determination of arsenicfinal report of the research projectcistup.iisc.ac.in/presentations/research...

Determination of arsenicFinal Report of

The Research Project

DEVELOPMENT OF DEDICATED SPECTROPHOTOMETRIC REAGENT KITS AND COOKBOOK FOR POLLUTION MONITORING OF BANGALORE LAKES AND WATERBODIES

Submitted to

The Chairman, CISTUP

Indian Institute of Science

Dr. J. R. Mudakavi

Department of Chemical Engineering,

Indian Institute of Science,

Bangalore 560 012

Contents

1. Introduction

2. Determination of Fluoride

3. Determination of Phenols

4. Determination of Arsenic

5. Determination of Free Chlorine

6. Determination of Boron

7. Determination of Cloride

8. Determination of Magnesium

9. Determination of Mercury

10. Determination of Nonionic surfactant

11. Determination of Iron

12. Determination of Phosphate

13. Determination of Nitrite

14. Determination of Manganese

15. Determination of Cadmium

16. Determination of Copper

17. Determination of Lead

18. Determination of Total hardness

19. Determination of Zinc

20. Determination of Nitrate

21. Determination of Chromium

22. Determination of Aluminum

23. Determination of Cyanide

24. Determination of Sulphate

25. Determination of Ammonium

26. Important improvements made

27. References

3

DEVELOPMENT OF DEDICATED SPECTROPHOTOMETRIC REAGENT KITS AND COOKBOOK FOR POLLUTION MONITORING OF BANGALORE LAKES AND WATERBODIES

Final Report of the Project during 1.1.2010 to 31.12.2011

1. Title of the Project

Development of Dedicated Spectrophotometric

Reagent Kits and Cookbook for Pollution

Monitoring of Bangalore Lakes and Water

bodies

2. Scheme Code No.

CiSTUP/RP/09-016/159

3. Principal Investigator-Name & Department

Dr. J. R. Mudakavi

Department of Chemical Engineering,

Indian Institute of Science,

Bangalore

Co-Investigator (If any)-Name & Department

Nil

4. Date of Commencement

01-01-2010

Project Duration

Two Years

Ending Date of the Project

31-12-2011

5. Discussion / Summary of work carried out

Highly sophisticated analytical techniques based on atomic absorption spectrophotometry, Inductively Coupled

Plasma Atomic Emission Spectrometry, flow injection analysis etc., have been developed for the determination of

water quality parameters. Such techniques are expensive and skilled personnel are required to operate, analyse

and interpret the results. Quite often these techniques are not within the reach of common man, NGOs and citizen

watchdog committees.

This project aims to develop simple analytical methods for the determination of anions, cations and organic

compounds commonly found in Bangalore lakes and water bodies. All in all, there are 32 parameters to be checked

for water quality which include anions, cations, surfactants, BOD, COD, coliform bacteria etc (Table 1). Therefore,

there is a long felt need for the development of simple, ready to use analytical kits for the determination of water

quality parameters. Over the past 3-4 years some multinational companies (MNCs) such as E.Merck, Hach etc.,

have forayed in these areas. However their analytical reagents and kits are coded to safeguard their business

interests. Therefore, the identities of the reagents are always in doubt, especially in the presence of interfering

concomitants. This also raises the question about the specificity of the readymade kits and their suitability of

application in complex matrices. These doubts can also spill over to the quantitative aspects.

In this project we have addressed such issues and made efforts to improve the existing spectrophotometric methods with respect to the following:

4

1. To record molecular absorption spectra of the complexes.

2. To minimize the use of all reagents and redesign the analytical procedures and limit

the dilution to a maximum of 10 ml.

3. To Present typical calibration plots.

4. To make the chosen analytical method to be compatible with green chemistry (shift to

easily biodegradable chemicals)

In this context we have reevaluated some standard APHA (American Public Health Association) methods and

modified some other spectrophotometric methods for the determination of several parameters. The parameters

and analytical reagents are mentioned below (Table 2). Twenty three parameters have been standardized till date.

Each method is standardized with respect to the choice of reagent, optimum pH, reaction conditions, colour

stability, spectral characteristics, Beer’s Lamberts Law range and typical interferences.

Methods have been suggested to overcome the interference of critical concomitants. Typical cookbook absorbance

for the method has been determined by taking average of three sets of determinations in which all the standards

and reagents are prepared afresh. All these have been used to prepare cookbook and reagent kits. Cookbooks for

the various parameters are presented in the following pages.

5

Table 1. Drinking water specifications

Sl. No Parameter Permissible limit (Max) as per IS: 10500/1991

Permissible limit in the absence of alternative source

1 Colour, Hazen Units 5 25

2 Odour Unobjetionable -

3 Taste Agreable -

4 Turbidity, NTU 5 10

5 pH 6.5-8.0 No relaxation

6 Total hardness as CaCO3, mg/l 300 600

7 Iron (Fe) ,mg/l 0.3 1

8 Chloride (Cl) mg/l 250 1000

9 Residual free chlorine (Cl2), mg/l 0.2 -

10 Total Dissolved solids, mg/l 500 2000

11 Calcium (Ca), mg/l 75 200

12 Copper (Cu), mg/l 0.05 1.5

13 Manganese (Mn), mg/l 0.1 0.3

14 Sulphate (SO4), mg/l 200 400

15 Nitrate (NO3), mg/l 45 No Relaxation

16 Fluoride (F), mg/l 1.0 1.5

17 Phenolic compounds as C6H5OH, mg/l 0.001 0.002

18 Mercury (Hg), mg/l 0.001 No Relaxation

19 Cadmium (Cd), mg/l 0.01 No Relaxation

20 Arsenic (As), mg/l 0.05 No Relaxation

21 Cyanide (Cn), mg/l 0.05 No Relaxation

22 Lead (Pb), mg/l 0.05 No Relaxation

23 Zinc (Zn), mg/l 5 15

24 Chromium (Cr+6), mg/l 0.05 No Relaxation

25 Alkalinity, mg/l 200 600

26 Aluminium (Al), mg/l 0.03 0.2

27 Boron (B), mg/l 1 5

28 Magnesium (Mg) mg/l 10 100

6

Table 2. Parameters and selected analytical reagents 1. Aluminum Eriochrome cyanine R 2. Chromium Diphenyl carbazone 3. Copper Bathocuproine disulphonic acid disodium salt 4. Fluoride SPADNS - zirconyl oxychloride 5. Iron 1,10 phenanthroline; Also Syzygium jabolana 6. Lead Pyridylazo resorcinol (PAR) 7. Magnesium Titian yellow 8. Nitrite Sulphanilic acid, N-1-naphthylethylene diamine Dihydrochloride

9. Nitrate Chromotropic acid

10. Chloride Mercury thiocyanate, Ferric sulphate

11. phosphate Stannous chloride reduced phosphomolybdate

12. Phenols 4-amino antipyrene, ferricyanide

13. Free Chlorine N, N-diethyl-p-phenylenediamine (DPD)

14. Cyanide Pyridine, Barbituric acid, chloramines T

15. Sulphate Barium chloride (Turbidimetric)

16. Manganese Pyridyl azo naphthol (PAN)

17. Arsenic Rhodamine B, molybdate

18. Cadmium Cadion

19. Zinc Zincon

20. Total hardness Calmagon

21. Nonionic surfactant Potassium iodide, iodine

22. Boron Carmine

23. Mercury Rhodamine 6G, iodide

7

1. DETERMINATION OF FLUORIDE

Fluoride in drinking water has beneficial effects on teeth at low concentrations, but excessive exposure can give rise to a number of adverse effects ranging from dental fluorosis to crippling skeletal fluorosis. The latter is a significant cause of morbidity in several parts of the world. Low Concentrations cause dental caries, whereas high fluoride concentrations results in fluorosis. The Bureau of Indian Standards (BIS) prescribes a limit between 1.0 and 1.5 ppm.

In the SPADNS ( 2, 4 sulphophenylazo) chromotrophic acid tri sodium salt) method, fluoride is reacted with the dark red Zirconyl-SPADNS dye lake, dissociating a portion of it into a colourless complex anion (ZrF6

2-) and the dye. As the amount of fluoride increases, the colour produced becomes progressively lighter. The change in the absorbance at 570 nm is related to the concentration of fluoride.

2, 4 sulphophenylazo) chromotrophic acid tri sodium salt (SPADNS)

Reagents

1. Stock Fluoride Solution (100 ppm): Dissolve 22.1 mg of anhydrous sodium fluoride in deionised

water and make up to 100 ml.

2. Standard Fluoride Solution: (5 ppm) Dilute 5 ml of the stock fluoride solution to 100 ml with deionised

water.

3. SPADNS Solution : Dissolve 95.8 mg of SPADNS in deionised water and dilute to 50 ml with deionised

water.

4. Zirconyl-Acid Reagent : Dissolve 13.3 mg of zirconylchloride octahydrate (ZrOCl2.8H2O) in about 2.5 ml of

deionised water and add 35 ml of concentrated HCl and dilute up to 50 ml using deionised water.

5. Acid-Zirconyl-SPADNS Reagent: Mix equal volumes of SPADNS solution and Acid-Zirconyl reagent to

obtain 50 ml of the Acid-Zirconyl-SPADNS Reagent.

Procedure

Transfer 0, 1, 2, 3, 4 ml of 5 ppm standard fluoride solutions into six different 10 ml volumetric flasks and add 2 ml

of Acid-Zirconyl-SPADNS reagent to each. Dilute to 10 ml with deionised water. After 10-15 minutes, measure the

absorbance of each solution in a spectrophotometer with 1 cm path length at a wavelength of 570 nm against the

blank. Subtract the absorbance values from that of the blank. Prepare a calibration curve of the difference

absorbance versus concentration of the fluoride and determine the concentration of the sample by referring the to

the calibration curve.

Recommended sample volume: 2 ml

Cookbook value : 10 µg of fluoride in 10 ml (1.0 ppm) gives an absorbance of 0.198 ± 0.01

8

Calibration curve

9

Efffect of Interfering Species

Interfering Species Concentration (µg/mL)

Absorbance Deviation from F- only

sample

Remarks

Nil (Fluoride .only) 1.50 0.209 Nil - Iodide(I-) 100.00 0.223 +0.014 NI Cobalt(Co) 100.00 0.226 +0.017 NI Manganese (Mn) 100.00 0.228 +0.019 NI Magnesium (Mg2+) 100.00 0.227 +0.018 NI Nitrate(NO3

-) 100.00 0.221 +0.012 NI Chloride(Cl-)

100.00

0.214

+0.005

NI SLS 100.00 0.225 +0.016 NI

Iron(III)(Fe3+) 100.00 0.224 +0.015 NI Copper(II)(Cu2+) 100.00 0.233 +0.024 NI Cadmium(Cd2+) 100.00 0.222 +0.013 NI Iron(II)(Fe2+) 100.00 0.216 +0.007 NI Vanadium(V) 100.00 0.212 +0.003 NI Antimony(Sb) 100.00 0.232 +0.023 NI Lead(Pb) 100.00 0.201 -0.008 NI Sulphate(SO4

2-) 100.00 0.196 -0.013 NI Citric Acid 100.00 0.194 -0.015 NI Nickel(Ni) 100.00 0.184 -0.025 NI DTPA 100.00 0.219 +0.010 NI Arsenic(As) 100.00 0.190 -0.019 NI Mercury(Hg) 100.00 0.185 -0.024 NI Zinc(Zn2+) 100.00 0.185 -0.024 NI

Interfering Species Concentration

(µg/mL) Absorbance Deviation

from F- only sample

Remarks

Tartaric Acid

100.00 0.265 +0.056 I 50.00 0.203 -0.006 NI

Phosphate (PO4

3-) 100.00 0.257 +0.048 I 50.00 0.223 +0.014 NI

EDTA

100.00 0.290 +0.081 I 50.00 0.224 +0.015 NI

Aluminium

(Al3+)

100.00 0.107 -0.102 Dark colour 50.00 0.124 -0.085 Dark colour 20.00 0.160 -0.049 I 10.00 0.216 +0.007 NI

Chromium(VI)

100.00 0.357 +0.148 Orange Red 50.00 0.263 +0.054 Light Red 20.00 0.196 -0.013 NI

Molybdenum (Mo)

100.00 0.163 -0.046 I 50.00 0.172 -0.037 I

10.00 0.200 -0.009 NI

Boron(B) 100.00 0.161 -0.048 I 50.00 0.171 -0.038 I 10.00 0.198 -0.011 NI

Bromide(Br-) 100.00 -0.057 +1.250 Colour change Silver(Ag+) 100.00 0.739 +0.530 Precipitate

(I-Interfering; NI- Not Interfering)

10

2. DETERMINATION OF PHENOLS

Phenols in water can form highly toxic chlorophenols on chlorination. The Bureau of Indian Standards (BIS)

prescribes a limit 0.001 ppm, relaxable to 0.002 ppm if no suitable alternative source is available.

phenols except p- substituted phenols react with 4-aminoantipyrene at pH 7.9 ± 0.1 in the presence of

ferricyanide to form antipyrene dyes. The absorbance at 500 nm is related to the concentration of phenol.

4-aminoantipyrene

Reagents

1. Stock Phenol Solution (1000 ppm): Dissolve 100 mg phenol in freshly boiled and cooled deionised


2. Standard Phenol Solution (10 ppm): Dilute 1 ml of the stock phenol solution to 100 ml with deionised

water.

3. 4-amino antipyrene Solution (2% : Dissolve 2.0 g of 4-amino antipyrene in deionised water and dilute to

100 ml with deionised water.(Stable for 1 day)

4. Ammonium hydroxide solution: Dilute 3.5 ml of concentrated ammoinium hydroxide

and dilute up to 100 ml using deionised water.

5. Citrate Buffer Solution (pH 6.8): Dissolve 1.92 g of citric acid in deionised water and make up to 100

ml. Dissolve 2.94 g of trisodium citrate in deionised water and make up to 100 ml. Add trisodium citrate

solution to citric acid solution slowly under a pH meter till the pH is 6.80

6. Potassium Ferricyanide Solution (80%: Dissolve 80 g of Potassium Ferricyanide and dilute up to 100 ml

using deionised water. Store in a brown bottle. (Stable for 1 week)

Procedure

Transfer 1, 2, 3, 4 and 5 ml of 10 ppm standard phenol solutions into five different 10 ml volumetric flasks and add

20 µl of ammonium hydroxide solution followed by 1 ml citrate buffer and 20 µl potassium ferricyanide to each.

Dilute to 10 ml with deionised water. After -15 minutes, measure the absorbance of each solution in a

spectrophotometer with 5 cm path length at 500 nm against the blank

Prepare a calibration curve of the absorbance versus concentration of phenol and determine the concentration of

the sample by referring the absorbance of the sample to the calibration curve.

Recommended sample volume : 5 ml. Cookbook value: 30 µg of phenol in 10 ml (3.0 ppm) in a 5 cm cell gives an absorbance of 0.20 ± 0.01.

11

Spectrum of phenol antipyrene reaction product

Calibration curve

12



Absorbance (1 cm path

length)

Deviation from phenol only sample

Remarks

Nil (phenol .only) 3.00 0.373 Nil - Iodide(I-) 100.00 0.355 -0.018 NI Bromide (Br-) 100.00 0.356 -0.017 NI Cobalt(Co) 100.00 0.382 +0.009 NI Manganese (Mn) 100.00 0.379 +0.006 NI Magnesium (Mg2+) 100.00 0.227 -0.146 I Nitrate(NO3

-) 100.00 0.371 -0.002 NI Chloride(Cl-)

100.00

0.349

-0.024

NI SLS 100.00 0.360 -0.013 NI

Iron(III)(Fe3+) 100.00 0.396 -.023 I Copper(II)(Cu2+) 100.00 0.395 +0.214 I Cadmium(Cd2+) 100.00 0.587 -0.157 I Iron(II)(Fe2+) 100.00 0.216 -0.012 NI Vanadium(V) 100.00

10.00 0.253 0.373

+0.003 0.000

NI NI

Antimony(Sb) 100.00 0.232 -0.141 I Lead(Pb) 100.00 0.380 0.007 NI Sulphate(SO4

2-) 100.00 0.369 -0.004 NI Citric Acid 100.00 0.194 -0.005 NI Nickel(Ni) 100.00 0.369 -0.004 NI DTPA 100.00 0.219 +0.010 NI EDTA 100.00 0.331 -0.042 I Arsenic(As) 100.00

10.00 0.149 0.389

-0.229 0.016

NI NI

Mercury(Hg) 100.00 10.00

0.274 0.394

-0.099 0.021

NI

Zinc(Zn2+) 100.00 0.185 -0.188 I Triton X-100 100.00 0.344 -0.029 NI

Calcium (Ca2+) 100.00 0.366 -0.007 NI

Chromium (Cr6+) 100.00 0.389 0.016 NI

Silver (Ag+) 100.00 0.408 0.035 I Molybdenum(Mo6+

) 100.00 10.00

0.302 0.391

-0.071 0.018

I

Tartaric Acid 100.00 0.386 +0.013 NI Citric acid 100.00 0.360 -0.013 NI Phosphate (PO4

3-) 100.00 0.397 +0.024 NI Aluminium (Al3+) 100.00 0.107 -0.266 I 20.00 0.386 +0.013 NI

NI = Not interfering I = interfering

13

3. DETERMINATION OF ARSENIC

Arsenic occurs naturally. It is the 20th most abundant element in earth’s crust. It is highly toxic and

carcinogenic. It occurs as arsenite (As3+) and arsenate (As5+). The Bureau of Indian Standards (BIS)

prescribes a limit of 0.05 ppm. The following method applies to arsenate but arsenite can be included by

oxidising arsenite to arsenate.

In the rhodamine B method, arsenate combines with molybdate and rhodamine B to form arsenomolybdate

rhodamine B ion pair at pH 4.0 resulting in a colour change from rose red to pink. The absorbance of the solution

measured at 595 nm is proportional to the concentration of arsenic.

Reagents

1. Stock arsenic Solution (100 ppm): Dissolve 41.7 mg of disodium hydrogen arsenate in deionised water and make up to 100 ml.

2. Standard arsenic Solution (1 ppm): Dilute 1 ml of the stock arsenic solution to 100 ml with deionised water.

3. Ammonium molybdate Solution (0.2%): Dissolve 0.2 g of ammonium molybdate tetrahydrate in deionised water and dilute to 100 ml with deionised water.

4. Rhodamine B solution (0.02%): Dissolve 20.0 mg of rhodamine B in deionised water and dilute to 100 ml with deionised water.

5. Sodium acetate Acetic Acid-Buffer pH 4.0 (0.1M) : Dissolve 1.5 ml glacial acetic acid (99.8%) and 0.3742ganhydrous sodium acetate in deionised water and make up to 250 ml. Adjust pH under a calibrated pH meter to 4.0 (by adding 0.1 N NaOH or 0.1N HCl as necessary).

6. Polyvinyl alcohol (0.1%): Dissolve 0.1g of polyvinyl alcohol in boiling deionised water, cool and dilute to 100 ml with deionised water.

Procedure

Transfer 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 ml of the standard arsenic solution into six different 10 ml volumetric flasks

and add1.0 ml ammonium molybdate solution and 2 ml of acetate buffer solution to each. Dilute to 6 ml with

deionised water and mix by swirling. Then add 1 ml rhodamine B solution followed by 1 ml polyvinyl alcohol

solution. Make up to mark and mix. Measure the absorbance of the solution against a blank at 595 nm. Prepare a

calibration curve of absorbance versus concentration of arsenic and determine the concentration of the sample by

referring the absorbance of the sample to the calibration curve.

Recommended sample volume 5 ml.

14

Cookbook value 0.5 µg of arsenic in 10 ml (0.05 ppm) gives an absorbance of 0.16 ± 0.01

Calibration curve

Wavelength (nm)

Abso

rban

ce

Spectrum of arsenomolybdate rhodamine B complex

15


Interfering Species Remarks

Lithium (Li+) NI

Magnesium(Mg2+) NI

Calcium (Ca2+) NI

Coppric (Cu2+) NI

Cobaltous (Co2+) NI

Manganous (Mn2+) NI

Nickelous (Ni2+) NI

Zinc (Zn2+) NI

Mercuric (Hg2+) NI

Chromic (Cr3+) NI

Aluminium (Al3+) NI

Ferric (Fe3+) NI

Arsenous (As3+) NI

Chloride (Cl-) NI

Bromide (Br-) NI

Iodide (I-) NI

Chlorate (ClO3-) NI

Sulphate (SO42-) NI

Nitrate (NO3-) NI

Nitrite (NO2-) NI

Silicate ( SiO3- (250) E

Phosphate (PO43-) (0.1) E

Fluoride (F-) (500) D

Oxalate (500) D

Citrate (500) D

Lead (Pb2+) (100) P

Antimony (Sb3+) P

Cerium (Ce4+) Bleached

Surfactant (cationic, nonionic or anionic) I

NI = Not interfering; I = Interfering; E = Enhaced absorbance above the limit in µg shown in parenthesis; D = decreased absorbance above the limit in µg shown in parenthesis.

16

4. DETERMINATION OF RESIDUAL FREE CHLORINE

Chlorine is added to drinking water to kill harmful organisms. Excess chlorine is then removed by aeration. Chlorine

is corrosive and poisonous. The Bureau of Indian Standards (BIS) prescribes a limit 0.1 ppm.

Chlorine reacts with N,N-diethyl-p-phenylenediamine (DPD) to produce a red colour. Bromine, iodine, NCl3 and

chlorine dioxide also give the reaction. Oxidized manganese also gives the reaction. Since chlorine solutions of

exactly known strength are difficult to make potassium permanganate is used for calibration. The absorbance at

515 nm is linearly related to the concentration of chlorine.

Reagents

1. Phosphate Buffer Solution (pH): Dissolve 6.0 g of anhydrous disodium hydrogen orthophosphate and 11.5

g of anhydrous potassium dihydrogen phosphate in in deionised water and make up to 100 ml. (Add 2

drops of toluene or 20 mg of HgCl2 to prevent mold growth)

2. Standard Potassium Permanganate Solution (equivalent of10 ppm chlorine): Dissolve 0.0891 g potassium

permanganate in deionised water and make up to100 ml.

3. DPD Solution: Dissolve 0.11 g of DPD sulphate in deionised water containing 0.25 ml concentrated

sulphuric acid and 200 mg EDTA disodium salt. Make up to 100 ml. Store in dark.

Procedure

Transfer 0.1, 0.5, 1, 2, 3 and 4 ml of 10 ppm standard potassium permanganate solution into six different 10 ml

volumetric flasks and add 0.5 ml of phosphate buffer solution followed by 0.5 ml DPD solution to each. Dilute to 10

ml with deionised water. Measure immediately the absorbance of each solution in a spectrophotometer at a

wavelength of 515 nm against the blank.

Prepare a calibration curve of the absorbance versus concentration of the chlorine and determine the concentration

of the sample by referring the absorbance of the sample to the calibration curve. (If the sample contains more than

4 ppm residual chlorine it should be diluted)

Recommended sample volume: 2 ml.

17

Cookbook value: 5 µg of chlorine in 10 ml (0.5 ppm) gives an absorbance of 0.18 ± 0.01

Calibration curve

18

5. DETERMINATION OF BORON

Boron (B) occurs in streams to the extent of 10 ppm and in groundwater to the extent of 0.01 to 10 ppm. Drinking

water rarely contains more than 1 ppm and generally contains less than 0.1 ppm. Ingestion of large amount of

boron can affect central nervous system. The Bureau of Indian Standards (BIS) prescribes a limit 1 ppm

relaxable if no suitable alternative source is available to 5 ppm.

In the carmine method, boron gives a bluish red colour with carmine in sulphuric acid. Ions commonly found in

water and wastewaters do not interfere. The method is applicable in the range 1 to 10 ppm B. The absorbance of

the solution measured at 585 nm after 45 to 60 minutes is proportional to the concentration of the surfactant.

Solutions are not stored in borosilicate glass to avoid contamination from glass.

Reagents

1. Stock Boron Solution (100ppm): Dissolve 0.0572 g of boric acid (H3BO3) in 50 ml of hot deionised water ,

cool and make up to 100 ml in a polyethylene volumetric flask.

2. Carmine reagent): Dissolve 46 mg of carmine in 100ml concentrated sulphuric acid in a polyethylene

volumetric flask.

3. Concentrated Hydrochloric acid

19

Procedure

Transfer 20, 40, 60, 80, 100 µl of the standard boron solution into six different polyethylene beakers. Add the

required amount of water to make the volume 1 ml. Add 50 µl Conc HCl. Then add 5.0 ml concentrated H2SO4 and

mix. Allow the solutions to cool. Then add 5.0 ml of the carmine reagent. Mix and transfer to dry 10 ml

polyethylene volumetric flasks. After 45 to 60 minutes measure the absorbance at 585 nm. Prepare a calibration

curve of absorbance versus concentration of boron and determine the concentration of the sample by referring the

absorbance of the sample to the calibration curve.


Cookbook value: 6µg of boron 1 ml sample gives an absorbance of 0.31 ± 0.01

Calibration curve

20

6. DETERMINATION OF CHLORIDE

Chloride in drinking water may impart a salty taste to drinking water if the water also contains much sodium. High

chloride content may harm metallic pipes and structures as well as growing plants. The Bureau of Indian

Standards (BIS) prescribes a limit of 250ppm relaxable if no suitable alternative source is available

to1000ppm.

In mercuric thiocyanate method chloride reacts with mercury thiocyante to liberate thiocyante ion. The

latter forms a red complex with the added ferric iron. The intensity of the red colour, measured at 460 nm is

proportional to the quantity of chloride in the sample of water.

Reagents

1. Stock Chloride Solution (1000 ppm): Dry about 1 g of sodium chloride at 105° C. Dissolve 0.1648 g of

anhydrous sodium chloride in deionised water and make up to 100 ml.

2. Standard chloride Solution (10 ppm): Dilute 10 ml of the stock chloride solution to 100 ml with deionised

water.

3. Mercuric thiocyante solution: Dissolve 0.417 g of mercuric thiocyante in isopropyl alcohol and make up to

100 ml with isopropyl alcohol.

4. Ferric ammonium sulphate solution (0.25M) : To 11.555 g of ferric ammonium sulphate add 6 ml of

concentrated nitric acid. Dissolve in 50 ml deionised water and make up to 100 ml.

21

Procedure

Transfer 1, 2, 3, 4, 5 ml of 10 ppm standard chloride solutions into five different 10 ml volumetric flasks and add 1

ml of Ferric ammonium sulphate solution and 1 ml of mercuric thiocyante solution to each. Dilute to 10 ml with

deionised water. After 10 minutes, measure the absorbance of each solution in a spectrophotometer at a

wavelength of 460 nmand 1 cm path length against the blank.

Prepare a calibration curve of the absorbance versus concentration of the chloride and determine the concentration

of the sample by referring the absorbance of the sample to the calibration curve.

Recommended volume : 0. 2ml

Cookbook value: 50 µg of chloride in 10 ml (1.0 ppm) in a 1 cm cell gives an absorbance of 0.19 ± 0.01

Spectrum of the colour produced by chloride

Calibration curve

22

Efffect of Interfering Species such as bromide, iodide, cyanide and substances that reduce ferric iron interfere.

7. DETERMINATION OF MAGNESIUM

Magnesium contributes to hardness of water. It is an essential element for plants and animals. Some magnesium

salts ar toxic by ingestion. At concentrations greater than 125 ppm it has cathartic and diuretic effects. In the Titan

Yellow method, magnesium reacts with titan yellow and sodium hydroxide (pH > 12) to give a red lake. Calcium

interferes by enhancing the colour intensity. This is over come by adding calcium to samples and standards. The

absorbance at 554 nm is related to the concentration of magnesium.

The Bureau of Indian Standards (BIS) prescribes a limit of 10 ppm relaxable up to 100 ppm where no

suitable alternative is available.

23

Reagents

1. Stock magnesium Solution (100 ppm): Dissolve 0.10141 g of magnesium sulphate heptahydrate (MgSO4 .

7 H2O) in deionised water and make up to 100 ml.

2. Standard magnesium Solution (10 ppm): Dilute 10 ml of the stock magnesium solution to 100 ml with

deionised water.

3. Polyvinyl alcohol (1%): Dissolve 1.0 g polyvinyl alcohol (PVA) in 25 ml deionised water by heating on a

hot plate, cool make up to 100 ml mark with deioised water.

4. Hydroxylamine hydrochloride Solution (5%): Dissolve 5.0 g of hydroxyl amine hydrochloride in deionised

water and dilute to 100 ml with deionised water.

5. Sodium hydroxide (4%): Dissolve 4.0 g of sodium hydroxide in deionised water and and dilute up to 100

ml using deionised water.

6. Titan Yellow Reagent (0.15%): Dissolve 0.15 g titan yellow in deionised water and dilute to 100 ml.

7. Calcium solution (1000ppm): Dissolve 0.025 g CaCO3 in minimum quantity of 0.1 N HCl (about 2.5 ml)

and make up to 100 ml with deionised water.

Procedure

Transfer 1, 2, 3, 4, 5 and 6 ml of 10 ppm standard magnesium solutions into six different 10 ml volumetric flasks

and add 1 ml of 1000 ppm calcium solution, 200 µl of hydroxylamine hydrochloride solution, 200 µl of titan yellow,

1 ml of polyvinyl alcohol and 1 ml of sodium hydroxide to each. Dilute to 10 ml with deionised water. After 10

minutes, measure the absorbance of each solution in a spectrophotometer at a wavelength of 554 nm against the

blank. Prepare a calibration curve of the absorbance versus concentration of the magnesium and determine the

concentration of the sample by referring the absorbance of the sample to the calibration curve.

Recommended sample volume: 1 ml Cookbook value : 40 µg of magnesium in 10 ml (4.0 ppm) gives an absorbance of 0.37 ± 0.01

24

Calibration curve

Stability: The colour is stable for one day



Absorbance Deviation from Mg

Remarks

Nil (magnesium .only) 2.50 0.249 Nil - Iodide(I-) 100.00 0.238 -0.009 NI Cobalt(Co) 100.00

50.00 25.00*

0.195 0.286 0.229

-0.054 +0.037 -0.020

I I

NI Manganese (Mn) 100.00

50.00 25.00*

0.344 0.298 0.238

+0.095 +0.049 -0.011

I I

NI Calcium (Ca2+) 100.00

50.00* 0.180 0.262

-0.069 +0.013

I NI

Nitrate(NO3-) 100.00 0.239 -0.010 NI

Chloride(Cl-)

100.00

0.249 0.000 NI SLS 100.00 0.238 -0.011 NI

Iron(III)(Fe3+) 100.00 0.135 -0.114 I Copper(II)(Cu2+) 100.00 0.098 (ppt) -0.151 I Cadmium(Cd2+) 100.00

50.00 -0.067 -0.008

-0. 316 - 0.257

I I

Vanadium(V) 100.00 0.202 -0.047 I

25

50.00 0.237 -0.012 NI Antimony(Sb) 100.00

50.0 0.070 0.153

-0.179 -0.096

I I

Sulphate(SO42-) 100.00 0.249 0.000 NI

Nickel(Ni) 100.00 0.195 -0.154 I Arsenic(As) 100.00 0.208 -0.041 I Zinc(Zn2+) 100.00

50.00 0.014 0.082

-0.235 -0.167

I

Triton X-100 100.00 0.233 -0.016 NI Tartaric Acid 100.00 0.102 -0.147 I

50.00 0.203 -0.046 I Phosphate

(PO43-)

100.00 0.235 -0.014 I

EDTA 100.00 0.239 -0.010 I

Aluminium(Al3+) 100.00 0.239 -0.010 NI

Chromium(VI) 100.00 0.095 -0.154 I 50.00

0.057 (ppt) -0.192 I

Boron(B) 100.00 0.245 -0.004 NI

I-Interfering; NI- Not Interfering; *In presence of 1 ml of 0.01M EDTA

8. DETERMINATION OF MERCURY Mercury (Hg) concentration in streams is 0.07 ppm and in groundwaters it is 0.5 to 1 ppm. It can form dimethyl

mercury which is very toxic. The Bureau of Indian Standards (BIS) prescribes a limit 0.001 ppm.

In the rhodamine 6G iodide method, mercury combines with rhodamine 6G and iodide to form ternary complex at

pH 4.0 resulting in a colour change from red to pink. The absorbance of the solution measured at 575 nm is

proportional to the concentration of mercury.

Reagents

26

1. Stock Mercury Solution (1000 ppm): Dissolve 0.1354 g of mercuric chloride in deionised water and make

up to 100 ml.

2. Standard mercuryc Solution (5 ppm): Dilute 0.5 ml of the stock mercury solution to 100 ml with

deionised water.

3. Bufferred iodide solutio: Dissolve 24 g of citric acid, 12 g of trisodium citrate, 5 g of potassium iodide, a

few crystals of sodium thiosulphate and 4.66 g of EDTA disodium salt in 200 ml of deionised water, adjust

pH to 4.0 under a standardized pH meter and make up to 250 ml.

4. Rhodamine 6G solution (0.005%): Dissolve 5.0 mg of rhodamine 6G in deionised water and dilute to 100

ml with deionised water.

5. Polyvinyl alcohol (0.1%): Dissolve 0.1g of polyvinyl alcohol in boiling deionised water, cool and dilute to

100 ml with deionised water.

Procedure

Transfer 0.1, 0.5, 1.0, 1.5, 2.0 ml of the standard mercury solution into five different 10 ml volumetric flasks and

add 0.5 ml buffered iodide solution. Dilute to about 7 ml with deionised water and mix by swirling. Then add 1 ml

rhodamine 6G solution followed by 0.5 ml polyvinyl alcohol solution. Make up to mark and mix. Measure the

absorbance of the solution in a 1 cm cuvette against a blank at 575 nm. Prepare a calibration curve of absorbance

versus concentration of mercury and determine the concentration of the sample by referring the absorbance of the

sample to the calibration curve.

Recommended sample volume: 5 ml Cookbook value: 10 µg of mercury in 10 ml (1.0 ppm) in 1 cm cell gives an absorbance of 0.32 ± 0.01

27

Calibration curve


Interfering species (1000µg) 10 µg Hg Remarks

Lithium (Li+) NI

Magnesium(Mg2+) NI

Calcium (Ca2+) NI

Strontium (Sr2+) NI

Barium (Ba2+) NI

Coppric (Cu2+) NI

Cobaltous (Co2+) NI

Manganous (Mn2+) NI

Nickelous (Ni2+) NI

Zinc (Zn2+) NI

Cadmium (Cd2+) NI

Silver (Ag+) I

28

Chromic (Cr3+) NI

Aluminium (Al3+) NI

Ferric (Fe3+) NI

Arsenous (As3+) NI

Chloride (Cl-) NI

Bromide (Br-) NI

Iodiate (IO3-) NI

Chlorate (ClO3-) NI

Sulphate (SO42-) NI

Nitrate (NO3-) NI

Nitrite (NO2-) NI

Silicate ( SiO3- NI

Phosphate (PO43-) NI

Fluoride (F-) NI

Oxalate NI

Citrate NI

Lead (Pb2+) NI

Antimony (Sb3+) NI

Cerium (Ce3+) NI

Surfactant (cationic, nonionic or anionic) I

Platinum Pt2+ I

Palladium (Pd2+) I

NI = Not interfering and I = interfering

9. DETERMINATION OF NONIONIC SURFACTANTS

Surfactants enter waters and wastewaters mainly by discharge of aqueous wastes from household and industrial

laundering and other cleaning operations Usually they are present in the range 1 to 20 ppm They interfere in the

analysis of some parameters in water analysis. The following method applies to nonionic surfactants.

In the iodide/iodine method, surfactants cause a reddish turbidity with the reagent in the solution. The absorbance

of the solution measured at 500 nm after 30 minutes is proportional to the concentration of the surfactant.

Reagents

1. Stock triton X-100 Solution (10%): Dissolve 10 g of Triton X-100 in deionised water and make up to 100

ml.

29

2. Standard triton X-100 Solution (1000 ppm): Dilute 1 ml of the stock arsenic solution to 100 ml with

deionised water.

3. Sodium acetate Acetic Acid-Buffer pH 4.0 (0.1M) : Dissolve 1.5 ml glacial acetic acid (99.8%) and 0.3742

g anhydrous sodium acetate in deionised water and make up to 250 ml. Adjust pH under a calibrated pH

meter to 4.0 (by adding 0.1 N NaOH or 0.1N HCl as necessary).

4. EDTA solution (0.05M): Dissolve by heating1.8615 g EDTA disodium salt in 80 ml deionised water, cool,

dilute to 100 ml mark and mix.

5. Iodine/iodide reagent: Dissolve 1.0 g finely ground iodine and 2.0 g of potassium iodide in 80 ml deionised


Procedure

Transfer 20, 40, 60, 80, 100, 120, 140 µl of the standard triton x-100 solution into six different 10 ml volumetric

flasks. Add 2 ml of acetate buffer solution and 1 ml 0.05 M EDTA solution to each. Dilute to 8 ml with deionised

water and mix by swirling. Then add 250 µl Iodine/iodide solution. Make up to mark and mix. Measure the

absorbance of the solution after 30 minutes against a blank at 500 nm. Prepare a calibration curve of absorbance

versus concentration of surfactant and determine the concentration of the sample by referring the absorbance of

the sample to the calibration curve.

.


Cookbook value: 40µg of surfactant in 10 ml (4.0 ppm) gives an absorbance of 0.32 ± 0.01

30

Calibration curve


Interfering Species Remarks

Lithium (Li+) NI

Magnesium(Mg2+) NI

Calcium (Ca2+) NI

Cuppric (Cu2+) NI

Cobaltous (Co2+) NI

Manganous (Mn2+) NI

Nickelous (Ni2+) NI

Zinc (Zn2+) NI

Mercuric (Hg2+) NI

31

Chromic (Cr3+) NI

Aluminium (Al3+) NI

Ferric (Fe3+) NI

Arsenous (As3+) NI

Chloride (Cl-) NI

Bromide (Br-) NI

Iodide (I-) NI

Chlorate (ClO3-) NI

Sulphate (SO42-) NI

Nitrate (NO3-) NI

Nitrite (NO2-) NI

EDTA NI

Silicate ( SiO3- (250) NI

Phosphate (PO43-) (0.1) NI

Fluoride (F-) (500) NI

Oxalate (500) NI

Citrate (500) NI

Lead (Pb2+) (100) NI

Antimony (Sb3+) NI

Anionic Surfactant NI

Cationic Surfactant I

NI = Not interfering / not interfering above the limit in µg shown in parenthesis and I = interfering

10. DETERMINATION OF IRON

Iron in water can cause stains. In drinking water it can impart objectionable taste and turbidity The Bureau of

Indian Standards (BIS) prescribes a limit 0.1 ppm and where there is no suitable alternative source of

drinking water the maximum limit is 1 ppm.

In the 1:10 phenanthroline method, ferric iron is reduced to ferrous iron by hydroxyl amine and then ferrous iron

reacts with 1:10 phenanthroline (also known as ortho phenenthroline) at pH 4.0 to form a red complex. The

absorbance at 510 nm is related to the concentration of iron. Three molecules of phenanthroline chelate each atom

of ferrous iron to form an orange-red complex. The colored solution obeys Beer’s law. Its intensity is independent

of pH from 3 to 9. A pH between 2.9 and 3.5 ensures rapid colour development in the presence of excess of

phenanthroline.

32

Reagents

1. Stock iron Solution (1000 ppm): Mix 0.7022 g of ammonium ferrous sulphate crystals in 2 ml

concentrated sulphuric acid, dissolve in deionised water, cool to room temperature and make up to 100

ml.

2. Standard iron Solution (50 ppm): Dilute 5. 0 ml of the stock fluoride solution to 100 ml with deionised

water.

3. Hydroxyl amine hydrochloride solution (10%): Dissolve 19 g of hydroxyl amine hydrochloride in deionised

water and dilute to 100 ml with deionised water.

4. Sodium acetate acetic acid buffer (pH 4.5): Dissolve 0.98 g of anhydrous sodium acetate in about 25 ml of

deionised water and add 35 ml of concentrated HCl

and dilute up to 50 ml using deionised water.

5. Reagent solution (0.1%): Dissolve 0.1g of 1, 10- phenanthroline monohydrate in 100ml of water by

stirring and heating.

Procedure

Pipette 0, 0.5, 1.0, 2.0, 3.0, 4.0 ml of 10ppm of iron solution into 10ml volumetric flask. Add 2 ml of sodium

acetate buffer, 0.2 ml of hydroxylamine solution and 2 ml of phenanthroline solution. Dilute to mark with water.

Mix thoroughly and allow a minimum of 10 min for maximum color development. Measure their absorbance at 510

nm in a 1 cm cuvette against a blank.

Recommended sample volume: 5 ml Cookbook value: 100 µg of iron in 10 ml (10.0 ppm) in a 1 cm cell gives an absorbance of 0.73 ± 0.01

33

Calibration curve

Interference The interference studies were carried out by determining the absorbance at 510 nm of 5 ppm iron and 100 ppm of

interfering species. Mg, Pb, F, Mn, I, Al, SO4, NO3, PO4, tartarate, Cl, V, Mo, BO3, sodium lauryl sulphate and triton

X-100 did not interfere but Sb, Ni, Br, Zn, EDTA, CA, DTPA, Cd, Co interfered.


Absorbance Deviation from Fe

Remarks

Nil (iron .only) 5 0.431 0 - Iodide(I-) 100 0.434 +0.003 NI Cobalt(Co) 100

50 Ppt

0.434 -

+0.003 NI

34

Manganese (Mn) 100 0.411 -0.02 NI Magnesium (Mg2+) 100 0.411 -0.02 NI Nitrate(NO3

-) 100 0.415 -0.016 NI Chloride(Cl-)

100 0.421 -0.01 NI

SLS 100 0.376 -0.055 I Fluoride (F-) 100 0.416 -0.017 NI Copper(II)(Cu2+) 100 Ppt - I Cadmium(Cd2+) 100

50 Ppt

0.428 -

-0.003 I

NI Vanadium(V) 100 0.398 -0.033 I Antimony(Sb) 100 0.396 -0.035 I Sulphate(SO4

2-) 100 0.425 -0.006 NI Nickel(Ni) 100 0.221 -0.21 I Bromide (Br-) 100 0.397 -0.34 I Zinc(Zn2+) 100 0.332 -0.099 NI

Triton X-100 100 0.424 -0.007 NI Tartaric Acid 100 0.428 -0.003 NI

Lead 100 0.422 -0.009 NI Phosphate

(PO43-)

100 0.412 -0.019 NI

EDTA 100 1.109 +0.678 I

Aluminium(Al3+) 100 0.425 -0.006 NI

Mercury (Hg2+) 100 ppt - I Molydate(Mo6+) 100 0.428 -0.003 NI

Boron(B) 100 0.427 -0.004 NI

Some of the interference was overcome by reducing the concentration: EDTA (100µg), DTPA (500µg), Co (50µg), Citrate (500µg), Ni 100 µg (1ml of citric acid), Cd 50 µg (1ml of citric acid).

35

IRON BY SYZYGIUM JAMBOLANA LEAF EXTRACT METHOD Syzygium jambolana leaf was dried at 110° C, ground in a porcelain pestle and mortar, passed through a 500

micron sieve (30mesh). Initially, 0.1 g of the powder was extracted into 10 ml distilled water by gentle shaking for

5 minutes and filtered through Whatman 42 filter paper. Latter, the extraction procedure was modified as follows

1 g of the leaf powder was shaken with 20 ml deionised water for 30 minutes and filtered through Wahtman No. 42

filter paper.

5 ml 1% or 1 ml 5% extract is required for 100 µg Fe (III)

36

Calibration curve

37

Interference studies

Fe 40 µg/10 ml 100 fold exess interfering ion

Absorbance 595nm

% Error Remarks

Nil 0.302

Co _ _ Ppt

Ni 0.349 15.6

Cu 0 (-100) No colour

Zn 0.186 -38.4

Mn 0.336 11.3

Cr 0.189 -37.4 Reddish brown colour

Ca 0.623 106.3 Ppt

Mg 0.303 0.3 NI

Pb _ 100% Black ppt

Sb 0.121 -59.9 Light brown

Bi _ 100% White ppt

Ag _ 100% Black ppt

Mo 0.103 -65.9 Yellowish brown

Nil 0.32 0.0%

AsO4 0.244 -23.8

BO3 0.318 -0.6 NI

PO4 0.139 -56.6

Cl 0.314 -1.9 NI

Br 0.298 -6.9 NI

I 0.326 1.9 NI

F 0.234 -26.9

SO4 0.318 -0.6 NI

NO3 0.334 4.4 NI

EDTA 0 -100.0

SLS 0.227 -29.1

CPC _ Ppt

Triton X-100 0.314 -1.9 NI

Tartaric acid -0.02 -106.3

Citric acid 0 -100.0

Al _ Ppt

Hg 0.279 -12.8

Nil 0.356 0.0

CO3 0.157 -55.9

NH4* 0.345 -3.1 NI

10 fold exess interfering ion

Absorbance 595nm

% Error Remarks

Nil 0.289 0.0

Ca 0.277 -4.2 Ppt

Cu 0.327 13.1

Zn 0.268 -7.3 NI

Cr 0.313 8.3 NI

Al _ Ppt

Hg 0.114 -60.6 RB colour

NO2 0.325 12.5 Ppt

Fe2+ 0.055 -81.0

Final pH 2.0, on rising pH to 4.5 by adding NaOAc, Fe2+ precipitated and gradually got oxidized to give high absorbance.

V 0.642 122.1

Equal amount of interfering ion

Absorbance 595nm

% Error Remarks

Al 0.306 6.3 NI

Hg 0.275 -4.5 NI

Mo 0.314 9.0 NI

Sb 0.196 -31.9 I

Bi 0.2 -30.6 I

F 0.253 -12.2 I

PO4 0.071 -75.3 I


Absorbance 595nm

% Error Remarks

F 0.34 -9.8 NI

PO4 0.082 -78.2 I

Citrate 0.284 -24.7 I

38

NO2 0.219 -38.5

CN 0.173 -51.4

NH2OH 0.316 -11.2

Vanadate 0.568 57.8

ClO4 0.324 -10.0 NI

Fe2+ 0.028 -92.1 Final pH= -1.2

In presence of 1 ml 0.1M glycine


Absorbance 595nm

% Error Remarks

Cu 0.66 112.9 Turbid

Co 0.817 163.5 Turbid

Cd 0.001 -99.7

Zn 0.252 -18.7 Turbid Equal amount of interfering ion

Absorbance 595nm

% Error Remarks

Cu 0.395 19.7 I

Co 0.343 3.9 NI

Cd 0.337 2.1 NI

Zn 0.324 -1.8 NI

Al 0.296 -10.3 I

Tartrate 0.321 -14.9 I

EDTA 0.002 -99.5 I

In presence of 5 ml 0.1M MgSO4

Equal amount of interfering ion

Absorbance 595nm

% Error Remarks

Nil 0.452 0.0

F 0.419 -7.3 NI

PO4 0.406 -10.2 NI

Citrate 0.413 -8.6 NI

EDTA 0.389 -13.9 I

39

11. DETERMINATION OF PHOSPHATE

Phosphorus is the eleventh most abundant mineral in the earth's crust. No national criteria have been

established for concentrations of phosphorus compounds in water. However, to control eutrophication, the

EPA makes the following recommendations Total phosphate should not exceed 0.05 mg/L (as phosphorus) in a

stream at a point where it enters a lake or reservoir, and should not exceed 0.1 mg/L in streams that do not

discharge directly into lakes or reservoirs.

Under acidic conditions, orthophosphate reacts with ammonium molybdate to form molybdophosphoric acid. It is

further reduced to molybdenum blue by adding a reducing agent such as stannous chloride. The intensity of the

blue colored complex is measured at 700 nm which is directly proportional to the concentration of phosphate

present in the sample.

Reagents

1. Stock Phosphate Solution (100 ppm) Dissolve 0.0439 g of KH2PO4 in deionised water and make up to 100

ml.

2. Standard Phosphate Solution (10 ppm) Dilute 10 ml of stock phosphate solution diluted to 100 ml with

deionised water.

3. Ammonium Molybdate Reagent Dissolve 2.5 g of (NH4)6Mo7O24.4H2O in 17.5 ml of deionised water.

Cautiously add 28 ml of conc. H2SO4 to 40 ml deionised water. Cool, add molybdate solution, and dilute to

100 ml.

4. Stannous Chloride Reagent Add 6 ml of conc. HCl to 44 ml of deionised water with slowly stirring then

boil. Then add 1.4 g of stannous chloride (SnCl2.2H2O), heat it until the solution becomes clear, cool and

dilute up to 100 ml using deionised water.

5. EDTA (0.01 M) Dissolve 3.7224 gm in deionised water and make up to 100ml.

Procedure

Transfer 0.1, 0.5, 1.0, 1.5, 2.0 ml of 10 ppm standard phosphate solution into five different 10 ml volumetric flasks

and add 1 ml of 0.1M EDTA followed by 0.2 ml of ammonium molybdate reagent, and 20 µl of stannous chloride

reagent to each. Dilute to 10 ml with deionised water. After 5-10 minutes, measure the absorbance at 700 nm in a

1 cm cuvette against the blank. Prepare a calibration curve of the absorbance versus concentration of the

phosphate and determine the concentration of the sample by referring the absorbance of the sample to the

calibration curve.

Recommended sample volume : 5 ml

Cookbook value: 0.5 µg of phosphate in a final volume of 10 ml measured with 1 cm cell gives an

absorbance of 0.39 + 0.01.

40

Spectrum of phosphomolybdate blue

Calibration curve

Interference Studies

41

Following ions do not cause interference in the phosphate determination

Concentration Ions Remarks

100 ppm

Iodide(I-), Chloride(Cl-), Fluoride(F-),

Sulphate(SO42-), Nitrate(NO3

-), Carbonate(CO3),

Perchlorate(ClO4), manganese(Mn), Citric acid,

Molybdenum(Mo), Cadmium(Cd), Aluminium(Al),

Ferrous(Fe2+), Sodium lauryl sulphate

Cobalt (Co)

NI

50 ppm Copper(Cu), Calcium (Ca2+) NI

50 ppm Bismuth (Bi) NI

10 ppm Vanadium(V), Silver(Ag), Mercury (Hg), CTAB NI

10 ppm Bismuth(Bi) NI

5 ppm Silver (Ag),Triton X- 100, CTAB NI

2 ppm Antimony (Sb), CTAB, NI

The ions that interfere in the determination of phosphate are

Concentration Ions Remarks

100 ppm Copper (Cu), Zinc (Zn), Bromide (Br-),

Vanadium(V), Antimony(Sb), Lead (Pb),

Bismuth(Bi), Arsenic(As), Chromium(Cr6+),

Ferric(Fe3+), Silver (Ag),TritonX-100,CTAB, nitrite

(NO2), Cetylpyridinium chloride.

I

0.5 ppm (with/without EDTA) Arsenic (As) I

42

12. Determination of Nitrite

Nitrite is normally not present in drinking water. However, it may be present if the water is contaminated with

sewage. Nitrite reacts with secondary amines to form nitrosamines. The latter are carcinogenic.

In acid solution nitrites react with primary aromatic amines to form diazonium salts. The latter give intensely

colored azo dye-stuffs with aromatic compounds containing an amino or hydroxyl group. The dyes are suitable for

photometry and the reaction is specific and sensitive.

Reage

nts

1. Stock nitrite solution (100 ppm): Dissolve 0.04928 g of A.R. sodium nitrite in 100 ml deionised water.

2. Working solution (1 ppm): Dilute 1 ml of the stock solution to 100 ml with deionised water

3. Reagent solution:

a) Dissolve 0.336 g sulphanilic acid and 2.72 g potassium hydrogen sulphate in deionised water and dilute to

100 ml water.

b) Dissolve N-(1-naphthyl)ethyleenediamine’dihydrochloride (0.04% aqueous solution)- store in dark

Procedure

Pipette 0 – 2.5 ml of 1ppm of nitrite solution into 10 ml volumetric flask. Add 1ml of sulfanlic acid solution and 1 ml

of N-(1-naphthyl)ethyleenediamine’dihydrochloride mix well and dilute to mark. Allow a minimum of 10 minutes for

maximum colour development. Measure their absorbance at 546 nm.


Cookbook value: 1 ppm Nitrite – N solution in1 cm cell gives an absorbance of 0. 32 + 0.01

43

Calibration curve

.

Interferences The interference studies were carried out by determining the absorbance of 1ppm nitrite and 1 ml of 1000ppm

concentration of interfering species at 546 nm.

Cu, SLS, Cr, Pb, Mo, V, Al, Cd, Fe, As, Hg, Br, SLS and DTPA interfered.

Some of the interference was overcome by reducing the concentration and by adding 0.05 M EDTA as follows

Al(500µg), Cd (500µg) Br (500µg), Fe (100µg), V(50µg), Mo (100µg), DTPA (500Μg), Hg (1 ml EDTA), Cr (1 ml

EDTA), Pb (1 ml EDTA), Br (1 ml EDTA), As ( 50µg)

Abso

rban

ce

µg Nitrite / 10 ml

44

13. DETERMINATION OF MANGANESE

Manganese is normally found in groundwaters at concentrations less than 0.1ppm. Elevated levels of manganese causes stains in plumbing, laundry, cooking utensils etc. It is an essential micronutrient for plants and animals but high doses are toxic. It is associated with Alzeimer’s disease.The Bureau of Indian Standards (BIS) prescribes a limit 0.1 ppm relaxable if no suitable alternative source is available to 0.3 ppm. Although many spectrophotometric methods have been reported, most of them require solvent extraction.

Spectrophotometric method for manganese determination with 4,2-pyridyl azo naphthol (PAN) and Triton X-100

has been selected for the present investigation due to high sensitivity and selectivity, and manganese can directly

determined in aqueous solution without separation.

Pyridyl azo naphthol forms a purple-red complex with manganese at pH 9.2 in the presence of Triton X-100, a non-

ionic surfactant. The intensity of the colour is measured at 562 nm which is directly proportional to the

concentration of manganese present in the sample. Masking-reagent solution which is a combination of

triethanolamine(TEA), potassium cyanide and sodium ascorbate overcomes most of the interferences.

Reagents

1. Stock Manganese Solution (1000 ppm): Dissolve 0.3076 g of manganous sulphate monohydrate in deionised water and make up to 100 ml.

2. Standard Manganese Solution (5 ppm): Dilute 0.5 ml of stock manganese solution to 100 ml with deionised water.

3. Triton X-100 (16%): Dilute 16 ml of pure Triton X-100 solution to 100 ml by stirring with deionised

water.

4. Triethanolamine Solution (16%): Dissolve 16 ml of Triethanolamine in deionised water and make up to 100 ml.

5. Buffer (pH 9.2): Dissolve 5.35g ammonium chloride and 7.0 ml concentrated ammonia solution (sp.gr. 0.91) in 100 ml deionised water.

6. Potassium cyanide solution (2%): Dissolve 2 g of potassium cyanide in deionised water and make up to 100 ml.

7. Methanolic PAN solution (0.1%): Dissolve 0.1 g pyridyl azo naphthol in methanoland make up to 100 ml.

8. Sodium ascorbate solution(0.5%): Dissolve 0.05 g of sodium ascorbate in deionised water and make up to 100 ml.

Procedure

Transfer 1,2,3,4 and 5 ml standard manganese solution (5 ppm) into 5 different 10 ml volumetric flasks and add 1

ml of sodium ascorbate solution, 0.5 ml triethanolamine solution, 0.5 ml triton X-100 solution, 0.5 ml potassium

cyanide solution and finally, 0.5 ml 4,2-pyridylazo naphthol solution to each. Dilute to 10 ml with deionised water.

Measure the absorbance of each solution in a spectrophotometer with a 1 cm path length at 562 nm against the

blank. Prepare a calibration curve of the absorbance versus concentration of the cadmium and determine the

concentration of the sample by referring the absorbance of the sample to the calibration curve.


Cookbook value: 5 µg of manganese solution gives an absorbance of 0.36 + 0.01

45

Calibration curve

46

Interference Studies

Following ions do not cause interferences in the determination of 5 µg manganese

Concentration Ions / species Remarks

1000 ppm Iodide(I-), Chloride(Cl-), Fluoride(F-) , Sulphate(SO42-), Nitrate(NO3

-),

Phosphate(PO4), Bromide (Br-), citrate, tartrate, triton X-100, SLS,

cupric (Cu2+), cobaltous(Co2+), nickelous(Ni2+), magnesium(Mg2+),

calcium(Ca2+), antimony(Sb3+), silver(Ag+), Molybdate(Mo7O246-)

NI

50 ppm Vanadium (V), Chromium (Cr6+), Aluminium (Al3+), mercury (Hg2+) ,

Arsenate (AsO43-)

NI

10 ppm Ferric (Fe3+), lead (Pb2+) NI

5 ppm Chromium (Cr6+), cadmium(Cd2+), zinc(Zn2+) NI

5 ppm EDTA I

NI = NI I = interferes

47

14. DETERMINATION OF CADMIUM

Cadmium is one of the poisonous elements. The average abundance of Cd in the earth’s crust is 0.16 ppm; in soils

it is 0.1 to 0.5 ppm; in streams it is 1 µg/l, and in ground waters it is from 1 to 10 µg/l. It often exists in

wastewaters discharged by the electroplating, metallurgical, chemical and other industries. Cadmium is extremely

toxic and causes itai itai or Ouch - Ouch disease. It accumulates in the kidneys and liver, with prolonged intake at

low levels sometimes leading to dysfunction of the kidneys. The Bureau of Indian Standards (BIS) prescribes

a limit 0.01 ppm.

Cadion, p-nitrobenzenediazoaminobenzene-p-azobenzene, is purple in alkaline alcoholic solution and forms an

orange-red complex with cadmium the intensity of which measured at 482 nm is directly proportional to the

concentration of cadmium present in the sample. Linear calibration graphs were obtained for 0.5-10 µg/ml of Cd.

Cadion (p-nitrobenzenediazoaminobenzene-p-azobenzene)

Reagents

1. Stock Cadmium Solution (100 ppm): Dissolve 0.2370 g of cadmium acetate (CH3COO)2Cd.2H2O in deionised water and make up to 100 ml.

2. Standard Cadmium Solution (5 ppm): Dilute 0.5 ml of stock cadmium solution to 100 ml with deionised water.

3. Triton X-100 (1%): Dissolve 1 g of pure Triton X-100 solution (102.0%) in 100 ml with deionised water.

4. Cadion Solution (0.02%): Dissolve 0.02 g of cadion and 1.12 g of potassium hydroxide (KOH) in 100 ml of ethyl alcohol.

5. NaOH (0.02 M) : Dilute 0.1 ml of 40 % NaOH in 50 ml of deionised water.

6. Mixed Reagent: Mix 5 ml Cadion, 5 ml Triton X – 100 10 ml NaOH

7. Masking Reagent: Dissolve 0.0074 g of triethanolamine(TEA), 0.0066 g of iminodiacetate (IDA) and 0.1470 g of sodium citrate in deionised water. Adjust pH 12 with 4 % sodium hydroxide (NaOH) and make up to 50 ml.

8. Rochelle Salt Solution(0.01 M): Dissolve 0.1411 g potassium sodium tartrate in deionised water and make up to 50 ml.

Procedure

Transfer 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 ml of 5 ppm standard cadmium solution into 8 different 10 ml

volumetric flasks and add 2 ml of mixed reagent to each. Add 0.5 ml of masking reagent and dilute to 10 ml with

deionised water. After 5-10 minutes, measure the absorbance of each solution in a spectrophotometer with 1 cm

path length at 482 nm against the blank. Prepare a calibration curve of the absorbance versus concentration of the

cadmium and determine the concentration of the sample by referring the absorbance of the sample to the

calibration curve.

Cookbook value: 0.5 ppm of cadmium solution gives an absorbance of 0.60+ 0.01.

48


Calibration curve

49

Efffect of Interfering Species Sl. No.

Species Conc. (µg/10

ml)

Conc. (ppm)

Absorbance Deviation Remarks

1. Cadmium (only) 1.5 0.15 0.181 Nil - 2. Iodide(I-) 1000.00 100 0.183 +0.002 NI 3. Chloride(Cl-) 1000.00 100 0.176 -0.005 NI 4. Fluoride(F-) 1000.00 100 0.176 -0.005 NI 5. Sulphate(SO4

2-) 1000.00 100 0.189 +0.008 NI 6. Nitrate(NO3

-) 1000.00 100 0.194 +0.013 NI 7. Bromide (B-) 1000.00 100 0.160 -0.021 NI 8. Phosphate (PO4

2-) 1000.00 100 0.176 -0.005 NI 9. Aluminium (Al) 1000.00 100 -0.135 -0.046 I, yellow & Turbid

100.00 10 0.185 +0.004 NI 10. Nickel (Ni) 1000.00 100 0.583 +0.402 I

100.00 10 0.204 +0.023 NI 11. Cobalt(Co) 1000 100 0.585 +0.404 I

20 2 0.582 +0.401 I

12. Copper(Cu)

1000 100 0.728 +0.547 I

20 2 0.607 +0.426 I 13. Zinc(Zn) 1000 100 0.894 +0.713 I

10 1 0.607 +0.426 I

14. Vanadium(V) 1000.00 100 0.232 +0.051 I 500.00 50 0.180 -0.001 NI

15. Antimony(Sb) 1000.00 100 0.224 +0.043 I

250.00 25 0.200 +0.019 NI

16. Lead(Pb) 1000.00 100 0.235 +0.054 I 500.00 50 0.203 +0.022 NI

17. Bismuth(Bi) 1000.00 100 -0.233 -0.052 I 100.00 10 0.177 -0.004 NI

18. Arsenic(As) 1000.00 100 -0.252 -0.071 I 100.00 10 0.184 +0.003 NI

19. Chromium VI(Cr6+) 1000.00 100 0.327 +0.146 I, brownish-red colour

500.00 50 0.208 +0.027 NI

20. Ferric(Fe3+) 1000.00 100 0.589 +0.408 I, orange colour

50.00 5 0.192 +0.011 NI 21. Ferrous(Fe2+) 1000.00 100 -0.185 -0.004 I, yellow colour

100.00 10 0.208 +0.027 NI 22. Silver(Ag)

1000.00 100 -- -- I, orange, turbid 100.00 10 0.210 +0.029 NI,

23. Mercury(Hg) 1000.00 100 0.107 -- I, yellow-orange 500.00 50 0.782 +0.601 I, orange 100.00 10 1.077 +0.896 I, orange

24. Molybdenum (Mo) 1000.00 100 --- --- I, precipitate

25. Manganese (Mn) 1000.00 100 0.857 +0.676 I

26. Mo + 1 ml of Masking Reagent + 1 ml of 0.01M Rochelle salt solution

50.00 5 0.201 +0.02 NI

27. Mn + 1 ml of Masking Reagent

10.00 1 0.201 +0.02 NI

50

NI = Not interfering and I = interfering.

15. DETERMINATION OF COPPER

Copper is considered an essential trace element for plants and animals but higher concentrations are toxic. The

Bureau of Indian Standards (BIS) prescribes a limit 0.05 ppm, relaxable if no suitable alternative

source is available to 1.5 ppm.

Cuprous ion forms water – soluble orange colored chelate with bathocuproine disulfonate salt. While the color

forms over the pH range 3.5- 11.0, the recommended pH range is between 4 and 5. The sample is buffered at a pH

of 4.3 and reduced with hydroxylamine hydrochloride. The absorbance is measured at 484 nm.

Reagents

1. Stock copper sulfate (1000 ppm): Dissolve 0.3929 g of copper sulfate (AR grade) in 100 ml water.

2. Working solution (10 ppm): Dilute 1.0 ml of the stock solution to 100 ml with deionised water.

3. Hydrochloric acid (1:1)

4. Hydroxylamine hydrochloride (11.11%): Dissolve 11.111 g Hydroxylamine hydrochloride in 100 ml water.

5. Sodium citrate solution (30%): Dissolve 30 g of sodium citrate in 100 ml water.

6. Reagent solution (0.1%): Dissolve 0.1g of Bathocuproine disulfonic acid disodium salt in 100 ml water.

Procedure

Pipette 0- 4 ml of 10 ppm copper solution into 10 ml volumetric flask.. Add HCl (1:1) 200 µg/l, 1 ml of

hydroxylamine hydrochloric acid, 1ml sodium citrate and 1 ml bathocuproine solution mix and dilute to mark. Allow

a minimum of 10 minutes for maximum color development. Measure their absorbance at 484 nm and 1 cm path

length. The colour is stable for 8 hours.

Cookbook value: 1 ppm of copper solution gives an absorbance of 0.21 + 0.01


51

Calibration curve

Interferences

The interference studies were carried out by determining the absorbance at 484 nm of 15µg of copper solution and 1 ml 1000 ppm concentration of interfering species.

Some of the interference was overcome by reducing the concentration: Fe (500µg), Cr(4 ml of Hydroxyl hydrochloride), EDTA ( 1 ml of 200 ppm Zn)

Spectrum of cuprous-bathocuproine complex

52

Sl. No.

Species Conc. (ppm)

Absorbance Deviation Remarks

1.Copper(only) 1.5 0.275 Nil -

2. Iodide(I-) 100 0.285 +0.01 NI

3. Chloride(Cl-) 100 0.279 +0.004 NI

4. Fluoride(F-) 100 0.273 -0.002 NI

5. Sulphate(SO42-) 100 0.283 +0.005 NI

6. Nitrate(NO3-) 100 0.271 -0.004 NI

7. Bromide (B-) 100 0.276 +0.001 NI

8. Phosphate (PO42-) 100 0.276 +0.001 NI

9. Aluminium (Al) 100 0.274 -0.001 NI

10. Nickel (Ni) 100 0.282 +0.007 NI

11. Cobalt(Co) 100 0.288 +0.013 NI

12. Triton X-100

100 0.278 +0.003 NI

13. Zinc(Zn) 100 0.283 +0.008 NI

14. Vanadium(V) 100 0.278 +0.003 NI

15. Antimony(Sb) 100 0.276 +0.001 NI

16. Lead(Pb) 100 0.294 +0.019 NI

17. Arsenic(As) 100 0.296 +0.021 I

18. Chromium VI(Cr6+) 100 0.325 +0.050 I

19. Ferric(Fe3+) 100 0.310 +0.035 I

20. Mercury(Hg) 100 0.291 +0.016 NI

21. Molybdenum (Mo) 100 0.280 +0.005 NI

22. Manganese (Mn) 100 0.276 +0.001 NI

23. EDTA+(200ppn)Zn 100 0.025 -0.250 I

24. Boron

100 0.274 -0.001 NI

25. Tartarate 100 0.278 +0.003 NI

53

16. DETERMINATION OF LEAD

Lead is toxic by ingestion and is a cumulative poision.Tap waters may contain leaddueto attack on lead pipes, brass

fixturesor solder pipe joints. In groundwaters it is usually less than 0.1 ppm. The Bureau of Indian Standards

(BIS) prescribes a limit between 0.05 ppm.

In the PAR (pyridyl azo resorcinol) method, lead reacts with the reagent at pH 10 to form a red water soluble

complex. The absorbance at 520 nm is proportional to the concentration of lead.

Reagents

1. Stock Lead Solution (1000 ppm): Dissolve 0.3996 g of lead nitrate in deionised water and make up to 100

ml.

2. Standard Lead Solution (10 ppm): Dilute 10 ml of the stock fluoride solution to 100 ml with deionised

water.

3. PAR Solution(0.4%): Dissolve 0.4 g of pyridyl azo resorcinol in deionised water and dilute to 100 ml with

deionised water.

4. Buffer solution (pH 10.0): Dissolve 17.5 g of ammonium chloride in 50 ml deionised water add 142 ml of

concentrated ammonia and make up to 250 ml using deionised water.

Procedure

Transfer 1, 2, 3, 4, 5 ml of 10 ppm standard lead solutions into five different 10 ml volumetric flasks and add 1 ml

of pyridyl azo resorcinol solution followed by 4 ml of buffer solution to each. Dilute to 10 ml with deionised water.

After 10 minutes, measure the absorbance of each solution in a 1 cm cuvette in a spectrophotometer at a

wavelength of 520 nm against the blank. Prepare a calibration curve of the absorbance versus concentration of

lead and determine the concentration of the sample by referring the absorbance of the sample to the calibration

curve.The colour is stable for 6 hours.


Cookbook value: 20 µg of lead in 10 ml (2.0 ppm) gives an absorbance of 0.36 ± 0.01

54

Spectrum of lead- PAR complex Calibration curve

55



Remarks

Nil (Lead .only) 1.00 - Iodide(I-)

100.00 50.00

I NI

Fluoride 100.00 5.00*

I NI

Cobalt(Co) 100.00 I Manganese (Mn) 100.00 I Magnesium (Mg2+) 100.00 I Nitrate(NO3

-) 100.00 NI Chloride(Cl-)

100.00 10.00

I NI

SLS 100.00 10.00

I NI

Iron(III)(Fe3+) 100.00 I Copper(II)(Cu2+) 100.00 I Cadmium(Cd2+) 100.00 I Iron(II)(Fe2+) 100.00 I Vanadium(V) 100.00 I Antimony(Sb) 100.00

2.00 I

NI Sulphate(SO4

2-) 100.00 NI Citric Acid 100.00 NI Nickel(Ni) 100.00 I Arsenic(As) 100.00 NI Mercury(Hg) 100.00 NI Zinc(Zn2+) 100.00 I Tartaric Acid

100.00 50.00

I NI

Phosphate (PO43-) 100.00

50.00 I

NI EDTA 100.00

50.00 I

NI Aluminium (Al3+)

100.00 50.00

I NI

Chromium(VI) 100.00 NI Molybdenum (Mo) 100.00

50.00 10.00

I I

NI

Boron(B) 100.00 10.00

I NI

Bromide(Br-) 100.00 I Silver(Ag+) 100.00 NI

I-Interfering; NI- Not Interfering *In presence of 0.01M citric acid

56

17. DETERMINATION OF TOTAL HARDNESS

Hardness of water is caused mainly by soluble calcium and magnesium salts. The Bureau of Indian Standards

(BIS) prescribes a limit 300 ppm as CaCO3. When no suitable alternate source of water is available the

limit may be relaxed up to 600 ppm.

In the calmagite () method, calcium and magnesium ions react with the reagent at pH 12.5 to form a complex,

changing the colour of the solution from blue to red-blue. The change in the absorbance at 520 nm is related to the

hardness of water.

Reagents

1. 2N HCl: Dilute 17.1 ml Concentrated HCl to 100 ml with deionised water.

2. Calcium Solution (100 ppm): Dry calcium carbonate at 105° C. Weigh out 0.1 g of calcium carbonate into

a 100 ml beaker. Add 2 N HCl slowly with swirling till all the white powder just dissolves. Shake well to

remove most of the dissolved carbondioxide gas. Transfer quantitatively to a 100 ml volumetric flask using

deionised water and make up to mark.

3. Magnesium Solution: Dissolve 0.2465 g magnesium sulphate hepta hydrate (MgSO4.7H2O) in deionised

water and make to 100 ml mark. SPADNS Solution Dissolve 95.8 mg of SPADNS in deionised water and

dilute to 50 ml with deionised water.

4. Calcium – magnesium Standard solution (100 ppm as Ca): Mix equal volumes of Calcium and magnesium

solutions.

5. Potassium hydroxide stock solution(1.5 N): Dissolve 8.415 g potassium hydroxide in deionised water and

make up to 100 ml. Store in a polyethlene bottle with a screw cap.

6. Potassium hydroxide working solution (0.015 N, pH 12.5±0.2): Dilute 1.0 ml of the potassium hydroxide

stock solution to 100 ml when required.

7. Calmagite reagent: Dissolve 0.0725 g Calmgaite in 100 ml deionised water

Procedure

Transfer 50, 100, 150, 200 and 250 µl of 100 ppm standard calcium – magnesium solution into five different 10 ml

volumetric flasks and add 1 ml of calmagite reagent to each. Add 1 ml 0.015N potassium hydroxide solution.

Dilute to 10 ml with deionised water. Measure the absorbance of each solution in a spectrophotometer at a

wavelength of 520 nm against the blank.

Prepare a calibration curve of the absorbance versus hardness of water and determine the hardness of the sample

by referring the absorbance of the sample to the calibration curve.

Recommended sample volume: 1 ml Cookbook value: 50 µg of CaCO3 in 10 ml (5.0 ppm) gives an absorbance of 0.53 ± 0.01

57

Absorption spectrum Calibration curve

58

18. DETERMINATION OF ZINC

Zinc is an essential micronutrient for plants and animals but at elevated levels it is toxic to some aquatic life The

Bureau of Indian Standards (BIS) prescribes a limit 5 ppm relaxable if no suitable alternative source is

available to 15 ppm.

Reagents

1. Standard Zinc solution (1000 ppm: Dissolve 0.4398 g of Zinc sulphate in 100ml of water.

2. Working solution (10 ppm): Dilute1 ml of the stock solution to 100 ml with deionised water.

3. Sodium Ascarbate solutio: Dissolve 10 g of L.Ascarbic acid in 50ml of water, then slowly add 5 g of

sodium bicarbonate and mix well. Then dilute to 100ml.

4. 1% KCN solutio: Dissolve 1g of Potassium Cyanide in 100ml of water.

5. Buffer solution (pH 9.0): Dissolve 2.1 g Sodium Hydroxide and 7.75 g Boric Acid in 250 ml of water.

6. Zincon Reagent: Dissolve 100mg of Zincon in 100 ml of methanol.

7. Cyclohexanone.

Procedure

Pipette out 0, 10, 20, 30, 40, 50 µg of 10 ppm Zinc solution into 10ml volumetric flask. Add 1.0 ml of Sodium

ascorbate solution, 1.5 ml of buffer solution, 0.5 ml of KCN solution 1.0 ml of Zincon solution and 0.2 ml of

Cyclohexanone, mix well and dilute to the mark. Allow to stand of 30 min for colour development. Measure the

absorbance of the solution in a spectrophotometer with a 1 cm cuvette at 620 nm.


Cookbook value: 10 µg Zn in 10 ml gives an absorbance of 0.34 ± 0.01

59

Calibration Curve

Interference The interference studies were carried out by determining the absorbance of 10 ppm Zinc solution at 620 nm and

different concentration of interfering species. 1000µg of CO2+, Ni2+, Ca2+, Mg2+, SO4, PO4, NO3, Ag, F, Cl, Br, I, Hg,

Tartaric Acid, Citric Acid did not interfere in the determination of 10 µg Zinc. Some of the interference was

overcome by reducing the concentration. 100 µg of Cr, Mo, Pb2+, Sb, SLS, Arsenic, and Triton X-100 did not

interfere in the determination of 10 µg Zinc. 50 µg of Al, Fe3+, Cu2+, and Cd2+ did not interfere in the determination

of 10 µg Zinc. 2 µg Vanadium did not interfere in the determination of 10 µg Zinc. 1 µg EDTA and Mn did not

interfere in the determination of 10 µg Zinc.

60

19. DETERMINATION OF NITRATE

Nitrate in drinking water above 45 ppm is harmful. It is associated with infant methaemoglobenia, cancer and

problems of digestive, circulatory and nervous systems. The Bureau of Indian Standards (BIS) prescribes a

limit of 45 ppm as nitrate.

In the Chromotropic acid method (Sodium–1,8–dihydroxy–3,6–naphthalene disulphonate) method, nitrate reacts

with the reagent in sulphuric acid medium to give a yellow coloured solution. The intensity of this colour measured

at 410 nm is proportional to the concentration of nitrate.

Chromotropic acid

Reagents

1. Stock nitrate Solution (1000 ppm): Dissolve 0.1371 g of anhydrous sodium nitrate in deionised water and

make up to 100 ml.

2. Standard nitrate Solution (100 ppm): Dilute 10 ml of the stock fluoride solution to 100 ml with deionised

water.

3. Chromotropic acid Solution(0.1%): Dissolve 0.1 g of chromotropic acid in concentrated sulphuric acid and

dilute to 100 ml with concentrate sulphuric acid.

4. Sulphite-urea solution: Dissolve 5 g of urea and 4 g anhydrous sodium sulphite) in about 50 ml of

deionised water and dilute up to 100 ml using deionised water.

5. Antimony solution: Heat 0.5 g of antimony powder with 80 ml concentrated sulphuric acid till all the metal

has dissolve. Cool and add to 20 ml of ice cold deionised water in a 250 ml beaker. Re dissolve any

precipite by heating. Cool and store in a container with air tight lid.

Procedure

Transfer 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 ml of 100 ppm standard nitrate solutions into six different 10 ml clean and

dry volumetric flasks and add I drop of sulphite-urea solution to each. Pipette 2.4, 2.3, 2.2, 2.1, 2.0 and 1.9 ml

deionised water to make the volume in each flask 2.5 ml. Add 2 ml of antimony solution followed by 1 ml

chromotropic acid to each. Dilute to 10 ml with concentrated sulphuric acid. After 45 minutes, measure the

absorbance of each solution in a1 cm cuvette in a spectrophotometer at a wavelength of 410 nm against the blank

(prepared from 2.5 ml deionised water). Prepare a calibration curve of the absorbance versus concentration of the

nitrate and determine the concentration of the sample by referring the absorbance of the sample to the calibration

curve.


Cookbook value: 50 µg of nitrate in 10 ml (5.0 ppm) gives an absorbance of 0.20 ± 0.01

61

Calibration curve

62


Interfering Species 10 fold excess

Concentration (µg/mL)

Remarks

Fluoride (F-) 200.00 NI Chloride(Cl-) 200.00 NI

Calcium(Ca) 200.00 NI

Manganese (Mn) 200.00 NI

Magnesium (Mg2+) 200.00 NI

Nitrite(NO2-) 200.00 NI

Chloride(Cl-)

200.00 NI Ammonium (NH4

+) 200.00 NI

Iron(III)(Fe3+) 200.00 NI

Copper(II)(Cu2+) 200.00 NI

Cadmium(Cd2+) 200.00 NI

Iron(Fe2+) 200.00 NI

Vanadium(V) 200.00 NI

Antimony(Sb) 200.00 NI

Zinc(Zn) 200.00 NI

Sulphide(S2-) 200.00 NI

Cobalt (Co2+) 200.00 NI

Nickel(Ni2+) 200.00 NI

Carbonate (CO32-) 200.00 NI

Arsenic(As) 200.00 NI

Mercury(Hg) 200.00 NI

I-Interfering; NI- Not Interfering Any concentration of barium, lead, iodide, iodate and >20.00 ppm of Chromium(III) interfered

63

20. DETERMINATION OF CHROMIUM

Chromium in hexavalent form (Cr6+) is carcinogenic. The Bureau of Indian Standards (BIS) prescribes a limit

between 0.05 ppm.

In the diphenyl carbazide method, Cr6+ reacts with diphenyl carbazide in acid medium to produce a red-violet

product. The absorbance of the solution at 545 nm is proportional to the concentration of chromium.

Diphenylcarbazide

Reagents

1. Stock chromium Solution (100 ppm) Dissolve 0.0283 g of potassium dichromate in deionised water and

make up to 100 ml.

2. Standard chromium Solution (5 ppm) Dilute 5 ml of the stock fluoride solution to 100 ml with deionised

water.

3. Diphenyl carbazide Solution Dissolve125 mg of diphenyl carbazide in acetone and dilute to 50 ml with

acetone.

4. Hydrochloric acid (1N) Dilute 8.5 ml of concentrated hydrochloric acid to 100 ml with deionised water.

Procedure

Transfer 0.5, 1, 1.5, 2, 3, 4 ml of 5 ppm standard chromium solutions into six different 10 ml volumetric flasks and

add 2 ml of 1N hydrochloric acid followed by 0.5 ml of diphenyl carbazide solution to each. Dilute to 10 ml with

deionised water. After 10 minutes, measure the absorbance of each solution in a 1 cm cuvette in a

spectrophotometer at a wavelength of 545 nm against the blank. The colour is stable for at least 8 hours.

Prepare a calibration curve of the absorbance versus concentration of chromium and determine the concentration

of the sample by referring the absorbance of the sample to the calibration curve.

Recommended sample volume: 1 ml Cookbook value: 10 µg of chromium in 10 ml (1.0 ppm) gives an absorbance of 0.71 ± 0.01

64

Calibration curve

65



Absorbance Deviation from F- only

sample

Remarks

Nil (chromium.only) 1.00 0.362 Nil - Iodide(I-) 100.00 0.000 -0.362 I Bromide 100.00 0.080 -0.282 I Cobalt(Co) 100.00 0.365 +0.003 NI Magnesium (Mg2+) 100.00 0.371 +0.009 NI Iron(III)(Fe3+) 100.00 0.307 -0.055 I Copper(II)(Cu2+) 100.00

5.00 0.297 0.332

-0.065 -0.030

I I

Antimony(Sb) 100.00 0.000 -0.362 I Nickel(Ni) 100.00 0.347 -0.015 NI TritonX-100 100.00 0.354 -0.008 Tartaric Acid

100.00 10.00 5.00

0.313 0.321 0.363

+0.056 0.041

+0.001

I I

NI Aluminium (Al3+)

100.00 0.371 +0.009 NI

I-Interfering; NI- Not Interfering

66

21. DETERMINATION OF ALUMINIUM

Aluminium is nonessential for plants and animals. Concentrations greater than1.5 mg / l constitutes a toxicity

hazard in the marine environment andlevels below 200 µg/ l present minimal risk. Aluminium levels in brain tissues

are suspected to be linked to Alzeimer’s disese. The Bureau of Indian Standards (BIS) prescribes a limit

0.03 ppm relaxable if no suitable alternative source is available to 0.2 ppm.

With Erichrome cyanine R dye, dilute aluminium solution s buffered to a pH of 6.0 produce a red to pink complex

that exhibits maximum absorption at 582 nm. The intensity of the developed colour is influenced by the aluminium

concentration, reaction time, temperature, pH and concentration of other ions in the sample.

Eriochrome cyanine R

Reagents

1. Stock aluminium solution (1000 ppm): Dissolve 0.8791 g of potassium. aluminium sulfate (potash alum)

(KAl(SO4)2.12H2O) in 50ml water.

2. Working solution (1 ppm): Dilute 0.1ml of the stock solution to 100 ml with deionised water

3. Sulfuric acid 0.02N H2SO4: Add 278 µl of concentrated sulphuric acid to 100 ml water to get approximately 1 N

sulphuric acid. Dilute 1 ml of this to 50 ml with deionised water.

4. Buffer Reagent (0.3M): Dissolve13.6 g of sodium acetate trihydrate NaC2H3O2.3H2O in 50 ml deionised water

and add 4 ml of glacial acetic acid. Make it up to 100 ml and adjust the pH to 6.

5. Erichrome cyanine R (0.015%): Dissolve 0.015 g of Erichrome cyanine R in 5 ml of deionised water and add

(1:1) acetic acid adjust the pH to 3.make it 100 ml with water.

6. Ascorbic acid(0.1%): Dissolve 0.1 g ascorbic acid in deionised water and make up the volume to 100 ml.

Prepare fresh every day.

Procedure

Pipette 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml of 1.0 ppm of aluminium solution into 10 ml volumetric flasks. Add 200 µl of 0.02 N sulfuric acid, 200 µl of ascorbic acid, 1.0 ml working dye reagent, 2.0 ml of buffer solution dilute to mark, mix, allow standing 5 minutes for colour development and measuring the absorbance at 535 nm in a 1 cm cuvette. Complete the measurement within 15 minutes of adding the buffer.

Recommended sample volume: 1 ml Cookbook value: 2 µg of aluminium in 10 ml (0.2 ppm) gives an absorbance of 0.25 ± 0.01

67

Spectrum of aluminium - Eriochrome cyanine R system

Calibration curve

Interferences The interference studies were carried out by determining the absorbance of 100 ppm aluminium at 535 nm in the presence of 1000 ppm of interfering species.

Co, F, Cu, Cd, Ni, Fe, SLS, Triton X-100, As, Tartarate, Mo, Cr, EDTA

Some of the interference was overcome by reducing the concentration Triton X-100 ( 500 µg), As (500µg), Ni (100µg), Cu (100µg)

68

22. DETERMINATION OF CYANIDE

Cyanide is highly toxic. The Bureau of Indian Standards (BIS) prescribes a limit 0.05 ppm. The following

method is applicable to cyanide amenable to chlorination in the absence of interfering substances like thiocyantes.

In the chloramieT - barbuturic acid method, cyanide reacts with chloramines T at pH < 8 to form CNCl which

then reacts with pyridine - barbituric acid reagent to form a red-blue colour, The absorbance of the solution at 578

nm is linearly related to the concentration of cyanide.

Reagents

1. Chloramine T Solution (100 ppm): Dissolve 1.0 g of chloramines t in deionised water and make up to 100 ml. (Stable for 1 week)

2. Sodiun hydroxide Solution (4 % ): Dilute 4 g sodium hydroxide in deionised water and make up to 100 ml with deionised water.

3. Sodiun hydroxide Solution (0.16 % ): Dilute 4 ml of 4% sodium hydroxide with deionised water to 100 ml. 4. Stock cyanide Solution (1000 ppm): Dissolve 0.2510 g of potassium cyanide in 4 ml 4% sodium

hydroxide solution and dilute to 100 ml with deionised water. (Stable for 1 week) 5. Standard solution of cyanide(20 ppm: Dilute 2.0 ml of the stock cyanide solution with deionised water to

100 ml. (Stable for 1 day) 6. Pyridine barbituric acid Reagent : To 15.0 g of barbituric acid in a 250 ml conical flask, add just enough

deionised water to wash the sides and wet the barbituric acid. Add 75 ml of pyridine (in a fume hood)and mix. Add 15 ml concentrated hydrochloric acid and mix. Cool to room temperature, dilute to volume and mix. Stable in dark for 6 months.

7. Acetate buffer (pH4.5) : Dissolve 41 g sodium acetate trihydrate and 50 ml glacial acetic acid in deionised water, adjust the pH to 4.5 under a calibrated pH meter,make up to 100 ml with deionised water.

Barbituric acid Pyridine

Procedure

Transfer 20, 40, 60, 80, and 100 µl of 20 ppm standard cyanide solutions into five different 10 ml volumetric flasks

and add 8 ml of 0.16% sodium hydroxide solution to each. Then add 200 l acetate buffer solution and 400 l of

chlorammine T solution. Mix and let stand for 2 minutes. Then add 1 ml pyridine – barbituric acid solution, dilute to

mark, mix and let stand for 8 minutes. Measure the absorbance of each solution in a spectrophotometer at a

wavelength of 578 nm against the blank. Prepare a calibration curve of the absorbance versus concentration of

cyanide and determine the concentration of the sample by referring the absorbance of the sample to the calibration

curve. The colour is stable for 15 minutes.


Cookbook value: 40 µg of cyanide in 10 ml (4.0 ppm) gives an absorbance of 0.29 ± 0.01

69

Calibration curve

70

23. DETERMINATION OF SULPHATE

Health concerns regarding sulphate in drinking water have been raised because of reports that diarrhoea and

dehydration may be associated with the ingestion of water with high levels of sulphate The Bureau of Indian

Standards (BIS) prescribes a limit 200 ppm relaxable if no suitable alternative source is available to

400 ppm.

As a precaution, water with a sulfate level exceeding 400 mg/L (400ppm) should not be used in the

preparation of infant formula. Beyond this, gastrointestinal irritation occurs when magnesium or sodium is

present. If sulfate in water exceeds 250 mg/L, a bitter or astringent taste may render the water unpleasant to

drink. High sulfate levels may also corrode plumbing, particularly copper piping. They can cause scale build-up

that can produce a black slime resulting in clogs and stains on clothes.

Sulfate ion (SO42-) is precipitated in an acetic acid medium with barium chloride so as to form barium sulphate

crystals of uniform size. Light absorbance of the BaSO4 suspension is measured by a spectrophotometer and the

SO42- concentration is determined by comparison of the reading with a standard curve.

Reagents

1. Sodium Chloride-Hydrochloric acid reagent: Dissolve 24 g of analytical reagent (A.R.) NaCl in 80 ml

distilled water. Add 2 ml of conc. HCl. Dilute to 100 ml in a volumetric flask.

2. Glycerol-alcohol solution: Dissolve 10 ml of gylcerol in 20 ml of isopropyl alcohol (or ethyl alcohol)

Procedure

Pipette out 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0 and 6.0 ml standard sulphate solution (100 ppm) into10

different 10 ml volumetric flasks. Add 1 ml Sodium Chloride-Hydrochloric acid reagent and then add 200 µl of the

glycerol-alcohol solution followed by1 ml (10%) barium chloride solution. Make up the volume to 10 ml by adding

deionized water. Close the lids and mix by inverting 20 times. Measure the absorbance of each solution at 420 nm

using a 1 cm cuvette, taking care to again shake the solutions vigorously just before taking readings.


Cook book value: 250 µg sulphate in 10 ml gives an absorbance of 0.206 ±0.04

71

Interference studies

S.No Interfering agent Absorbance Inference

1 pure sulphate (reference)200µg

0.144 -

2 Oxalate 2000 µg 1000µg

0.198 0.181

I NI

3 Bicarbonate2000 µg 0.118 NI

4 phosphate2000 µg 0.116 NI

5 EDTA2000 µg 1000 µg

0.104 I I

6 citrate2000 µg 0.115 NI

7 tartrate2000 µg 0.150 NI

8 anionic surfactant2000 µg 0.386 I

9 cationic surfactant2000 µg 1000 µg

0.198 0.166

I NI

10 non-ionic surfactant2000 µg 0.160 NI

11 calcium2000 µg 1000 µg

0.146 NI

72

23. DETERMINATION OF AMMONIUM

Ammonia reacts with hypochlorite to form chloramines which reacts with phenol to form indophenols blue.

Reagents

1. Sodium hydroxide (40%): Dissolve 40 g Sodium Hydroxide in water and dilute to 100 ml.

2. Phenol solution :Dissolve 11.1ml liquefied phenol and 5 g Sodium Hydroxide dilute to 100 ml.

3. Sodium Nitroprusside (0.5%): Dissolve 0.25 g Sodium Nitroprusside in 50 ml of water.

4. Alkaline Citrate: Dissolve 20 g Sodium Citrate and 1g Sodium hydroxide in water and dilute of 100 ml.

5. Sodium Hypochlorite: Dissolve 25 ml commercial Sodium Hypochlorite with 1.0ml 40% NaoH, dilute it to

100 ml.

6. Stock Ammonium Solution (1000 ppm N): Dry 5 g Ammonium Chloride at 100oC. Weigh out 0.3819 g,

dissolve in water and dilute to 100ml.

7. Standard Ammonium Solution (10 ppm N): Dilute 250 µl of 1000 ppm N solution to 25 ml.

Procedure

Pipette out o, 2, 4, 6, 8, 10 µg of 10ppm Ammonium solution into 10ml volumetric flask. Add 200 µl of Phenol, 200

µl Sodium Nitroprusside, 500 µl Alkaline Citrate and 500 µl Sodium Hypochlorite. Mix well. Let stand for 30 min.

Avoid direct sunlight. Read the absorbance in a 1 cm cuvette at 640 nm.


Cookbook value: 5 µg ammoniacal N in 10 ml gives an absorbance of 0.50 ± 0.01

73

Absorption spectrum of indophenols blue

Calibration Curve

Determination of Ammoniumy = 0.1043x - 0.021 R2 = 0.9987

-0.20

0.20.40.60.8

11.2

0 5 10 15

(µg/10 ml Ammonium N)

Abso

rban

ce

74

Important improvements made in the existing procedures

Species Modificaton Advantage(s) gained

Magnesium PVA added 80% higher sensitivity (slope of

calibration)

Mercury Phthalate buffer replaced by citrate

buffer and combined with KI and

EDTA

PVA added instead of gelatin

Much higher tolerance of interfering

species, simplified procedure and

stable reagent.

Stable reagent

Iron Alternative new method developed Cheap biodegradable natural

product reagent. Applicable to ferric

iron

6. References

1) Standard Methods for the Examination of Water and Wastewater. 20thEdition 1998 Ed. L. S. Clesceri, A. E.

Greenberg and A. D. Eaton American Public Health AssociationWashington DC.

2) P. W. West and T. P. Ramachandran (1966) Spectrophotometric Determination of Nitrate using

Chromotropic Acid. Anal. Chim. Acta, 35, 317-324.

3) K. Goto, S. Taguchi, Y. Fukuke and K. Ohta (1977) Spectrophotometric Determination of Manganese with

1-(2-pyridylazo)-2-naphthol and a nonionic surfactant. Talanta, 24, 752 – 753.

4) R. M. Dagnall, T. S. West and P. Young (1965) Determination of lead with 4-(2-pyridylazo)-resorcinol-I

Spectrophotometry and solvent extraction. Talanta, 12, 583 – 588.

Signature of the Principal Investigator

determination of arsenicfinal report of the research projectcistup.iisc.ac.in/presentations/research...

Documents