SOP's

DETERMINATION OF SLUDGE VOLUME INDEX, MIXED LIQUOR SUSPENDEDSOLIDS (MLSS) AND MIXED LIQUOR VOLATILE SUSPENDED SOLIDS (MLVSS)INTRODUCTION:

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

PROCEDURE:

1.Draw the sample from aeration tank.

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

3.Set the laboratory timer for 30 minutes.

4.Immediately after 30 minutes read the settled volume.

5.Record volume of settled sludge for determining SVI.

6.Pour off the supernatant.

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

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

104oC.

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

10.Weigh the filter paper together with sludge.

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

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

CALCULATION:

volume occupied by sludge after 30 min settling

SVI=

Volume of sample taken in Imhoff Cone/cylinder

( Y - X )

MLSS=

Volume of sample taken (1 litre)

Where,

Y=Weight of filter paper with sludge (MLSS)

X=Weight of filter paper alone

Y X=Weight of sludge alone

(Y - X) - Z}

MLVSS=

Volume of sample taken (1 litre)

Where,

Y=Weight of filter paper with sludge (MLSS)

X=Weight of filter paper alone

Z=Weight of Ash or Residue left in crucible

DETERMINATION OF FIXED AND VOLATILE SOLIDSPRINCIPLE:

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 + 50oCHave 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, mLWhere:A = Weight of residue + Dish before ignition, mg,

B = Weight of residue +Dish or filter after ignition, mg, and

C = Weight of dish or filter, mgDETERMINATION OF TOTAL SOLIDSPRINCIPLE:

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 dishesb. Muffle furnace for operation at 550 + 50oC.c. Steam bath.

d. Desiccator.

e. Drying oven

f. Analytical balance.

PROCEDURE:

a. Preparation of evaporating dish:

If the volatile solids are to be measured ignite clean evaporating dish at 550 + 50oC for 1 h in a muffle Furnace. If only total solids are to be measured, heat clean dish to 103 to 105oC for 1 h. Store dish in dessicator until needed. Weigh immediately before use.

b. Sample analysis:Choose sample volume that will yield a residue between 2.5 mg and 200 mg. Transfer a measured volume of well mixed sample to preweighed dish and evaporate to dryness on a steam bath or in a drying oven. If necessary, add successive sample portions to the same dish after evaporation. When evaporating in a drying oven, lower temperature to approximately 2oC below boiling to prevent splattering. Dry evaporate sample for at least 1 hr in an oven at 103 to 105oC, cool dish in dessicator to balance temperature, and weigh. Repeat cycle of drying, cool, desiccating, and weighing until weight loss is less than 4% of previous weight or 0.5 mg, whichever is less. CACULATION:

(A B) 1000

Mg total solids/ L =

Sample volume, mL Where: A = Weight of drying residue + dish, mg, and B = Weight of dish, mgDETERMINATION 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 dishesb. 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 ovenPROCEDURE:a. Preparation of glass filter disk:Insert disk with wrinkled side up into filtration apparatus. Apply vacuum and wash disk with three successive 20- ml volumes of distilled water. Continue suction to remove all traces of water. Discarded washing.b. Preparation of evaporating dish:

If the volatile solids are to be measured ignite clean evaporating dish at 550 + 50oC for 1 hr in a Muffle Furnace. If only total solids are to be measured, heat clean dish to 180+2oC for1 hr. Store dish in dessicator until needed. Weigh it immediately before use.

c. Selection of filter and sample sizes: Choose sample volume to yield between 2.5 and 200mg dried residue, If more than 10 min are required to complete filtration, increase filter size or decrease sample volume but not less than 2.5 mg residue.

d. Sample analysis: Filter the measured volume of the well mixed sample through glass fiber filter paper, wash with three successive 10ml volume of distilled water, allowing complete drainage between washing, and continue suction for about 3 min after filtration is complete. Transfer filtrate to a weighed evaporating dish and evaporate to dryness on a steam bath. If filtrate volume exceeds dish capacity add successive portion to the same dish after evaporation. Dry for at least 1h in an oven at 180+ 2oC, cool in a dessicator to balance temperature, and weigh. Repeat drying cycle of drying, cooling, desiccating, and weighing until a constant weight is obtained or until weight loss is less than 4% of previous weight or 0.5 mg, whichever is less.CALCULATION:

(A B) 1000

Mg total dissolved solids/L =

Sample volume, mL Where: A = weight of dried residue + dish, mg, and B = weight of dish, mgDETERMINATION OF TOTAL SUSPENDED SOLIDSPRINCIPLE:

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. 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 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.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:

*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.

DETERMINATION OF SETTEABLE SOLIDSGRAVIMETRIC METHODINTRODUCTION:

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 sampleb. 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 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/LDETERMINATION 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 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. 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.

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

APPARATUS:a. Self-contained conductivity instrument.b. Thermometer.c. Conductivity cell.

REAGENT:a. Conductivity water:

Pass the distilled water through a mixed-bed deionizer and discard first liter. Conductivity should be less than 1mho/cm.b. Standard potassium chloride solution:KCL, 0.0100 M: Dissolved 745.6 mg anhydrous KCL in conductivity water and dilution to 1000ml at 25oC has conductivity of 1413 mhos/cm. It is satisfactory for most sample when the cell has a constant between 1 and 2. For other cell constant, use the stronger and weaker solution store in a glass-stoppered borosilicate bottle.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 ) ] Where:

K = Conductivity, 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.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.

CALCULATION:

V, NaOH (Methyl Orange) x 50 x 1000 x N

Mineral acidity mg/l=

(as CaCO3)

ml of sample (50)

V, NaOH (Phenolphthalein) x 50 x 1000 x N

Total acidity mg/l=

(as CaCO3)

ml of sample (50)

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).

A x B

Normality, 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.

CALCULATIONS:

V x N x 50 x 1000

Total 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.

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.)

e. Stock ammonium solution:dissolve 381.9 mg anhydrous NH4Cl, dried at 100oC, in water, and dilute to1000mL; 1.00mL=100g N =122g NH3 .

f. Standard ammonium solution: Dlute 5.00mL stock ammonium solution to 1000 mL with water; 1.00mL= 0.500 g N =0.607mg NH34) PROCEDUREProcedure Treatment of sample: To a 10.0mL sample in a 50-mL beaker, add 1 drop (0.05 mL) MnSO4 solution. Place on a magnetic stirrer and add 0.5mL hypochlorous acid reagent. Immediately add, a drop at a time, 0.6 mL phenate reagent. Add reagent without delay using a bulb pipet or a buret for convenient delivery. Mark pipet for hypochlorous acid at the 0.5mL level and deliver the phenate reagent from a pipet or buret that has been calibrated by counting the number of drops to 0.6 mL. Stir vigorously during addition of reagents. Because color intensity is affected by age of reagents, carry a blank and a standard through the procedure with each batch of samples.Measure absorbance using reagent blank to zero the spectrophotometer. Color formation is complete in 10 min and is stable for at least 24 h. although the blue color has a maximum absorbance at 630 nm, satisfactory measurements can be made in the 600- to 660-nm region.Preparation of standards: Prepare a calibration curve in the NH3-N range of 0.1 to 5 g, treating standards exactly as the sample. Beers Law governs.

5. CALCULATION Calculate ammonia concentration as follows: mg NH3-N/L (11.1 mL final volume) =A x B X D

C x S EWhere A= absorbance of sample,

B = NH3-N in standard, g,

C= absorbance of standard,

S= volume of sample used, mL,

D= volume of total distillate collected, mL, including acid absorbent, neutralizing agent, and ammonia-free water added, and

E= volume of distillate used for color development, mL.The ratio D/E applies only to distilled samples.

DETERMINATION OF CHLORIDESARGENTOMETRIC METHOD -4500 Cl- B

( Titrimetric )

PRINCIPLE:

In neutral or slightly alkaline solution, potassium chromate indicates the end point of AgNO3 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-).

4.Standard AgNO3 (0.0141 N) solution, titrant:

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

10 x 0.0141

Normality of AgNO3=

ml of AgNO35.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 Hydroxide1 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.

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 103Cl- mg/litre=

ml sample

Where,

A=ml titration for sample

B=ml titration for blank

N=Normality of AgNO3 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 2oC. 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.

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.

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

3.Set absorbance to zero on the spectrophotometer at 570 nm using reference solution as blank.

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

COLOUR DEVELOPMENT:

Use a 50 ml sample or a portion diluted to 50 ml with distilled water. Adjust sample temperature to that prevalent at the time of making the Standard curve. Add 10 ml of mixed Acid Zirconyl and SPADNS reagent. Mix well. Set the absorbance to zero with the reference solution and read absorbance with the sample at 570 nm.

CALCULATIONS:

g of F- read from curve

B

Mg/l of the fluoride=

X

ml of sample

C

Where,B=ml of final volume of diluted sample

C=ml of diluted sample taken for colour development

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

D. Sodium sulphide solutionDissolved 5.0 g of Na2S.9H2O or 3.7 g in 100 ml of distilled water. Tightly close the bottle to prevent oxidation.

PROCEDURE:1. Take 50ml of sample in a conical flask. If sample is having higher calcium, take a smaller volume and dilute to 50 ml.

2. Add 1 ml of buffer solution.

3. If the sample is having higher amount of heavy metals add 1 ml of Na2S solution.

4. Add 100 200 mg of Eriochrome Black T indicator; the solution wine red.

5. Titrate the contents against EDTA solution. At the end point colour change from wine red to blue.CALCULATION:

ml EDTA used 1000

Hardness as mg/L CaCO3 =

ml of sampleDETERMINATION DISSOLVED OXYGENPRINCIPLE: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 solution1.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 solutionDissolved 100 g of KOH and 50 g of KI in 200ml of boiled distilled water and filter. d. Starch solutionDissolved 1 g of starch in 100 ml of warm ( 80o C 90o C ) distilled water and filtere. Sulphuric Acid (H2SO4) conc.

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 Na2S2O3DO, mg/l = ml of sample taken When only part of the contents has been titrated:

Burette reading 8000 N of Na2S2O3DO, mg/l = V1 - V V2 ( )

V1Where, 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)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.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.

CALCULATION:Kjeldahl N- mg/l = a b 0.01 100014 D / ml sample distilled Where,

a = ml of HCL used with sample.b= ml of HCL used with blank.D = dilution factor (2.5). the original volume ( 40 ml) of sample has been made to

100 ml after digestion.ORGANIC NITROGEN

Organic nitrogen can be determined by subtracting the concentration of ammonia from the Kjeldahl nitrogen values.

Organic N = Kjeldahl-N NH3NDETERMINATION 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 NO3-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

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:

1Preparation 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: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.

CALCULATION:

g NO3-N

mg/l Nitrate N =

ml sample

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

DETERMINATION OF NITRITEPRINCIPLE: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.

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.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.

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.

EXTRACTION:

REAGENTS FOR EXTRACTION:

1Benzene-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 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 = -------------------------------------

x1000

ml sample

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):

2Barium chloride solution (10%):Dissolve 100 gm of BaCl2.2H2O in 1 litre distilled water.

3Silver 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.

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 SO4SO4 mg/lit=mg Residue x

x

ml of sample

mol. wt of BaSO4OR

mg BaSO4 x 411.5

SO4 mg/l =

ml of sample

DETERMINATION OF BROMIDE

PHENOL RED COLORIMETRIC METHOD

PRINCIPLE:

When sample contain bromide (Br-) is treated with diluted solution of chloroamine T in the presence phenol red occur readily. If the reaction is buffered to pH 4.5 to4.7, the colour of brominated compound will range from riddish to violet, depending on the bromide concentration. Thus a sharp differentiation can be made among veriou concentration of bromide. The concentration of chloroamine T and timing of the reaction before dechlorination are critical.

APPRATUS:SpectrophotometerAcid 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.d. Sodium thiosulfate 2 MDissolved 49.6 g Na2S2O3.5H2O OR Na2S2O3 and dilute to 100 ml with distilled water.

e. Stock bromide solution

Dissolved 744.6 mg anhydrous KBr in distilled water and make up 1000 ml: 1.00ml = 5.00gBr-.

f. Standard bromide water solutionDiluted 10.00 ml stock bromide solution to 1000 ml with distilled water: 1.00 ml = 5.00 g Br-.

PROCEDURE:a. Preparation of bromide water standards:

Prepare at least six standard, 0 , 0.20, 0.40, 0.60, 0.80, and 1.00 mg Br- / L by diluting 0.0, 2.00, 4.00, 6.00, 8.00, and 10.00 ml standard bromide solution to 50.00 ml with distilled waterb. 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.DETERMINATION RESIDUAL CHLORINEPRINCIPLE: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 solutionDissolved 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

CALCULATION: (ml N) of titrant 1000 35.5Residual chlorine, mg/l = ml of sampleDETERMINATION OF CARBON DIOXIDEPRINCIPLE: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 indicatorDissolve 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 sampleDETERMINATION OF CYANIDECOLORIMETRIC METHODPRINCIPLE: CN - in the alkaline distillate from preliminary treatment is converted to CNCl by reaction with chloramines Tat pH < 8 without hydrolyzing to CNO - .1 (Caution CNCl is a toxic gas; avoid inhalation.) After the reaction is complete, CNCl forms a red- blue dye on addition of a pyridine barbituric acid reagent . If the dye is kept in an aqueous solution, the absorbance is read at 578 nm. To obtain colour of comparable intensity, have the same salt content in sample and standards.

INTERFERENCE:All known interferences are eliminated or reduce to a minimum by distillation.REAGENTS:a. Chloramine-T solution

Dissolved 1.0 g white, water-soluble powder in 100ml water. Prepare weekly and store in refrigerator.b. Stock cyanide solution:

Dissolved approximately 1.6 g NaOH and 2.51 g KCN in 1 litre distilled water (CAUTION - KCN is highly toxic; avoid contact or inhalation) Standardize against standard silver nitrate (AgNO3) using KCN solution. Check titer weekly because the solution gradually loses strength;1 ml = 1 mg CN-.c. Standard cyanid solution

Base on the concentration determine for the KCN stock solution calculate volume requried to prepar 1 L of 10 g CN-/ ml solution. Dilute with the NaOH dilution solution. Dilute 10 ml of the 10 g CN -/ml solution to 100ml with the NaOH solution 1.0 ml = 1.0 g CN-/ml prepare fresh daily and keep in a glass-stoppered bottle.d. Pyridine-barbituric acid reagent:

Place 15 g barbituric acid in a 250-ml volumetric flask and add just enough water to wash sides of flask and wet barbituric acid acid. Add 75 ml pyridine and mix. Add15 ml conc hydrochloric acid (HCl), mix, and cool to room temperature. Dilute to mark with water and mix. This reagent is stable for up one month; discard if a precipitate develops.e. Sodium dihydrogen phosphate, 1 M

Dissolved 138 g NaH2PO4. H2O in 1 L distilled water. Refrigerate.f. Sodium hydroxide dilution solution

Dissolved 1.6 g NaOH in 1 L distilled water.

PROCEDURE:a. Preparation of calibration curve

Prepare a blank of NaOH dilution solution. From the standard KCN solution prepare a series of standards containing from 0.2 to 6 g CN- in 20ml solution using the NaOH dilution solution for all dilution. Plot absorbance of standards against CN- concentration.Recheck the calibration curve periodically and each time a new reagent prepare.

On the basis of the first calibration curve, prepare additional standards containing less than 6 g CN- to determine the limits measurable with the Photometer being used.b. Colour development

Adjust photometer to zero absorbance each time using a blank consisting of the NaOH dilution solution and all reagent. Take a portion of absorption liquid obtained in method c, such that the CN- concentration fall in the measurable range, and dilute to 20ml with NaOH dilution solution. Place in a 50 ml volumetric flask. Add 4 ml Phosphate buffer solution and and mix thoroughly. Add 2.0 ml Chloramine T solution and swirl to mix. Immediately and 5ml pyridine-barbituric acid solution and swirl gently. Dilute to mark with water; mix well by inversion.

Measure absorbance with the photometer at 578 nm after 8 min but within 15 min form the time of adding the pyridine-barbituricacid reagent. Even with the specified time of 8 to 15 min there is a slight change in absorbance. To minimize this, standardize time for all readings. Using the calibration curve and the formula in below, determine CN- concentration in original sample.CALCULATION:

A B

CN- , mg/L = C D Where:

A = g CN- read form calibration curve (50 ml final volume)

B = Total volume of absorbing solution from the distillation, ml.

C = Volume of original sample used in sample distillation, ml. and D = Volume of absorbing solution used in colorimetric test, ml.

PRECISION:The analysis of mixed cyanide solution containing sodium zinc, copper, and silver cyanides in tap water gave a precision within the designated range as follows:ST = 0.115 X + 0.031

Where:

ST = overall precision and

X = CN- concentration, mg/L.

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.

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. 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 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 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.

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 insensitiveto 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.

MINIMUM DETECTABLE CONCENTRATION10 g I asI2/L

REAGENTS:a. Iodinedemandfree water

Prepare a 1-m ion exchange column of 2.5 to 5 cm diam, containing strongly acid cation strong basic anion exchange resins. If a commercial analytical- grade mixed bed resin is used, verify that compound that react with iodine are removed. Pass distilled water at aslow rate through the risin bed and collect in clean container that will protect the treated water form unduse exposure to the atmosphere. Prepare all stock Iodine and solution with iodine demand free water. Prepare all stock Iodine and solution with iodine demand free water.b. Stock Iodine solution

Prepare the saturated iodine solution by dissolving 20 g elemental iodine in 300 ml. water Let stand several hours. Decent iodine solution and dilute 170 ml to 2000 ml. Prepare a working solution of g I as I2 / ml by appropriate dilution of the standardized stock solution.

c. Citric buffer solution, pH 3.8 1. Citric acid

Dissolved 192.2 g C6H8O7, or 210.2 g C6H8O7. H2O and dilute to 1 L with water.

2. Ammonium hydroxide, 2 NAdd 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

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

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 5.0f. 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.DETERMINATION OF SULFITEIODOMETRIC METHODPRINCIPLE: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 Cu2+, 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-/ lREAGENTS:a. Sulfuric acid: H2SO4, 1+1b. 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 g SO32 -.

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.