Where:

A = Weight of drying residue + dish, mg, and

B = Weight of dish, mg

4

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 180oC 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

5

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.

6

CALCULATION:

(A – B) × 1000Mg total dissolved solids/L =

Sample volume, mL

Where:

A = weight of dried residue + dish, mg, and

B = weight of dish, mg

7

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 105oC. 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.

8

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 105oC 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 fiber-

filter.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.

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

9

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.

10

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 (NH2)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:

11

* Turbidity determination is important from 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 de-

accelerated, which causes the reduction in the rate of production of D.O. in water.

12

DETERMINATION OF SETTEABLE SOLIDSGRAVIMETRIC 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

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

13

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

14

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 hexa-

hydrate (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. Protect standards against evaporation and

contamination when not in use.

PROCEDURE:

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

15

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 exceeds 70 units, then

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

range.

CALCULATION:

Color unit = A x (50/B) Hazen units

where,

A = estimated color of a dilute sample and

B = ml. of sample taken for dilution.

16

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.

17

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 1µmho/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 µmhos/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.

18

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 ± 0.1oC. 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 25.0oC, 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 =

R m [ 1 + 0.0191 ( t – 25 ) ]

19

Where:

K = Conductivity, µ mhos/ cm,

C = Cell constant, cm-1,

Rm = Measured resistance of sample, ohms, and

t = 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.

20

DETERMINATION OF ACIDITY

The acidity of water is usually due to the presence of uncombined CO2 and the mineral acids and

salts of strong acid and weak bases. It can be distinguished as follows:

a) The equivalent point of titration of mineral acids occurs at a pH of 4.4.

b) The equivalence point of titration of uncombined CO2 to Na2CO3 is completed at a

pH of 8.3.

Accordingly, if different end point indicators are employed w.r.t. the end points

corresponding to these pH values, it shall be thus possible to determine these acidities.

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.

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.

21

CALCULATION:

V, NaOH (Methyl Orange) x 50 x 1000 x NMineral acidity mg/l = (as CaCO3) ml of sample (50)

V, NaOH (Phenolphthalein) x 50 x 1000 x NTotal acidity mg/l = (as CaCO3) ml of sample (50)

22

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

Industrial waste or complex system 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).

23

A x BNormality, N =

53 x C

A = Na2CO3 in g weighed into one litre i.e. 2.5 g

B = Volume (in ml) of Na2CO3 solution taken for titration

C = Volume (in ml) acid used

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.

24

CALCULATIONS:

V x N x 50 x 1000Total alkalinity mg/l = (as CaCO3) ml of sample Where,

V = ml of H2SO4.

N = 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 control, Industrial waste

treatment and biological treatment.

25

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.)

26

e. Stock ammonium solution:

dissolve 381.9 mg anhydrous NH4Cl, dried at 100oC, in water,

and dilute to1000mL; 1.00mL=100µg N =122µg NH3 .

f. Standard ammonium solution:

Dlute 5.00mL stock ammonium solution to 1000 mL with water; 1.00mL= 0.500 µg N

=0.607µmg 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 µg, treating standards exactly as the

sample. Beer’s Law governs.

5. CALCULATION

Calculate ammonia concentration as follows:

mg NH3-N/L (11.1 mL final volume) =A x B X D C x S E

27

Where

A= absorbance of sample,

B = NH3-N in standard, µg,

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.

28

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 is precipitated quantitatively before red silver chromate is

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 AgNO3 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 140oC, in distilled water and dilute to exactly 1000 ml (1

ml = 500 ug Cl-).

29

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 0.0141Normality 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 1 N

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.

30

Titration:

Titrate samples in the pH range 7 to 10. Adjust pH in this range with H2SO4 or NaOH solution.

Take 100 ml of sample or diluted sample. Add 1 ml K2CrO4 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 sample

B = ml titration for blank

N = Normality of AgNO3

31

DETERMINATION OF FLUORIDE(BY SPADNS METHOD 4500-F D)

PRINCIPLE:

The SPADNS colorimetric method is based on the reaction between fluoride and a zirconium-

dye lake. Fluoride reacts with the dye lake, dissociating a portion of it into a colorless complex

anion (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.

32

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-para- sulfophenylazo- 1,8-dihydroxy- 3,6-

naphthalene 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 water and add 350

ml Conc. HCl and dilute to 500 ml with distilled water.

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 F- by diluting appropriate

quantities of standard fluoride solution to 50 ml. with distilled water. Note the

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

33

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:

µg of F- read from curve BMg/l of the fluoride = X

ml of sample CWhere,

B = ml of final volume of diluted sample

C = ml of diluted sample taken for colour development

34

DETERMINATION OF TOTAL HARDNESSEDTA 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 ( NH4CL) 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

35

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 × 1000Hardness as mg/L CaCO3 =

ml of sample

DETERMINATION DISSOLVED OXYGEN

36

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.

37

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

When only part of the contents has been titrated:

38

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)

39

PRINCIPLE:

The digestion of the sample with H2SO4 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.

40

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.

41

CALCULATION:

Kjeldahl N- mg/l = a – b ×0.01 ×1000×14 ×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

42

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 NO2-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. Colorimetric equipment, spectrophotometer, for use at 410 nm providing a light path of

2.5 cm.

2. Water bath, reaction test tubes with racks

43

REAGENTS:

1. Stock nitrate solution:

Dissolve 721.8 mg anhydrous potassium nitrate, KNO3 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:

44

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 reaction conditions by running at least two

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.

45

CALCULATION:

µg NO3-Nmg/l Nitrate N =

ml sample

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

46

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 NO2-N). Dilute this solution 250

times (4 1000 ml) to prepare standard solution having 1 mg/l NO2-N.

47

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.

48

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. Spectrophotometer : For use at 690 nm.

2. Acid washed glassware (since detergents have phosphorous)

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 (NH4)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 (SnCl2. 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.

49

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. Activated carbon: Analytical grade activated carbon.

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.

50

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 (NH4)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 Reagent-

II, 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

51

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 = ------------------------------------- x 1000

ml sample

52

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. Ashless filter papers.

4. Crucible - silica or porcelain

5. Glass wares

REAGENTS:

1. Hydrochloric acid (1+1):

2 Barium chloride solution (10%):

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

3 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.

53

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 750oC for at least 30 minutes in weighed

crucible. Cool in dessicator and weigh.

CALCULATIONS:

1000 mol. wt of SO4

SO4 mg/lit = mg Residue x x ml of sample mol. wt of BaSO4

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

54

DETERMINATION OF BROMIDEPHENOL 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 + 6HNO3 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.

55

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 =

5.00µgBr-.

f. Standard bromide water solution

Diluted 10.00 ml stock bromide solution to 1000 ml with distilled water: 1.00 ml = 5.00 µg

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.

56

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

57

CALCULATION:

(ml × N) of titrant 1000 × 35.5Residual chlorine, mg/l =

ml of sample

58

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 CO2 free distilled water (boiled) to make 1

litre of solution. Dilute 50 ml of 1.0 N NaOH to 1 litre. Standardize it with H2SO4, 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 × 44Free CO2, mg/l = ml sample

59

DETERMINATION OF CYANIDECOLORIMETRIC 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 µg CN-/ ml solution. Dilute with the NaOH dilution solution. Dilute 10 ml

of the 10 µg CN -/ml solution to 100ml with the NaOH solution 1.0 ml = 1.0 µg CN -/ml

prepare fresh daily and keep in a glass-stoppered bottle.

60

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 µg 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

µg 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

61

readings. Using the calibration curve and the formula in below, determine CN- concentration

in original sample.

CALCULATION:

A × BCN- , mg/L =

C × D

Where:

A = µg 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.

62

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

titrimetric, colorimetric or poteniometric procedures.

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) Heating element, adjustable.

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 MgCl2. 6H2O in water and dilute to

one litre.

c) Sulfuric acid, H2SO4 , 1+1.

d) Lead carbonate, PbCO3, powdered.

e) Sulfamic acid. NH2SO3H.

63

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.

64

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

65

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.)

d) Pyridine barbituric acid reagent : Place 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 and mix thoroughly. Add 2.0 mL

66

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 barbituric acid 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 readings. Using the calibration curve and formula below determine CN

concentration in original sample.

5. CALCULATION:

CN, mg/L = (AxB)/ (CxD) Where

A = ug CN read from calibration curve (50 mL final volume).

B = ml of total absorbing solution from distillation

C = ml original sample used in the distillation.

D = ml absorbing solution used in colorimetric test.

67

DETERMINATION OF IODINELEUCO 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.

68

MINIMUM DETECTABLE CONCENTRATION

10 µg I asI2/L

REAGENTS:

a. Iodine–demand–free 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 µg 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

69

Measure 200 ml water and 3.2 ml conc. Sulphuric acid (H2SO4) 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 (HgCl2) 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 I2/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 µg 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 Beer’s 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.

70

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 Sample volume Required ml

6.0 – 12.0 25.0 12.0 – 30 10.0 30 – 60 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 Na2S2O3 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.

71

DETERMINATION OF SULFITEIODOMETRIC 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 SO32 -. 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 KIO3 (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

µg SO32 -.

72

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 ZnCl2 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 (< 50oC)

Immediately by adding 1 ml EDTA solution/100 ml sample. Cool hot sample to 50oC 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 MgSO3

2- / L = mL sample

Where:

A= ml titrant for sample,

B= ml titrant for blank,

M= molarity of KI-KIO3

73

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.

74

CALCULATION:

One milliliter 0.0250N iodine solution react with 0.4 mg S2- :]

[ (A × B) – (C × D) ] × 16000Mg S2- /L =

ml sample

Where:

A = ml Iodine solution,

B = normality of iodine solution,

C = ml Na2S2O3 solution, and

D = normality of Na2S2O3 solution.

75

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. Mercuric Sulfate [HgSO4] crystals: (Used to mask the chlorides)

6. Silver Sulfate, [Ag2SO4] Crystals: (Used as a catalyst)

76

PROCEDURE:

1. Quantity of the chemicals according to the sample size is indicated in the

following table.

Sample Size 0.25N

Standard

Dichromate

Conc. H2SO4

with Ag2SO4

HgSO4 Normality of

Fe (NH4)2-

(SO4)2

Dilution Qty.

after Reflux

ml ml nk g N ml

10 5 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.

77

CALCULATION:

(B-S) x 8000 x N

COD in mg/litre =

ml of sample taken

where,

B = ml of FAS used for Blank

S = ml of FAS used for Sample

N = 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.

78

DETERMINATION BIOLOGICAL OXYGEN DEMAND

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 KH2PO4, 21.75 g of Dipotassium

Hydrogen phosphate K2HPO4, 33.4 g of Disodium Hydrogen phosphate Na2HPO4 .7H2O and

1.7 g of ammonium chloride NH4Cl in distilled water and dilute to 1 L. The pH should be

7.2 Magnesium sulfate solution: Dissolve 22.5 g MgSO4.7H2O 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.

79

Alkali- iodide – azide solution.:

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 270C.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. H2SO4. Shake well. Take

80

out 100ml from this bottle and titrate against 0.01 N sodium thiosulfate solution using starch as

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) 5 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.

81

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. Hydrochloric acid [ 1:1 ]. 3. Sodium sulfate

2. Petroleum ether 60o C - 80o C 4. Sodium chloride

PROCEDURE:

a) Take known quantity of sample.

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) Wash the upper layer by small quantity of distilled water.

e) Again shake well for two minutes.

f) Allow the layers to separate.

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.

82

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 1000Oil and Grease mg/lit = ml of sample

= {(X - Y)/ V} x 1000

Where,

X = Weight of residue of extracted sample

Y = Weight of the residue of blank

83

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

84

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.

85

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. Hydrochloric acid, HCl, conc.

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.

86

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. Sodium sulfate, anhydrous Na2SO4, granular:

14. Potassium iodide, KI, crystals:

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).

87

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 H3PO4 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.

88

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

89

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 = (A/B) x 1000 Where,

A = micro-gm phenol in sample, from calibration curve.

B = ml original sample.

90

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 µS/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.

91

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 µS/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 µS/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.

92

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 – 4100C. 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) Sulphuric acid (Conc.), sp. Gr. 1.84

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) Sodium hydroxide solution (40 %):

93

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 blue in 50 ml of 95 % ethyl or isopropyl alcohol. Mix the two solutions.

f) Boric acid + mixed indicator solution:

Dissolve 20g boric acid (H3BO3) 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 H2SO4 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 (105oC) 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

94

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 H2SO4 (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.

95

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) Conical flask, 250ml

b) Hot plate

c) Nessler’s tube (50ml).

d) Spectrophotometer for use at 690 nm.

REAGENTS:

a) Ammonium molybdate solution:

Dissolve 25 g (NH4)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 Sncl2.2H2O 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 (HNO3), 1ml of Sulphuric acid H2SO4) and 4ml of 60%

Perchloric acid HClO3) in a conical flask.

96

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

Dissolve 0.439 g anhydrous Potassium hydrogen phosphate (KH2PO4) 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 (105oC) 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 (HNO3-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 Nessler’s 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.

97

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.

98

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.

99

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.

100

CALCULATION:

( ml ×N ) of AgNO3 ×35.5

% OF Chlorides =

ml of soil solution × 2

To convert the values in mg/100 g, multiply the value in % with 1000.

101

ESTIMATION OF CALCIUM CONTENT IN A SOIL SAMPLE

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 (NH4 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. Take 2 gm of finely sieved soil.

2. Add 100 ml Ammonium Acetate Extractant.

3. Keep it for one hour with intermittent mixing.

4. Decant and filter suspension through filter paper.

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.

102

CALCULATIONS:

0.4008 V 100

Calcium % = A x --------- x ----- x ------ Where,

1000 Vt S

A = Volume of EDTA required in ml

V = Total volume of extract prepared in ml

Vt = Volume of extract taken for titration in ml

S = Weight of soil taken for extraction in grams

103

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. Also determine the EDTA required during, Calcium determination.

CALCULATIONS:

0.4008 V 100

Magnesium % = (B - A) x ---------- x ----- x ------ Where,

1000 Vt S

A = Volume of EDTA used in Calcium titration, ml

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B = Volume of EDTA used in above titration, ml

V = Total volume of soil extract prepared in ml

Vt = Volume of extract taken for titration in ml

S = Weight of soil sample used for extraction, g

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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 (10→1000ml) 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. Nessler’s 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.

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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 H2SO4 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 Nessler’s 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.

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CALCULATION:

a. Colorimetric Method: %ammonia-N = NH3-N mg/l soil extract x V X 100 10000 X S 100-M

Where,

M = % Moisture content of soil

V = volume of total extract prepared (ml ) S =Weight of soil taken (g)

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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).

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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 × 61c. 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.

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