doctor of philosophy in chemistryshodhganga.inflibnet.ac.in/bitstream/10603/21/12/chand...sufiyan...

STUDIES ON NEW REAGENTS FOR THE SPECTROPHOTOMETRIC

DETERMINATION OF ANIONS, METAL IONS AND DRUGS

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN CHEMISTRY

TO

THE FACULTY OF SCIENCE AND TECHNOLOGY OF

MANGALORE UNIVERSITY

BY

Mr. CHAND PASHA

DEPARTMENT OF POST- GRADUATE STUDIES AND RESEARCH

IN CHEMISTRY


MANGALAGANGOTHRI – 574 199

INDIA

September - 2008

2

TO MY BELOVED PARENTS,

BROTHERS, SISTERS, WIFE, SUFI & MAHIN

3

CONTENTS

___________________________________________________________________________

Page No

DECLARATION i

CERTIFICATE ii

ACKNOWLEDGEMENT iii

SUMMARY vi

CHAPTER 1: INTRODUCTION TO SPECTROPHOTOMETRY 14

CHAPTER 2: SPECTROPHOTOMETRIC DETERMINATION OF IODATE 32

IN IODIZED TABLE SALT AND SEA WATER SAMPLES

CHAPTER 3: SPECTROPHOTOMETRIC DETERMINATION OF 61

HYPOCHLORITE IN ENVIRONMENTAL SAMPLES

CHAPTER 4: NEW REAGENTS FOR THE SPECTROPHOTOMETRIC 85

DETERMINATION OF VANADIUM IN ALLOYS, SYNTHETIC

AND PHARMACEUTICAL SAMPLES

CHAPTER 5: SPECTROPHOTOMETRIC DETERMINATION OF CHROMIUM 123

USING TOLUIDINE BLUE AND SAFRANINE O AS NEW REAGENTS

CHAPTER 6: SPECTROPHOTOMETRIC DETERMINATION OF ARSENIC 158

IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES

4

CHAPTER 7: SPECTROPHOTOMETRIC DETERMINATION OF SELENIUM 186

IN ENVIRONMENTAL, BIOLOGICAL AND PHARMACEUTICAL

SAMPLES.

CHAPTER 8: SPECTROPHOTOMETRIC DETERMINATION OF 213

CEPHALOSPORINS IN PHARMACEUTICAL SAMPLES

CHAPTER 9: SPECTROPHOTOMETRIC DETERMINATION OF MOSAPRIDE 247

BY DIAZOTIZATION METHOD

LIST OF PUBLICATIONS

5

DECLARATION

I hereby declare that the matter presented in this thesis is the result of

investigations carried out by me in the Department of Post-Graduate Studies and

Research in Chemistry, Mangalore University, Mangalagangothri-574 199,

Karnataka, India, under the guidance of Dr. B. Narayana, Professor of Chemistry,

Mangalore University and the same has not previously formed the basis for the award

of any degree or diploma.

Whenever the work described is based on the findings of other researchers,

due acknowledgement is made in keeping with the general practice of reporting

scientific observations. However, errors and unintentional oversights, if any are

regretted.

Place: Mangalore University

Date: 23rd

September 2008 Chand Pasha

6


DEPARTMENT OF CHEMISTRY

[Post- Graduate Studies and Research] Phone: +91-824-2287262 (O)

MANGALAGANGOTHRI – 574 199 Fax : +91-824-2287367

KARNATAKA, INDIA Email : [email protected]

_______________________________________________________________________________

Dr. B. Narayana Ref. MU/CHEM/BN/2008-09

Professor of Chemistry Date: September 2008

CERTIFICATE

This is to certify that this thesis entitled “STUDIES ON NEW REAGENTS

FOR THE SPECTROPHOTOMETRIC DETERMINATION OF ANIONS,

METAL IONS AND DRUGS” submitted by Sri. Chand Pasha to the Faculty of

Science and Technology of Mangalore University for the award of the degree of

Doctor of Philosophy in Chemistry is the result of bona fide research work carried out

by him in the Department of Post Graduate Studies and Research in Chemistry,

Mangalore University under my guidance and direct supervision. I further certify that

this thesis or part thereof has not previously formed the basis for the award of any

degree, diploma, associate ship of any other University or Institution.

Dr. B. Narayana

7

ACKNOWLEDGEMENT

It gives me a great pleasure to place on record my deep sense of gratitude and

heartfelt thanks to my supervisor Dr. B. Narayana, Professor, Department of Post-

Graduate Studies and Research in Chemistry, Mangalore University,

Mangalagangothri, who gave me a chance to work with him, extended all facilities,

sustained interest in me and provided inspiring guidance for the successful

completion of my research work. I deem it as my privilege to work under his able

guidance. I ever remain grateful to him.

I am thankful to Prof. J. Ishwara Bhat, Chairman, Department of Post

Graduate Studies and Research in Chemistry, Mangalore University for providing

me an opportunity and making available the department facilities. The inspiration,

help and suggestions received from Prof. A. M. Abdul Khader, Prof. B. Thimme

Gowda, Prof. Balakrishna Kalluraya, Dr. Suresh P Nayak, Dr. B.

Vishalakshi, Dr. Boja Poojary, Dr. Jagadeesh Prasad and all other staff members

are beyond evaluation.

I wish to express my sincere thanks to my colleague Dr. B. K Sarojini,

Professor & Head, Department of Chemistry, P. A. College of Engineering,

Nadupadav, Mangalore, for the support and constant encouragement extended

throughout the investigations.

I am thankful to Prof. Syed Akheel Ahmed, Vice Chancellor, Yenepoya

University, Mangalore, Prof. K. Basavaiah, Prof. P. G. Ramappa and the faculty

members of the Department of Post Graduate Studies and Research in Chemistry,

Mysore University for the moral support and constant encourangement extended

throughout the investigation.

I also record my appreciation to the senior researchers and fellow researchers

of our group, Dr. A. Joseph, Dr. R. A Nazareth, Dr. N. V. Sreekumar, Dr. N. G.

Bhat, Dr. Tom Cherian, Dr. M. Mathew, Dr. K. K Vijay Raj, Dr. B. V.

8

Ashalatha, Sri. K. Sunil, Smt. K. Veena, Smt. T. V. Sreevidya and Sri. S.

Samshuddin for their pleasant association and help in various forms. My thanks are

also to Dr. N. S. Rai, Sri. M. Ashok and Sri. V. Prakash for their timely help and

association.

I remain grateful to the Head, Microtron Centre of Mangalore Univesity for

permitting me to use the spectrophotometer and I thank Dr. Ganesh and Sri.

Dayanand for providing all the assistance during the spectrophotometric analysis of

the metal cations and anions.

I was fortunate enough to avail the ungrudging help and assistance from all

our laboratory and non-teaching staff particularly, Sri. M. Manohar and Sri.

Chitharanjan.

I am thankful to the librarian Dr. M. K. Bhandi, Asst. librarian Dr. N. V.

Gowda and staff of Mangalore University Library for their valuable co-operation.

I express my sincere thanks to the Director of Computer Centre, Mangalore

University, and the staff of the computer centre for the computer facilities.

I wish to express my heartful thanks to Dr. S. A. Khan, Principal, P. A.

College of Engineering, Nadupadav, Mangalore, for his continuous support and

constant encourangement extended throughout the investigation.

I also wish to express my thanks to my other colleagues Sri Abdulla K. P,

Sri. Mohd. Eliyas, Sri. U. Farroq, Smt. Suhana, Sri. Hakeem and Sri. Kiran of

P. A. College of Engineering, Nadupadav, Mangalore.

I am thankful to Dr. Mohammed Asif, Professor and Head, Department of

Biotechnology, P. A. College of Engineering, Nadupadav, Mangalore, for his support

and constant encourangement extended throughout the investigation.

9

I am grateful to Sri. Saleem Ahmed (Gust Faculty, CPDM, I.I.Sc.

Bangalore), Sri. Abhishek, Sri. Wilson (J. H. Bio, Bangalore), Smt. Sunitha (Delnet,

Delhi) and Staffs of I.I.Sc. Library, Bangalore for their support I received at various

stages of the investigations.

I also thank to my Brothers (Sri. Ghaffar Jan, Aleem Pasha and Afsar

Nadeem) for their special interest, love and affection.

At this juncture I think of my Parents, Brothers and Sisters with profound

sense of gratitude whose selfless sacrifice and their great efforts with pain and tears

and unceasing prayers has enabled me to reach the present position in life,

I express my sincere thanks to my beloved wife Mrs. Shabreen Taj and little

darlings Mohd. Sufiyan Pasha and Noor Maheen Taj, who have been a source of

inspiration to me. Her loving concern, care, understanding and sacrifice made my

task much easier. Words fail me to express the sort of heavenly benediction they have

been to me.

Finally I thank all those who have helped me directly or indirectly in the

successful completion of my thesis. Above all I humbly thank God Almighty, whose

sustaining grace has been sufficient for me to complete this endeavor.

CHAND PASHA

10

SUMMARY

In the past few decades, a number of elegant instrumental techniques are

reported which are rapid, selective and having a high degree of accuracy.

Spectrophotometric methods of analysis is an excellent tool, which is widely used for

the determination of wide variety of materials. The technique is the most commonly

used method for metal analysis. Achievement of high accuracy and precision coupled

with the cost effectiveness is the most important advantage of visual

spectrophotometry. Therefore the availability of spectrophotometer made this

technique indispensable to the modern analytical chemists.

Analytical chemistry plays a vital role in many research areas in chemistry,

biochemistry, biology, geology, and other sciences and analytical chemist has a very

important role in modern industrialized society. Thus most industries rely upon both

qualitative and quantitative chemical analysis to ensure that the raw materials used

meet certain specifications, and also to check the quality of the final product. Analysts

have developed large number of instrumental techniques and these techniques are

extremely sensitive and can yield results rapidly to a high degree of accuracy. Among

these instrumental analytical techniques, spectrophotometric technique occupies a

unique position because of its simplicity, sensitivity, accuracy and rapidity. The

availability of spectrophotometer made this technique indispensable to the modern

analytical chemists. It is the most important method for determining metals in alloys,

minerals and complexes, owing to its sensitivity and selectivity. Spectrophotometry

offers the advantage of having calibration graphs that are linear over a wide range.

These instruments are with digital readouts or connected to a computer, which

provides a high degree of accuracy and an excellent way to determine the metal ion in

parts per million levels. The limitation to its use is set by the degrees of interference

from other metal ions.

Absorption spectrophotometry in the ultra-violet and visible regions is

considered to be one of the valued techniques for the quantitative analysis. The basis

of spectrophotometric methods is the simple relationship between the color of a

substance and its electronic structure. A molecule or an ion exhibits absorption in the

visible or ultra-violet region when the radiation causes an electronic transition in

11

molecules containing one or more chromophoric groups. The colour of a molecule

may be identified by substituents called auxochromic groups, which displace the

absorption maxima towards longer wavelength (bathochromic shift). The colour

determining factors in many molecules is the introduction of conjugated double bonds

by means of electrons donor and electron acceptor groups. The importance of the

coloured solution lies in the fact that the radiation absorbed is characteristic of the

material responsible for absorption. Any soluble coloured material can be determined

quantitatively as well as qualitatively. In addition, a substance that is not coloured

may often be determined by adding a chromogenic reagent that will convert to an

intensively coloured species showing strong absorbance in the visible region. This

absorbance data can be used for the determination of metal ions and anions in the

suitable concentration range in accordance with Beer’s law. Proper selectivity can be

achieved by controlling the pH, using masking agents etc. It provides an excellent

way to determine the metal ions and anions in parts per million.

Cephalosporins are penicillinase-resistant antibiotics with significant activity

against both gram-positive and gram-negative bacteria. The key intermediate for

semisynthetic production of a large number of cephalosporins is

7-aminocephalosporanic acid, which is formed by hydrolysis of cephalosporin C

produced by fermentation. A few thousand semisynthetic cephalosporins have been

described in the scientific literature, but only a small number of these has shown

clinical importance. One of the current objectives of research on new semisynthetic

cephalosporins is the preparation of compounds with ß-lactamase resistance plus acid

stability, in order to make possible effective absorption from the gastro-intestinal

tract.

Mosapride citrate, chemically 4-amino-5-chloro-2-ethoxy-N-[[4-[(4-fluoro-

phenyl)methyl]-2-morpholinyl]-methyl benzamide citrate is a potent gastroprokinetic

drug. Gastroprokinetic agents have an important role to play in conjunction with life

style modifications in the short and long term medical management of

gastroesophageal reflux disease (GERD). The prevalence of GERD and dyspepsia is

increasing in many Asian countries. It behaves as a selective 5-HT4- receptor agonist

and enhances only upper gastroprokinetic motor activity. Now it is proposed to have

12

new analytical reagents for the spectrophotometric determination of cephalosporins

and mosapride in pharmaceutical preparations.

The present investigation is the studies on new reagents for the

spectrophotometric determination of anions, metal ions and drugs. The work included

in the thesis is divided into 9 chapters

Chapter 1 provides an introduction to spectrophotometry.

Chapter 2 describes the spectrophotometric determination of iodate using methylene

blue, rhodamine B and leuco xylene cyanol FF as chromogenic reagents. The effects

of diverse ions are studied and the method is applied for the determination of the

iodate in iodized table salt and sea water samples.

Chapter 3 deals with the spectrophotometric determination of hypochlorite using

methylene blue and rhodamine B as chromogenic reagents. The interference of

various cations and anions are studied and the method is applied for the determination

of the hypochlorite in various samples of tap water, natural water and milk.

Chapter 4 describes three new reagent for the spectrophotometric determination of

vanadium using toluidine blue, safranine O and leuco xylene cyanol FF as

chromogenic reagents. The method has been applied for the determination of

vanadium in steel, pharmaceutical, environmental and biological samples. The effect

of diverse ions is also discussed.

Chapter 5 describes the spectrophotometric determination of chromium using

toluidine blue and safranine O as reagents. The method has been applied for the

determination of chromium in steel, pharmaceutical and environmental samples. The

effect of foreign ions is presented.

Chapter 6 presents spectrophotometric determination of arsenic using toluidine blue

and safranine O as chromogenic reagents. The interference of various cations and

13

anions are studied. The method has been applied for the determination of arsenic in

various environmental and biological samples.

Chapter 7 describes the spectrophotometric determination of selenium using

toluidine blue and safranine O. The method has been applied for the determination of

selenium in environmental, biological and pharmaceutical samples. The effect of

various diverse ions is also discussed.

Chapter 8 deals with the spectrophotometric determination of cephalosporins using

variamine blue and thionin as the selective reagents. The developed method has been

successfully applied to the cephalosporins in pharmaceutical samples.

Chapter 9 presents spectrophotometric determination of mosapride by diazotization

method using sodium nitrite and coupled with acetylacetone or ethyl acetoacetate. The

developed method has been successfully applied to the determination of mosapride in

pharmaceutical samples.

14

CHAPTER 1

INTRODUCTION TO SPECTROPHOTOMETRY

1.1 SPECTROPHOTOMETRY

1.2 CALIBRATION CURVE

1.3 CHOICE OF THE WAVELENGTH

1.4 COLOR DEVELOPMENT

1.5 SENSITIVITY OF THE SPECTROPHOTOMETRIC METHODS

1.6 ACCURACY AND PRECISION

1.7 LIMITATIONS

1.8 APPLICATIONS

1.9 PRESENT INVESTIGATIONS

1.10 REFERENCES

15

1.1 SPECTROPHOTOMETRY

Analysts have developed large number of instrumental techniques and these

techniques are extremely sensitive and can yield results rapidly to a high degree of

accuracy. Among these instrumental analytical techniques, spectrophotometric

technique occupies a unique position, because of its simplicity, sensitivity, accuracy

and rapidity. Spectrophotometry is the quantitative measurement of the reflection or

transmission properties of a material as a function of wavelength [1]. While relatively

simple in concept, determining the reflectance or transmittance involves careful

consideration of the geometrical and spectral conditions of the measurement. The

national scales for reflectance and transmittance in the ultraviolet, visible and near-

infrared spectral regions are arise from 250 nm to 2500 nm. The availability of

spectrophotometer made this technique indispensable to the modern analytical

chemists.

Spectrophotometric method is the most important for determining metals in

alloys, minerals and complexes, owing to its selectivity. In comparison with atomic

emission spectroscopy, atomic absorption spectroscopy and similar techniques, it

offers the advantage of having calibration graphs that are linear over a wider range. A

very extensive range of concentration of substances (10-2

–10-8

M) may be covered.

Identifying materials based on the color was probably one of the earliest examples of

qualitative molecular absorption spectrophotometry. Also, the first recognition that

color intensity can be the indicator of concentration was probably the earliest

application of employing molecular absorption spectroscopy for quantitative

estimation. Using the human eye as the detector and undispersed sunlight or artificial

light as the light source made the first measurements, later it was found that the

accuracy and the precision could be improved by isolating specific frequencies of

light using optical filters.

Further improvement of the measurement came with the use of prism and

grating monochromators for wavelength isolation. Photoelectric detectors were soon

developed, but were quickly replaced with phototubes and photomultiplier tubes. The

development of solid state microelectronics has now made available a wide range of

detector type which are coupled with the computers, provide highly sophisticated

16

readout electronic systems. Absorption spectrophotometries in the ultra-violet and

visible regions are considered to be one of the valued techniques for the quantitative

analysis.

The basis of spectrophotometric methods is the simple relationship between

the color of a substance and its electronic structure. A molecule or an ion exhibits

absorption in the visible or ultra-violet region when the radiation causes an electronic

transition in molecules containing one or more chromophoric groups. The color of a

molecule may be intensified by substituents called auxochromic groups, which

displace the absorption maxima towards longer wavelength (bathochromic shift). The

color determining factors in many molecules is the introduction of conjugated double

bonds by means of electron donor or electron acceptor groups [2]. The quantitative

applicability of the absorption method is based on the fact that the number of photons

absorbed is directly proportional to the number or concentration of atoms, ions or

molecules. The sequence of events in a spectrophotometer is as follows:

i) The light source shines through the sample.

ii) The sample absorbs light.

iii) The detector detects how much light the sample has absorbed.

iv) The detector then converts how much light the sample absorbed into

a number.

v) The numbers are either plotted straight away or are transmitted to a

computer to be further manipulated (e.g. curve smoothing, baseline

correction)

1.1.1 Deviation from Beer’s Law

Beer’s law describes that the plot of absorbance ‘A’ against concentration, a

straight line passing through the origin should be obtained. It generally holds over a

wide range of concentrations if the structure of the colored ion or of the colored non-

electrolytes in the dissolved state does not change with concentration. The presence of

small amount of colorless electrolytes, which do not react chemically with the colored

components, normally does not affect the light absorption.

17

Large amounts of electrolytes may result in a shift of the maximum absorption

and may also change the value of the extinction coefficient. When the colored species

ionizes or dissociates or associates in solution, Beer’s law will usually not be obeyed

as the nature of species in solution will vary with the concentration. The law does not

hold good when the colored solute forms complexes, the composition of which

depends upon the concentration. Deviation from Beer’s law may also occur when

monochromatic light is not used. Electrolytes that react chemically with the colored

components affect the absorption.

1.2 CALIBRATION CURVE

The spectrophotometer requires the construction of a calibration curve for the

constituents being determined. For the purpose, suitable quantities of the constituents

are taken and treated in exactly the same way as the sample solution for development

of the color, followed by the measurement of the absorption at the optimum

wavelength. The absorbance is then plotted against concentration of the constituents.

A straight line is obtained if Beer’s law is followed. This calibration curve may then

be used in future determinations of the constituents under the same conditions. The

calibration curve needs checking at intervals.

1.3 CHOICE OF THE WAVELENGTH

It is important to avoid making measurements in a region where the molar

absorptivity (ε) changes rapidly with the wavelength. In such a region even a small

error in setting the wavelength scale will result in a large change in the apparent molar

absorptivity [3]. Therefore, it is necessary to select the wavelength corresponding to

the maximum of ε. When the transmittance of the solution increases continuously

over the wavelength range covered by the light filter, Beer’s law will not be obeyed.

1.4 COLOR DEVELOPMENT

If the development of color is linked to the concentration of a substance in

solution then that concentration can be measured by determining the extent of

18

absorption of light at the appropriate wavelength [4]. There are few elements, which

give sufficient intense absorption by themselves and are spectrophotometrically

measurable. Majority of the substances are generally determined indirectly in a

variety of ways, such as

i) Substances may be converted by a suitable reagent to an absorbing product

changing oxidation state to a colored valence state

ii) Adding complexing agent to get colored complexes and so on.

iii) Organic complexing agents are found to be more selective and sensitive

color developing agents

1.4.1 Requirements of a Color Developer

A color developer should possess a high molar absorptivity, high selectivity

and the spectrum of the complex should be significantly different from that of the

reagent.

1.4.2 The Criteria for Satisfactory Spectrophotometric Analysis

Spectrophotometric methods are often versatile in nature, in order to have

successful and satisfactory results, the process of analysis needs careful operations.

Since the color development in spectrophotometry involves diverse type of reactions,

a number of points need to be ensured before applying the method for a particular

application. Some of the points have to be considered are discussed in the following

sections.

1.4.3 Specificity of the Color Reactions

A very few reactions are specific for a particular substance, but may give

colors for a small group of related substances only. Because of this selective character

of many colorimetric reactions, it is important to control the operational procedure so

that the color is specific for the component being determined. This may be achieved

by isolating the substance by the normal methods of inorganic analysis. But these

separation methods are often tedious and time consuming. Further there is every

possibility of appreciable loss of the analyte during these separations.

19

The specificity in colorimetric reactions can be achieved by introducing other

complex forming compounds. These are required to suppress the action of interfering

substance by formation of complex ions or of non-reactive complexes. When the

colorimetric reaction takes place within well-defined limits of pH, adjustment of pH

may also sometimes help to achieve the desired specificity in certain cases. The

methods of selective absorption, chromatographic separations and ion exchange

separations are also of use in certain cases.

Solvent extraction method also finds its application in achieving specificity in

the spectrophotometric determinations. The interfering substances are removed by

extraction with an organic solvent, sometimes after suitable chemical treatment.

Alternatively the substance to be determined can also be isolated from the interfering

species by converting it into an organic complex, which is then selectively extracted

into a suitable organic solvent.

1.4.4 Proportionality between Color and Concentration

For colorimeters, it is important that color intensity should increase linearly

with concentration of the compound to be determined. This is not necessary for

photoelectric colorimeters or spectrophotometers. Since a calibration curve may be

constructed relating the instrumental reading of the color with the concentration of the

solution. It is desirable that the system follows Beer’s law even when photoelectric

colorimeters are used.

1.4.5 Stability of the Color and Clarity of the Solutions

The color produced must be stable so as to allow accurate readings to be

taken. The period over which maximum absorbance remains constant must be long

enough for precise measurement to be made. Stability of the color is influenced by

experimental conditions like temperature, pH etc. The solution must be free from

precipitate if comparison is to be made with a clear standard. Turbidity scatters as

well as absorbs the light.

20

1.4.6 Reproducibility and Sensitivity

The colorimetric procedure must give reproducible results under specific

experimental conditions. The reaction need not necessarily represent a

stoichiometrically quantitative chemical change. It is desirable, particularly when

minute amounts of substances are to be determined, that the color reactions be highly

sensitive. It is also desirable that the reaction product absorbs strongly in the visible

rather than in the ultraviolet region, as the interfering effect of other substances is

usually more pronounced in the ultraviolet region.

1.5 SENSITIVITY OF SPECTROPHOTOMETRIC METHODS

The sensitivity is often described in terms of the molar absorptivity (ε, Lmol-1

cm-1

)

of the metal ligand complex. The awareness of the sensitivity is very important in

spectrophotometric determination of trace metals. The numerical expression [5-7] is the

molar absorptivity (εmax

) of the colored

species.

ε =

A

c l

Sensitivity depends on the monochromaticity of the radiation. With

monochromatic light of very narrow bandwidth corresponding to the wavelength of

λmax

, the maximum value of molar absorptivity is obtained.

Savvin [8] suggested a relation between sensitivity and molar absorptivity. He

suggested the following criteria for describing the sensitivity.

Low sensitivity, ε < 2 × 104

, Lmol-1

cm-1

Moderate sensitivity ε = 2 – 6 × 104

, Lmol-1

cm-1

High sensitivity ε > 6 × 104

, Lmol-1

cm-1

It is generally stated [9] that the molar absorptivity will not exceed

approximately 105

. Other ways of specifying sensitivity are as specific absorptivity

[10] or the Sandell’s sensitivity [11]; both methods give the sensitivity in terms of

mass of analyte per unit volume of solution. Such an approach is perhaps more

convenient than using molar absorptivities as a basis of comparison. The Sandell’s

21

sensitivity is the concentration of the analyte (in µgmL-1

) which will give an

absorbance of 0.001 in a cell of path length 1 cm and is expressed as µgcm-2

.

Organic reagents with high molecular weights furnish maximum sensitivity if

used as chromogenic agents. Detection limits can be reduced to somewhat by solvent

selection because molar absorptivities depend on the solvent system. Another

technique used to increase the detection limit is to use indirect determinations, where

a stoichiometric gain in the number of chromophores may result or the newly formed

chromophore may have a higher molar absorptivity. Reaction rate methods can

sometimes have lower detection limits than do conventional spectrophotometric

measurements.

1.6 ACCURACY AND PRECISION

The accuracy and precision of spectrophotometric method depends on three

major factors.

i) Instrumental limitations

ii) Chemical variables

iii) Operator’s skill.

Instrumental limitations are often determined by the quality of the instruments,

optical, mechanical and electronic systems. Chemical variables are determined by

purity of standards, reagents and chromophore stability, reaction rates, reaction

stoichiometry, pH and temperature control. These factors are usually determined by

the methodology chosen for the analysis. Under ideal conditions it is possible to

achieve relative standard deviations in concentrations as low as about 0.5%, which

enables the determination of microquantities of components. The precision of

spectrophotometric method also depends on concentration of the determinant. Visual

methods generally give results with a precision of 1-10 %. The precision of the

photometric method is of course, higher and varies from 0.5–2 % under suitable

measuring conditions.

22

Precision describes the reproducibility of results where accuracy denotes the

nearness of a measurement to its accepted value. The precision attainable is a function

of the absorbance measured. The error observed is, as expected, very large on lower

side of concentrations. When intensely colored solutions are measured, only an

insignificant part of the radiation is transmitted and on the logarithmic absorbance

scale the gradations are so close that the reading error is very high.

Precision is conveniently expressed in terms of the average deviation from the

mean or in terms of standard deviation. When applied to small sets of data with which

the analytical chemists work, the standard deviation is the most reliable estimate of

the indeterminate uncertainty. When the standard deviation turns out to be

approximately proportional to the amount present in the formation on the precision

can be expressed in percent by using the coefficient of variation. Mathematical

equation for the coefficient of variation is

C.V =_

100 x s

x

s = Standard deviation and

_

x = Arithmetic mean of a series of measurements.

Standard deviation is given by

n-1

s = (x - x )

2

1.6.1 Detection Limit

Detection limit is the smallest concentration of a solution of an element that

can be detected with 95 per cent certainty [12, 13]. This is the quantity of the element

that gives a reading equal to twice the standard deviation of a series of any least ten

determinations taken with solutions of concentrations, which are close to the level of

the blank. Several approaches for determining the detection limit are possible,

depending on whether the procedure is a non-instrumental or instrumental. Based on

the standard deviation of the blank samples and the slope of the calibration curve of

the analyte, the detection limit (DL) may be expressed as:

23

S

3.3D

L =

Where = the standard deviation of the reagent blank

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte. The

1.6.2 Quantitation Limit

The quantitation limit is generally determined by the analysis of samples with

known concentrations of analyte with those of blank samples and by establishing the

minimum level at which the analyte can be quantified with acceptable accuracy and

precision [14, 15]. Based on the standard deviation of the blank samples and the slope

of the calibration curve of the analyte, the quantitation limit (QL) may be expressed

as:

S

QL =

10

where = the standard deviation of the reagent blank

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte. The

1.6.3 Comparison of the Results

The comparison of the values obtained from a set of results with either (a) the

true value or (b) other sets of data makes it possible to determine whether the

analytical procedure has been accurate and / or precise, or if it is superior to another

method. There are two common methods for comparing results: (a) Student’s t-test

[16,17] and (b) the variance test (F-test).

These methods of test require knowledge of what is known as the number of

degrees of freedom. In statistical terms this is the number of independent values

24

necessary to determine the statistical quantity. Thus a sample of n values has n

-

_

x )2

is considered to have n-1 degree of

freedom, as for any defined value of

_

x only n-1 values can be freely assigned, the

nth being automatically defined from the other values.

1.6.4 (a) Student’s t-test

Student’s t-test is used for small samples; its purpose is to compare the mean

from a sample with some standard values and to express some level of confidence in

the significance of the comparison [17]. It is also used to test the difference between

the means of the two sets of data x

1 and x

2.

t =

_

x( ) n

s

Where s = Standard deviation,

_

x = Arithmetic mean of a series of

measurements, µ is the true value and n is the number of trials of the measurements.

It is then related to a set of t-tables [16,17] in which the probability p of the

t-value falling within certain limits is expressed, either as a percentage or as a

function of unity, relative to the number of degrees of freedom.

1.6.5 Comparison of the Means of Two Samples.

(a) t – test

This method is also used to compare the values of the mean and precision of

the test method with those of the reference methods [16, 17]. The value of t when

comparing two sample means x

1 and x

2 is given by the expression:

Sp1

n2

1

n1

t =

x1

x2

where SP

is the pool standard deviation, is calculated from the two standard

deviation s1 and s

2 as follows :

25

(n1-1)s

1

2

+(n2-1)s

2

2

n1+n

2-2

Sp =

n1 and n

2 are the number of trials of first and second method.

(b) F–test (Variance Ratio Test)

F-test is used to compare the precisions of two sets of data of two different

analytical methods are calculated from the following equation [16, 17]:

F =

sB

2

sA

2

The larger value of s is always used as the numerator so that the value of F is

always greater than unity. The value obtained for F is then checked for its significance

against values in the F–table calculated from an F–distribution [16,17] corresponding

to the numbers of degrees of freedom for the two sets of data.

1.7 LIMITATIONS

The common but unrecognized problem in measuring the absorbance is stray

light error. All wavelength isolation devices tend to produce some low intensity

radiations at wavelengths other than the desired one. This is usually due to the optical

imperfections, or simply from scattered light due to dust particles on optical surface.

Because one has usually selected a wavelength at which the compound of interest

absorbs most strongly, the stray light falling on the sample is of wavelengths at which

the compound does not absorb strongly. Thus the stray light errors will result in a

negative bias for absorbance readings which can be represented in the equation

Ttrue

+ p

Tobs

=

1 + p

The fraction of all the light coming from the wavelength isolation device,

which is stray light, and Tobs

and Ttrue

are the observed and true transmittances,

26

respectively. Normally the absolute amount of stray light tends to be relatively

constant with respect to the wavelength. But the fraction of stray light is highly

wavelength dependent because the amount of energy of the selected wavelength

depends on the source intensity at that wavelength. Thus, stray light errors are most

predominant at long and short wavelengths and when high absorbance are measured.

When the measurement is made, finite slit width effect is a common error that we

frequently encountered with. The exit slit of the monochromator subtends a portion of

the dispersed continuum from the grating or prism. If any light is to pass through the

slit it must have a finite width. However, due to its width, more than one wavelength

of light, called the bandwidth, emerges.

Although most of the energy emerging from the slit is of selected or nominal

wavelength, a small percentage is of adjacent wavelengths, called spectral bandwidth.

This is simply the wavelength span, centered on the nominal wavelength, containing

75% of the radiant energy emerging from the slit. Thus, the narrower the spectral

band width, the better conformity to Beer’s law. If the spectral bandwidth is too wide,

negative deviation from the Beer’s law occurs, resulting in a false absorbance reading.

Errors also occur when distilled water blank is used instead of a true blank for 100%

transmittance or baseline reading. Even though there are no known absorbing species

in distilled water as well as in the blank reagent solution, the difference in the

refractive indices between the sample solution and the reference solution must be kept

reasonably close or reflective loses at the cell windows may not be the same.

When the incident light is highly collimated and falls on the cell window at

normal incidence, a small fraction of the light is reflected back at each interface where

there is a refractive index difference, at the two air-window interfaces, and the two

window-solution interfaces. Because the sample and the reference cells are of the

same composition, reflections from the air-window interfaces are compensated for.

However, reflections from the solution-window interfaces may be different if the

refractive indices of the sample and the blank are not nearly the same.

27

1.8 APPLICATIONS

The greatest use of spectrophotometry lies in its application to quantitative

measurements. The reasons for this stem from the ease with which most

spectrophotometric measurements can be made, their sensitivity and precision, and

the relatively low cost of instrument purchase and operation. A variety of techniques

have been developed for different types of samples. Direct determinations are made

when the analyte molecule contains a chromophore, thus allowing the direct

measurement of its absorbance. Standards must be used to determine the absorptivity

so that concentration can be calculated by using the equations or by establishing a

calibration plot from which the concentration can be determined by graphic

interpretation or by regression analysis. Indirect determinations are commonly used

when the analyte molecule does not contain a suitable chromophore. In this instance

the analyte is made to quantitatively react with a molecules containing a chromophore

and correlating the diminution of absorbance with the concentration of the analyte or

by reacting with a reagent, which produces a chromophoric groups.

Spectrophotometric analysis continues to be one of the most widely used

analytical techniques available. Many methods are available for a variety of analytes

(such as colored, colorless, natural, synthetic, inorganic and organic analytes) and

sample types ranging from in-situ biological assays to the determination of trace

elements in steels. Many medical diagnostic test kits use photometric measurements.

Diabetics commonly use blood-glucose analysis kits based on the glucose oxidase

enzyme reaction that secondarily produces a colored product. In the food industry,

winemakers have long recognized the effect of iron levels on the taste of wines and

consequently are one the largest users of 1, 10-phenanthroline for determining iron

spectrophotometrically. A common field test for chlorine in swimming pools and

drinking water is based on the color produced by the action of chlorine on o-tolidine.

Many compilations of methodology for a variety of analytes and sample types

that are regularly updated are available [18-20]. Other general sources for

spectrophotometric analysis are commonly consulted and found helpful [21-23].

Methods specific for metals [24], and nonmetals [25] should be consulted when

dealing with these analytes. Standard methods specific to certain industries and areas

28

of study are very useful sources when specific sample types are being considered,

such as water and wastewater [26] and pharmaceuticals [27].

1.9 PRESENT INVESTIGATIONS

The work presented in chapters 2–9 deals with new reagents for the

spectrophotometric determination of iodate, hypochlorite, vanadium, chromium,

arsenic, selenium, cephalosporins and mosapride. Chapter 2 describes the

spectrophotometric determination of iodate using methylene blue, rhodamine B and

leuco xylene cyanol FF as chromogenic reagents. Chapter 3 deals with the

spectrophotometric determination of hypochlorite in environmental samples using

methylene blue and rhodamine B as chromogenic reagents. Again chapter 4 describes

the spectrophotometric determination of vanadium in alloy, synthetic and

pharmaceutical samples using toluidine blue, safranine O and leuco xylene cyanol FF

as chromogenic reagents. In chapter 5 the spectrophotometric determination of

chromium using toluidine blue and safranine O as new reagents is explained.

Chapter 6 presents spectrophotometric determination of arsenic in environmental and

biological samples using toluidine blue and safranine O as chromogenic reagents.

Chapter 7 consists of the spectrophotometric determination of selenium in

environmental, biological and pharmaceutical samples using toluidine blue and

safranine O as reagents. Chapter 8 deals with the spectrophotometric determination

of cephalosporins in pharmaceutical samples using variamine blue and thionin as the

selective reagents. Chapter 9 presents the spectrophotometric determination of

mosapride by diazotization method using sodium nitrite and coupled with

acetylacetone or ethyl acetoacetate. The structures of the reagents used are given

below.

29

SL. NO REAGENT STRUCTURE

1. Methylene blue

N

S+

Cl -

N(CH3

)2

(CH3

)2

N

2. Rhodamine B

(H5C

2)2N N(C

2H

5)2

COOH

Cl-�O

+

3. Xylene cyanol FF

SO3Na

SO3H

CH3

CH3

NHHN

CH3

CH3

4. Toluidine blue

N

S

+

CH3

NH2

(CH3)2N

Cl

-

5. Safranine O

N

N

+

CH3

NH2

NH2

CH3

Cl

-

6.

Variamine blue

NH

NH2

H3CO

7.

Thionin

S

N

N

+

H2

NH2

Cl-�

8. Acetylacetone

CH3

O O

CH

3

9. Ethyl acetoacetate OO

C2H

5OCH

3

30

1.10 REFERENCES

1. P. Dehahay, “Instrumental Analysis”, The Macmillan Company, New York

(1967).

2. W. J. Blaedel and V. M. Meloche, “Elementary Quantitative Analysis - Theory and

Practice”, 2nd

Edn., Harper and Row, New York, (1964).

3. I. M. Kolthoff and E. B. Sandell, “Text Book of Quantitative Inorganic Analysis”,

3rd

Edn., The Macmillan Company, New York, (1965).

4. Experimental Biosciences, Introductory Lab., Rice University, Houston, (2005).

5. A. B. Blank, Z. Anal. Chem., 17 (1962) 1040.

6. I. S. Mustafin, Zavod. Lab., 28 (1962) 664.

7. I. E. Banney, Talanta, 14 (1967) 1363.

8. S. B. Savvin, Crit. Rev. Anal. Chem., 8 (1979) 55.

9. H. Muller, Anal. Chem., 13 (1982) 313.

10. A. H. Ayres and B. D. Narang, Anal. Chem. Acta, 24 (1961) 241.

11. E. B. Sandell, Colourimetric Determination of Traces of Metals, 3rd

Edn., Inter

Science, New York, (1959) 83.

12. J. M. Green, Anal. Chem., News & Features, (1996) p.305.

13. B. Renger, H. Jehle, M. Fischer and W. Funk, J. Planar Chrom., (1995) p.269.

14. J. Vessman, J. Pharm. Biomed. Anal., 14 (1996) 867.

15. D. Marr, P. Horvath, B. J. Clark and A. F. Fell, Anal. Proceed., 23 (1986) 254.

16. D. A. Skoog, D. M. West and F. J. Holler, Fundamentals of Analytical Chemistry,

Saunders College Publishing, Philadelphia, 7th

Edn., (1996).

17. G. H. Jeffery, J. Bassett, J. Mendham and R. C. Denney, Vogel’s Text Book of

Quantitative Chemical Analysis, 6th

Edn., (2000).

18. R. A. Storer, Annual Book of ASTM Standard (66 volumes in 16 sections),

American Society for Testing and Materials, Philadelphia, 1987.

19. S. Williams, Edn., Official Methods of Analysis of the Association of Official

Analytical Chemists, 16th

Edn., Arlington, 1995.

20. L. G. Hargis, J. A. Howell and R. E. Sutton, Anal. Chem., 68 (1996) 169.

21. D. Eckroth, Edn., Encyclopedia of Chemical Technology, 3rd

Edn., Wiley, New

York, (1984).

22. L. C. Thomas and G. J. Chamberlin, Colorimetric Analytical Methods, 9th

Edn.,

Tintometer press, Salisbury, England, (1980).

31

23. Z. Marczenko, Separation and Spectrophotometric Determination of Elements; E.

Horwood, Halsted Press: Chichester New York, 1986.

24. F. D. Snell, Photometric and Fluorimetric Methods of Analysis, part 1 & 2,

Wiley, New York, (1978).

25. D. F. Boltz and J. A. Howell, Colorimetric Determination of Nonmetals, 2nd

Edn., Wiley, New York, (1978).

26. American Public Health Association, American Water Works Association and

Water Environment Federation, Standard Methods for the Examination of

Water and Wastewater, 21st

Edn., Wasington, D.C, (2005).

27. United States Pharmacopoeial Convention, United States Pharmacopoeia, 23rd

Rev., New York, (1995).

32

CHAPTER 2

SPECTROPHOTOMETRIC DETERMINATION OF IODATE IN IODIZED TABLE

SALT AND SEA WATER SAMPLES

2.1 INTRODUCTION

2.2 ANALYTICAL CHEMISTRY

2.3 APPARATUS

2.4 REAGENTS AND SOLUTIONS

2.5 PROCEDURES

2.6 RESULTS AND DISCUSSION

2.7 APPLICATIONS

2.8 CONCLUSIONS

2.9 REFERENCES

33

2.1 INTRODUCTION

Iodine appears to be the trace element essential to plants and animal. Iodine

occurs naturally not only as iodide but also as iodate in the form of minerals such as

lautarite Ca(IO3)2

and dietzeite 7Ca(IO3)28CaCrO

4. Iodine is an essential part of the

thyroid hormones that play an important role in the development of brain function and

cell growth. Deficiency of iodine causes serious delay in neurological development.

On the other hand, an excess of iodine or iodide can cause goiter and hypothyroidism

as well as hyperthyroidism [1]. Table salt is iodized by iodate as a source of iodine, in

order to prevent iodine deficiency. The recommended concentration of the iodate in

the salt is 40 ppm [2]. Truesdale et al. reported that the iodate is also present in sea-

water in the range 0--1

[3]. Iodine essentially, in the iodine cycle iodo-methane

and other iodo-alkanes are produced in near surface water of the sea and pass into the

atmosphere where the iodine forms a number of iodine compounds. These are taken

into cloud water where, after several reactions, the iodine appears in rainwater at

ground level as iodate and iodide-iodine. The iodate and iodide-iodine are then

involved in other cycles within soil, lake and seawater, which involve uptake and

regeneration from plant tissue.

Several isotopes of iodine, an element with multiple oxidation states, are of

radiological health importance in relation to nuclear weaponry and the nuclear fuel

cycle. 131

I has been a major concern in fall out from atmospheric nuclear testing,

releases from fuel reprocessing and the Chernobyl accident. However, due to the

short half life of 131

I (8 days), its environmental fate is determined more by decay time

than geochemical processes. In contrast, another fission product 129

I has a half life of

1.6-107 years. Long-term (105–109 years) models of radionuclide release to the

biosphere from high-level waste repositories show 129

I to contribute a significant

fraction to the population dose [4].

Iodate is more stable than iodide and most health authorities preferentially

recommend iodate as an additive to salt for correcting iodine deficiency. Even though

-1

, doubts have recently been raised

whether the safety of iodate has been adequately documented. In humans and rats,

oral bioavailability of iodine from iodate is virtually equivalent to that from

34

iodide [5]. When given intravenously to rats, or when added to whole blood or tissue

homogenates in vitro or to foodstuff, iodate is quantitatively reduced to iodide by

nonenzymatic reactions and thus becomes available to the body as iodide. Therefore

except for the gastrointestinal mucosa, exposure of tissues to iodate might be minimal.

At much higher doses given intravenously (i.e., above 10 mgkg-1

), iodate is highly

toxic to the retina. Ocular toxicity in human has occurred only after exposure to doses

of 600 to 1200 mg per individual. Oral exposures of several animal species to high

doses, exceeding the human intake from fortified salt by orders of magnitude, pointed

to corrosive effects in the gastrointestinal tract, hemolysis, nephrotoxicity, and hepatic

injury. The studies do not meet current standards of toxicity testing, mostly because

they lacked toxicokinetic data and did not separate iodate specific effects from the

effects of an overdose of any form of iodine. With regard to tissue injury, however,

the data indicate a negligible risk of the small oral long-term doses achieved with

iodate-fortified salt. Genotoxicity and carcinogenicity data for iodate are scarce or non

existing. The proven genotoxic and carcinogenic effects of bromate raise the

possibility of analogous activities of iodate. However, iodate has a lower oxidative

potential than bromate, and it did not induce the formation of oxidized bases in DNA

under conditions in which bromate did, and it may therefore present a lower genotoxic

and carcinogenic hazard. This assumption needs experimental confirmation by proper

genotoxicity and carcinogenicity data. These in turn will have to be related to

toxicokinetic studies, which take into account the potential reduction of iodate to

iodide in food, in the intestinal lumen or mucosa, or eventually during the liver

passage.

The element iodine is dissolved in sea water at concentrations of about

0.45 mM with some as iodide in near surface waters, but largely as iodate in deeper

ones. It has been assigned a quasi-nutrient status because of the similarity in the

distribution of nitrate, phosphate and iodate. In disproportionation iodine [6] in a

given oxidation state produces compounds with both higher and lower oxidation

states. For example, hypoiodous acid forms iodate and iodide. This reaction has been

studied because of its possible importance in formation of iodide and iodate in

rainwater and at the sea-surface. The reaction is also of incidental interest in

decreasing the volatility of radio iodine present in fission reactors at the time of

35

catastrophic failure. Thus, when alkaline waters are sprayed into the reactor any

iodine present converts to iodate and iodide, which are both non-volatile. Of course,

without such measures the radio-iodine will escape as a cloud from the ruptured

reactor and is able to contribute to the formation of thyroid cancer.

Iodate is the thermodynamically stable species of iodine in seawater [7,8].

However, iodide has been shown to be the dominant iodine species in many marine

surface waters [9,10]. Some postulated mechanisms for the observed abundance of

iodide in surface seawaters and other systems include inorganic reducing agents such

as bisulfide [11], sulfite [12] or ferrous iron [13]. Other studies suggest that the

reduction of iodate to iodide may be microbial enzymatically mediated in soils and

water. Tsunogai and Sase [14] and Hackett [15] using sterilized seawater media

spiked with iodate and amended with bacteria possessing the enzyme nitrate

reductase, observed increases in iodide accompanied by decreases in iodate in the

amended samples. It was further noted by Hackett, that the iodate reduction only

occurred at low (<0.25 mM) oxygen levels.


Several methods have been reported for the determination of iodate, such as

GC-MS [16], ion-chromatography [17], chemiluminescence [18], flow injection-

amperometry [19], potentiometric titrations [20], differential pulse-polarography [21],

spectro-fluorimetry [22], flow injection- spectrophotometry [23, 24], coulometry [25],

photometric analysis [26, 27] and gravimetry [28].

Fuchs et al. reported the spectrophotometric determination of iodate and

iodide with p-aminophenol [29]. Salinas et al. reported 2-oximinodimedone

dithiosemicarbazone as a reagent for the spectrophotometric determination of iodate

and bromate [30]. The reagent produced colored solution with iodate and bromate

ions acid medium. Beer’s law was obeyed in the concentration range 0.24–5.00

of iodate and 0.16– of bromate. The maximum absorption was at

400 nm. Reagents such as 3,4-dihydroxybenzal-dehydeguanylhydrazone(3,4-DBGH)

[31], 1,3-diphenyl-3-hydroxyamino-1-pro-panoneoxime [32], dithizone [33],

36

isonicotinicacidhydrazide and 2,3,5-triphenyl tetrazoliumchloride [34] and N, N’-

-hydroxypropyl)-o-phenylene diamine [35] were also used for the

spectrophotometric determination of iodate.

In some of the other spectrophotometric methods iodate was determined after

prior oxidation to periodate [36, 37]. The ion-associate of periodate with a suitable ion

pairing agent was then extracted into an organic solvent and determined by

spectrophotometric methods. Most of the proposed methods are either not sensitive

enough, or require complicated and expensive instruments, or are time consuming.

Kamburova reported iodonitrotetrazolium chloride [38] as a new reagent for

the spectrophotometric determination of iodate and periodate. The calibration plot

was linear within the range of 0.02--1

of IO3

-

or IO4

-

.

Rosa Lina et al. [39] reported a new method for the determination of iodate in

table salt. Potassium iodate in table salt was spectrophotometrically determined at two

well defined UV absorption maxima (352 and 288 nm), after being converted to I3

-

by

reaction with iodide in the presence of phosphoric acid. The molar absorptivity of the

methods were found to be 7.320×104

and 1.103×105

Lmol-1

cm-1

at 352 and 288 nm

respectively at 22°C. Typical results of 37.39 (±0.15) and 63.67 (±0.16) mg KIO3 per

Kg of salt were obtained with samples of 0.15-0.21 g, comparable with results from a

standard.

Wei-Xing et al. reported rhodamine-6G as a reagent for the

spectrophotometric determination of iodate in table salt [40]. It was based on color

reaction of rhodamine-6G with iodate and potassium iodide in HCl medium. The

maximum absorption of the reaction product was exhibited at 560 nm and Beer's law

was obeyed within the concentration range of 0-2.0 mgL-1

of KIO3 with the regression

coefficient r=0.9998.

Weixing described a simple and sensitive spectrophotometric method for the

determination of micro amounts of iodate in table salt [41]. The method was based on

chromogenic reaction of crystal violet on I3

-

produced from iodate reacting on KI in

HCl medium. Kang et al. used 3, 3’, 5, 5’-tetramethylbenzidine(TMB) [42] as a new

37

reagent for the spectrophotometric of iodate in table salt. In the acid medium, TMB

was oxidized by iodate. The yellow imine formed showed strong absorption at 450

nm with an apparent molar absorptivity 2.13×105

Lmol-1

cm-1

. Beer's law was obeyed

in the range 0-0.7 mgL-1

for iodate.

Ensafi and Dehaghi described a simple and accurate procedure for

simultaneous spectrophotometric determinations of iodate and periodate in aqueous

media [43]. In this method periodate and iodate react with iodide to produce iodine,

which was determined by spectrophotometric detection at 349 nm. The stream was

treated with iodide and sulfuric acid and then passed through the flow cell of the

spectrophotometer. The increase in absorbance at 349 nm was due to periodate and

iodate. The influences of the acid concentration, reagent concentration and manifold

variables were studied. The effect of diverse ions on the determination of periodate

and iodate by the proposed method was also investigated. Within the detection limit

(3 /s) was 3.5×10-6

M for periodate and 1.0×10-6

M for iodate, respectively. Iodate

and periodate in artificial fresh-water samples were determined by this method.

Afkhami and Zarei reported a spectrophotometric determination of periodate

and iodate by differential kinetic method [44]. The method was based on their

reaction with iodide in the presence of methylene blue. The reactions can be

monitored spectrophotometrically by measuring the decrease in absorbance at

665 nm. Two sets of conditions were established. In the first set of conditions only

periodate reacted with iodide but in the other set both the ions reacted with iodide

during the first 180 s after the initiation of the reaction. The data were evaluated by

proportional equations. The method was allowed the determination of periodate and

, respectively.

The method was applied to the determination of periodate and iodate in tap water and

spring water with satisfactory results. Afkhami et al. also described

spectrophotometric determinations of periodate, iodate and bromate based on the

reaction with iodide ion at different pH values [45].

Afkhami and Mosaed described a simple, precise, sensitive and accurate

method for rapid determination of trace quantities of iodate [46]. The method was

based on the accelerating effect of iodate on the reaction of bromate and chloride acid

38

in the presence of hydrazine in acidic medium. The decolorization of methyl orange

with the reaction products was used to monitor the reaction spectrophotometrically at

525 nm. Iodate was determined in the concentration ranges of 0.03-1.2 µgmL-1

. The

relative standard deviation for ten replicate determinations of 0.3 µgmL-1

of iodate

was 1.65 %. The reported method was applied to the determination of iodate in table

salts with satisfactory results.

Afkhami and Zarei reported a simultaneous kinetic spectrophotometric

determination iodate–bromate by the H-point standard addition method (HPSAM)

[47]. The method was based on the difference between the rates of their reactions with

iodide in acidic medium. The results showed that simultaneous determinations could

be performed with the ratio 15:1–1:15 for iodate–bromate. The proposed method was

successfully applied to the simultaneous determination of iodate–bromate in water

and synthetic samples.

Barzegar et al. used molybdosilicic acid blue as a reagent for the kinetic

spectrophotometric determination of trace amounts of iodate in table salt and water

[48]. Xu et al. describes a method for the determination of iodate was developed by

reversed-phase high-performance liquid chromatography [49] with UV detection.

Iodate was converted to iodine, which was separated from the matrix using a

reversed-phase Ultrasphere C18

column (250×4.6 mm, 5 m) with methanol (1M)

H3PO

4 (1:4) as mobile phase at 1.00 mLmin

–1

and UV detection at 224 nm. The

calibration graph was linear from 0.05 gmL–1

to 5.00 gmL–1

for iodine with a

correlation coefficient of 0.9994 (n=7). The detection limit was 0.01 gmL–1

. The

method was successfully applied to the determination of iodate in iodized salt. The

recovery was from 96% to 101% and the relative standard deviation was in the range

of 1.5% to 2.9%.

Dian-Wen et al. described a method for the spectrophotometric determination

of iodine based on the decoloration of arsenazo-III by iodate in H2SO4 medium [50].

The maximum absorption was at 530 mm. Beer's law was obeyed from 0-4 mgL-1

for

iodine. The apparent molar absorptivity was 2.07×103

Lmol-1

cm-1

. This method was

used for the determination of iodine in celery salt with satisfactory results.

39

Ghasemi et al. used pyrogallol red reagent for the simultaneous kinetic

spectrophotometric determination of iodate and periodate in sulfuric acid medium

[51]. The reaction was monitored spectrophotometrically by measuring the decrease

in absorbance of pyrogallol red at 470 nm. The calibration curve was linear over the

concentration ranges of 0.1–12 and 0.1–14 for iodate and periodate,

respectively. The experimental calibration matrix for partial least squares (PLS) and

orthogonal signal correction (OSC–PLS) method was designed with 35 mixtures.

Abbas et al. reported a simple kinetic spectrophotometric method for the

simultaneous determination of binary mixtures of iodate and bromate in water

samples [52]. The method was based on the mean centering of ratio kinetic profiles,

allows rapid and accurate determination of bromate and iodate. The analytical

characteristics of the method such as detection limit, accuracy, precision, relative

standard deviation and relative standard error for the simultaneous determination of

binary mixtures of iodate and bromate were calculated. The results show that the

method was capable of simultaneous determination of 0.05--1

each of

iodate and bromate. The results allowed simultaneous determination with the ratio

30:1-1:30 for iodate-bromate. The proposed method was successfully applied to the

simultaneous determination of iodate and bromate in several water samples.

Ghasemi et al. reported a simultaneous spectrophotometric determination of

iodate and iodide by partial least squares regression (PLS) using original and derivate

data named as absorbance and rate data [53]. The method was based on the catalytic

effect of the cited anions on the reaction rate between Ce(IV) and As(III) in 2 M

sulfuric acid medium. The Savitzky-Golay convolution method is used for calculating

and smoothing the rate data. Results show that PLS is an excellent calibration method

to resolve the mixtures of two anions by first-order or pseudo first-order kinetic

procedures without any previous knowledge about rate constant values. The 26

calibration solutions were made of iodide and iodate in the range of 10-48 and

55-235 ngmL . The application of the method was confirmed by the analysis of these

anions in real matrix samples.

Chen et al. described a non-suppressed ion chromatography (IC) with

inductively coupled plasma mass spectrometry (ICP-MS) for simultaneous

40

determination of trace iodate and iodide in seawater [54]. An anion-exchange column

was used for the separation of iodate and iodide with an eluent containing 20 mM

NH4NO

3 at pH 5.6, which reduced the build-up of salts on the sampler and skimmer

cones. The influences of competing ion (NO3

) in the eluent on the retention time and

detection sensitivity were investigated to give reasonable resolution and detection

limits. Linear plots were obtained in a concentration range of 5.0-500-1

and the

detection limit was 1.5-1

for iodate and 2.0-1

for iodide. The proposed

method was used to determinate iodate and iodide in seawaters without sample pre-

treatment with exception of dilution.

Cherian and Narayana described a simple spectrophotometric method for the

determination of iodate in table salt samples using thionin and azure B [55]. The

method was based on the reaction of iodate with potassium iodide in an acid medium

to liberate iodine. The liberated iodine bleaches the violet colour of thionin or azure

B, which are measured at 600 and 644 nm, respectively. This decrease in absorbance

is directly proportional to the initial iodate concentration and obeys Beer’s law in the

range of 1–12 µgmL–1

of iodate with thionin and 0.2–16 µgmL–1

of iodate with azure

B. The molar absorptivity and Sandell’s sensitivity of the methods using thionin and

azure B were found to be 2.7×104

Lmol–1

cm–1

, 7.9×10–2

µgcm–2

and 2.06×104

Lmol–1

cm–1

, 0.85×10-2

µgcm–2

respectively. The proposed method was successfully

used for the determination of iodate in table salt samples. Most of the proposed

methods are either not sensitive enough or required complicated and expensive

instruments, or are time consuming, or provide high detection limits. Thus the need

for a simple and sensitive spectrophotometric method for the determination of iodate

is therefore clearly recognized.

The present chapter describes simple and sensitive spectrophotometric method

for the determination of iodate using methylene blue, rhodamine B and leuco xylene

cyanol FF(LXCFF) as new reagents. The proposed method has been successfully

applied for the determination of iodate in table salt samples and sea water samples.

41

2.3 APPARATUS

A Secomam Anthelie NUA 022 UV-Visible spectrophotometer with 1

cm quartz cell was used. A WTW pH 330 pH meter was used.


All chemicals were of analytical reagent grade or chemically pure grade and

distilled water was used throughout the study. Iodate stock solution (1000 µgmL-1

)

was prepared by dissolving 1.2220 g of potassium iodate in 1000 mL of water and

standardized using standard sodium thiosulphate solution [28]. The following reagents

were prepared by dissolving appropriate amounts of reagents in distilled water:

methylene blue (0.01 %), rhodamine B (0.01 %), leuco xylene cyanol FF(LXCFF)

(0.1%) (prepared by dissolving 100 mg of xylene cyanol FF in 25 mL of water

containing 30 mg of zinc dust and 2 mL of 1M acetic acid, stirred well and kept aside

for 20 minutes. The resulting solution was then filtered and diluted to 100 mL with

water), potassium iodide (2 %), acetate buffer (1 M),

sulfuric acid (0.05 M) and

hydrochloric acid (2 M).

2.5 PROCEDURES

2.5.1 Using Methylene Blue as a Reagent

Aliquots of sample solution containing 0.5–14 µgmL-1

of iodate were

transferred into a series of 25 mL calibrated flasks and potassium iodide (2 %, 1 mL)

then hydrochloric acid (2 M, 1 mL) were added and the mixture was gently shaken

until the appearance of yellow color, indicating the liberation of iodine. Methylene

blue (0.01 %, 0.5 mL) and 2 mL of acetate buffer solution were added and the

reaction mixture was shaken for 2 minutes. The contents were diluted to 25 mL with

distilled water and mixed well. The absorbance of the resulting solutions were

measured at 665.6 nm against distilled water. Reagent blank was prepared by

replacing the analyte (iodate) solution with distilled water. The absorbance

corresponding to the bleached color which in turn corresponds to the analyte (iodate)

concentration was obtained by subtracting the absorbance of the blank solution from

42

that of test solution. The amount of the iodate present in the volume taken was

computed from the calibration graph (Figure IIB1).

2.5.2 Using Rhodamine B as a Reagent

Aliquots of sample solution containing 0.5–7.0 µgmL-1

of iodate were

transferred into a series of 25 mL calibrated flasks and potassium iodide (2 %, 1 mL)

then hydrochloric acid (2 M, 1 mL) were added and the mixture was gently shaken

until the appearance of yellow color, indicating the liberation of iodine. Rhodamine B

(0.01 %, 0.5 mL) and 2 mL of acetate buffer solution were added and the reaction

mixture was shaken for 2 minutes. The contents were diluted to 25 mL with distilled

water and mixed well. The absorbance of the resulting solutions were measured at

553 nm against distilled water. Reagent blank was prepared by replacing the analyte

(iodate) solution with distilled water. The absorbance corresponding to the bleached

color which in turn corresponds to the analyte (iodate) concentration was obtained by

subtracting the absorbance of the blank solution from that of test solution. The

amount of the iodate present in the volume taken was computed from the calibration

graph (Figure IIB2).

2.5.3 Using Leuco Xylene Cyanol FF as a Reagent

Aliquots of sample solution containing 0.4-14 µgmL–1

of iodate were

transferred in to a series of 10 mL calibrated flasks. Volumes of 0.5 mL each of the

0.05 M H2SO

4 and 0.1 % LXCFF were added, and the mixture was kept in a water

bath (≈90°C) for 15 minutes, after being cooled to room temperature (27±2°C), the

contents were diluted to the mark with acetate buffer of pH 4, and mixed well. The

absorbance of the xylene cyanol FF dye formed was then measured at 620 nm against

the reagent blank prepared in the same manner, without iodate. The amount of the

iodate present in the volume taken was computed from the calibration graph

(Figure IIB3).

2.5.4 Determination of Iodate in Table Salt Samples

A Table salt sample (2.9215g) was dissolved in water and diluted up to the

mark in a 25 mL volumetric flask. A 0.5 mL portion of this solution was transferred

into a 10 mL volumetric flask. Then the procedure described above for the standard

43

solution was followed. The absorbance was measured and the iodate concentration

was calculated using the calibration graph and proposed method was also compared

with reference method [56]. The results are listed in Table 2B1, 2B2 and 2B3.


2.6.1 Absorption Spectra

2.6.1.1 Using methylene blue as a reagent

This method is based on the reaction of iodate with potassium iodide in acid

medium to liberate iodine. This liberated iodine bleaches the blue color of methylene

blue. The decrease in absorbance at 665.6 nm is directly proportional to the iodate

concentration. The absorption spectrum of the colored species of methylene blue is

presented in Figure IIA1 and reaction system is presented in Scheme II.

2.6.1.2 Using rhodamine B as a reagent

Similarly this method is also based on the reaction of iodate with potassium

iodide in acid medium to liberate iodine. This liberated iodine bleaches the color of

the rhodamine B. The decrease in absorbance at 553 nm is directly proportional to the

iodate concentration. The absorption spectrum of the colored species of rhodamine B

is presented in Figure IIA2 and reaction system is presented in Scheme II.

2.6.1.3 Using leuco xylene cyanol FF as a reagent

In this method iodate quantitatively oxidized leuco xylene cyanol FF

into its blue color xylene cyanol FF dye in a sulfuric acid medium (pH 1.4 – 3.9) in a

boiling water bath (∼90°C for 15 min), the resulting colored dye shows a maximum

absorbance at 620nm in an acetate buffer medium. The reagent blank has negligible

absorbance at this wavelength. The absorption spectrum of the colored species of

LXCFF against reagent blank is presented in Figure IIA3 and reaction system is

presented in Scheme II.

44

2.6.2 Using Methylene Blue and Rhodamine B as a Reagents

2.6.2.1 Effect of iodide concentration and acidity

The oxidation of iodide to iodine is effective in the pH range 1.0 to 1.5, which

could be maintained by adding 1 mL of 2 M HCl in a final volume of 25 mL. The

liberation of iodine from KI in an acid medium is quantitative. The appearance of

yellow color indicates the liberation of iodine. Although any excess of iodine in the

solution will not interfere. It is found that 1 mL of each 2 % KI and 2M HCl are

sufficient for the liberation of iodine from iodide by iodate and 0.5 mL of 0.01 %

methylene blue and 0.5 mL of 0.01% rhodamine B are used for the decolorization

reaction. The bleached reaction system is found to be stable for more than a week for

both methylene blue and rhodamine B reagents.

The variation of absorbance of known concentration of the iodate with pH of

the medium is studied. A series of buffer solutions differing by pH=0.5 is prepared,

and using these buffers the system is studied. The maximum absorbance value is

found at pH= 4±0.2 Hence, the pH is maintained at pH= 4±0.2 throughout the study

by using acetate buffer (pH= 4). Effect of pH on color stability is presented in Figure

IIA4 and IIA5.

2.6.3 Using Leuco Xylene Cyanol FF as a Reagent

2.6.3.1 Effect of the acidity and temperature

The oxidation of LXCFF by iodate is studied. Of the various acids (sulfuric,

hydrochloric and phosphoric) studied, sulfuric acid is found to be the best acid for the

system. Constant absorbance readings were obtained in the range (0.1-1.5 mL) of 0.05

M sulfuric acid (pH 1.4-3.9) at a temperature 90°C for 15 minutes. An increase of the

pH above 3.9 markedly affected the stability and sensitivity of the dye. Color

development did not take place below pH 1.4. Hence a volume of 0.5 mL of 0.05M

sulfuric acid (or maintained pH=2) in a total volume of 10 mL is used in all

subsequent work.

2.6.3.2 Effect of reagent concentration and buffer media

The optimum concentration of LXCFF leading to maximum color stability is

45

found to be 0.5 mL of 0.1 % reagent per 10 mL of the reaction mixture. The

absorbance values are measured in the pH range of 3.5-4.6. This could be achieved by

adding 3 mL of acetate buffer of pH 4. Appreciable results are obtained when the

entire reaction mixture is diluted with the same acetate buffer solution of pH 4. A

change in the pH of the final reaction mixture is affected by the intensity of the

colored dye. The formed colored dye is stable for more than 24 hours.

2.6.4 Analytical Data


In this method adherence to Beer’s law is studied by measuring the absorbance

values of solutions varying iodate concentration. A straight line graph is obtained by

plotting absorbance against concentration of iodate. Beer’s law is obeyed in the

concentration range of 0.5-14 µgmL-1

of iodate (Figure IIB1). The molar obsorptivity

and Sandell’s sensitivity of the system is found to be 1.24×104

Lmol-1

cm-1

and

1.41×10-2

µgcm-2

respectively. The detection limit (DL

= 3.3σ/S) and quantitaion limit

(QL = 10σ /S) [where σ is the standard deviation of the reagent blank (n=5) and S is

the slope of the calibration- curve] of iodate determination is found to be 0.048 µgmL-

1

and 0.145 µgmL-1

respectively.


In rhodamine B method also adherence to Beer’s law is studied by measuring

the absorbance values of solutions varying iodate concentration. A straight line graph

is obtained by plotting absorbance against concentration of iodate. Beer’s law is

obeyed in the range of 0.5–7.0 µgmL-1

of iodate (Figure IIB2). The molar

absorptivity and Sandell’s sensitivity of the system is found to be 1.406×105

Lmol-1

cm-1

and 1.23×10-3

µgcm-2


= 3.3σ/S)

and quantitaion limit (QL = 10σ/S) [where σ is the standard deviation of the reagent

blank (n=5) and S is the slope of the calibration- curve] of iodate determination is

found to be 0.132 µgmL-1

and 0.400 µgmL-1

respectively.

46


In this method also adherence to Beer’s law is studied by measuring the

absorbance values of solutions varying iodate concentration. A straight line graph is

obtained by plotting absorbance against concentration of iodate. Beer’s law is obeyed

in the range of 0.4 to 14 µgmL-1

of iodate (Figure IIB3). The molar absorptivity and

Sandell’s sensitivity of the colored system is found to be 1.71×104

Lmol-1

cm-1

and

1.02×10-2

µgcm-2


= 3.3σ/S) and quantitation

limit (QL = 10σ/S) [where σ is the standard deviation of the reagent blank (n=5) and S

is the slope of the calibration curve] of iodate determination is found to be 0.026

µgmL-1

and 0.0806 µgmL-1

respectively.

2.6.5 Effect of Divers Ions

The effect of a various ions at microgram levels on the determination of iodate

is examined. The tolerance limits of the interfering species are established as those

concentrations, which cause not more than ± 2.0 % changes in the absorbance value

during the determination of a fixed amounts of iodate. The tolerance limits of various

foreign ions are given in Table 2A1 and 2A2. In this reaction system, various

oxidants such as Fe3+

, Ce4+

, V5+

and Cr6+

are found to interfere. The interference of

chromium was removed by extracting with methyl isobutyl ketone. However, the

tolerance limit of iron, cerium and vanadium can be increased by the addition of

appropriate (2% NaF) amount of sodium fluoride.

2.7 APPLICATIONS

The method developed is applied to the quantitative determination of traces of

iodate in table salt and sea water samples. The results are listed in the Table 2B1,

2B2, 2B3 and 2C1, 2C2, 2C3, compare favorably with those from a reference method

[56]. Statistical analysis of the results by the use of t–test and F–tests show that, there

is no significant difference between the accuracy and precision of the proposed and

reference method. The precision of the proposed method is evaluated by replicate

analysis of samples containing iodate at five different concentrations.

47

2.8 CONCLUSIONS

1. The reagents provide a simple and sensitive method for the spectrophotometric

determination of iodate.

2. The reagents have the advantage of high sensitivity and low absorbance of reagent

blank (LXCFF).

3. Common ions do not interfere seriously.

4. The developed method does not involve any stringent reaction conditions and

offers the advantages of high stability of the reaction system for both methylene

blue, rhodamine B (more than a week) and leuco xylene cyanol FF (more than 24

hours).

5. The statistical analysis of the results by t and F- tests show that, there is no

significant difference in accuracy and precision between the proposed method and

reference method.

6. The proposed method has been successfully applied to the determination of traces

of iodate in table salt and sea water samples. A comparison of the method reported

is made with earlier methods and is given in Table 2C4.

FIGURE IIA1

48

ABSORPTION SPECTRUM OF COLORED SPECIES OF METHYLENE BLUE

W ave leng th (nm )

300 400 500 600 700 800 900

Ab

so

rb

an

ce

0 .0

0 .1

0 .2

0 .3

0 .4

FIGURE IIA2

ABSORPTION SPECTRUM OF COLORED SPECIES OF RHODAMINE B

W avelength (nm)

300 400 500 600 700 800 900

Ab

so

rb

an

ce

0.0

0.5

1.0

1.5

2.0

2.5

FIGURE IIA3

49

ABSORPTION SPECTRA OF COLORED SPECIES OF LEUCO XYLENE

CYANOL FF (IO3

-

, 2 µgmL-1

) Vs REAGENT BLANK (a) AND REAGENT

BLANK Vs DISTILLED WATER (b)

W avelength (nm )

580 600 620 640 660

Ab

so

rb

an

ce

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

a

b

FIGURE IIA4

EFFECT OF pH ON COLOR INTENSITY USING METHYLENE BLUE AS A

REAGENT

pH

1 2 3 4 5 6 7 8

Ab

so

rb

an

ce

0.00

0.05

0.10

0.15

0.20

0.25

FIGURE IIA5

50

EFFECT OF pH ON COLOR INTENSITY USING RHODAMINE B AS A

REAGENT

pH

1 2 3 4 5 6 7 8

Ab

so

rb

an

ce

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

FIGURE IIB1

ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF IODATE

USING METHYLENE BLUE AS A REAGENT

C oncentra tion o f Iodate (µgm L

-1

)

0 2 4 6 8 10 12 14 16 18 20

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

FIGURE IIB2

51


USING RHODAMINE B AS A REAGENT

Concentration of Iodate (µgmL

-1

)

0 2 4 6 8 10 12

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

FIGURE IIB3


USING LEUCO XYLENE CYANOL FF AS A REAGENT

Concentration of Iodate (µgmL

-1

)

0 5 10 15 20

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

SCHEME II

52

SCHEME OF REACTIONS

KIO3

+ 5 KI + 6 HCl 3 I2

+ 3 H2O + 6 KCl

(CH3

)2

N

N

S+ N(CH

3

)2

Cl

-

I2

, H

+

N(CH3

)2

(CH3

)2

N

N

H

S

Methylene blue (colored) Methylene blue (colorless)

(H3CH

2C)

2N N(CH

2CH

3)2

O

COOH

Cl

I2

(H3CH

2C)

2N N(CH

2CH

3)2

O

COOH

Rhodamine B (colored) Rhodamine B (colorless)

SO3Na

SO3H

CH3

CH3

NHHN

CH3

CH3

IO3

-

+ + 4H

+

SO3Na

SO3H

CH3

CH3

NHN

CH3

CH3

+ I

-

+ 3H2O

Xylene cyanol FF Xylene cyanol FF

(Leucoform) (Blue color)

TABLE 2A1

53

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF IODATE

(1.0 µgmL-1

) USING METHYLENE BLUE AND RHODAMINE B AS REAGENTS

Foreign ions Tolerance limit in µgmL-1

Ca2+

, Br-

, Cl-

2000

Mn2+

, Mg2+

, Zn2+

1500

Gd3+

, PO4

3-

, Yb3+

, Sm3+

, Eu3+

, 1000

Cr3+

, NO2

-

, La3+

, Al3+

, SCN-

500

*Cr2O7

2-

, *Fe3+

, *Ce4+

, *VO3

-

, oxalate,

citrate, tartarate

≤ 1

MoO4

2-

, AsO4

3-

, Co2+

, WO4

2-

≤ 100

* Masked with masking agents.

TABLE 2A2

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF IODATE

(1.0 µgmL-1

) USING LEUCO XYLENE CYANOL FF AS A REAGENT

Foreign ions Tolerance limit in µgmL-1

Ca2+

, Br-

, Cl-

2500

Mn2+

, Mg2+

, Zn2+

2000

Sm3+

, Eu3+

, Gd3+

, PO4

3-

, Yb3+

1500

Cr3+

, NO2

-

, La3+

, Al3+

, SCN-

1000

*Cr2O

7

2-

, *Fe3+

, *Ce4+

, *VO3

-

, oxalate,

citrate, tartarate

≤ 1

AsO4

3-

, Co2+

, MoO4

2-

, WO4

2-

≤ 500


TABLE 2B1

54

DETERMINATION OF IODATE IN TABLE SALT SAMPLES USING

METHYLENE BLUE AS A REAGENT

a. Iodate concentration expressed in mgKg-1

(n=5)

b. Tabulated t-value for 8 degree of freedom at P (0.95) is 2.306

c. Tabulated F-value for (4, 4) degree of freedom at P (0.95) is 6.39.

TABLE 2B2

DETERMINATION OF IODATE IN TABLE SALT SAMPLES USING

RHODAMINE B AS A REAGENT


(n=5)



TABLE 2B3

Proposed method Reference method [56]

Table

Salt

Samples

Iodate

founda

mgKg-1

Std

deviation

Iodate

found

mgKg-1

Std.

deviation

b

t-testc

F-test

1 25.67 0.02 25.69 0.01 2.00 4.00

2 29.09 0.02 29.11 0.02 2.00 4.00

3 34.23 0.01 34.24 0.01 1.67 1.00


Table

Salt

Samples

Iodate

founda

mgKg-1

Std

deviation

Iodate

found

mgKg-1

Std.

deviation

b

t-testc

F-test

1 25.67 0.01 25.68 0.02 1.00 4.00

2 30.80 0.03 30.83 0.02 1.87 2.25

3 34.20 0.02 34.22 0.03 1.25 2.25

55

DETERMINATION OF IODATE IN TABLE SALT SAMPLES USING LEUCO

XYLENE CYANOL FF AS A REAGENT


(n=5)



TABLE 2C1

DETERMINATION OF IODATE IN SEA WATER SAMPLES USING

METHYLENE BLUE AS A REAGENT


Sea

water

Samples

Iodate

added

µgmL-1

Iodate

founda

µgmL-1

Std

deviation

Iodate

found

µgmL-1

Std.

deviation

b

t-testc

F-test

1 2.00 2.00 0.02 2.01 0.04 0.50 4.00

2 4.00 4.02 0.03 4.04 0.05 0.77 2.78

3 8.00 8.04 0.05 8.05 0.07 0.26 1.96

4 12.00 12.02 0.10 12.04 0.14 0.26 1.96

a. Iodate concentration expressed in µgmL-1

(n=5)

b. Tabulated t-value for four degree of freedom at P (0.95) is 2.306

c. Tabulated F-value for (4,4) degree of freedom at P (0.95) is 6.39.

TABLE 2C2


Table

Salt

Samples

Iodate

founda

mgKg-1

Std

deviation

Rel. Std

deviation

Iodate

found

mgKg-1

Std.

deviation

Rel. Std

deviation

b

t-testc

F-test

1 23.48 0.67 2.86 23.49 0.65 2.76 0.02 1.06

2 27.64 0.35 1.26 27.67 0.36 1.30 0.14 1.06

3 33.76 0.62 1.83 33.77 0.64 1.88 0.02 1.06

56

DETERMINATION OF IODATE IN SEA WATER SAMPLES USING

RHODAMINE B AS A REAGENT


Sea

water

Samples

Iodate

added

µgmL-1

Iodate

founda

µgmL-1

Std

deviation

Iodate

found

µgmL-1

Std.

deviation

b

t-testc

F-test

1 2.00 2.05 0.03 2.01 0.03 2.11 1.00

2 4.00 4.07 0.04 4.03 0.05 1.42 1.56

3 6.00 6.04 0.03 6.04 0.04 1.81 1.78


(n=5)



TABLE 2C3

DETERMINATION OF IODATE IN SEA WATER SAMPLES USING LEUCO



Sea

water

samples

Iodate

added

µg mL-1

Iodate

founda

µg mL-1

Std

deviation

Iodate

found

µg mL-1

Std.

deviation

b

t-testc

F-test

1 4.00 4.00 0.007 4.01 0.009 2.02 1.51

2 8.00 8.01 0.009 8.02 0.007 2.02 1.51

3 12.00 11.99 0.009 12.01 0.01 0.55 1.35


(n=5)



TABLE 2C4

57

COMPARISON OF THE METHOD REPORTED WITH EARLIER METHODS

ε = Molar absorptivity, ss = Sandell’s sensitivity

Reagent Method Beer’s

law

-1

)

ε in (Lmol-1

cm-1

)

-2

)

λmax

(nm)

Ref.

No.

Phosphoric acid Spectrophotometry -----

------

ε = 7.320×104

--------

ε = 1.103×105

---------

352

288

39

39

3, 3’, 5, 5’-

Tetramethylbenzidine

Spectrophotometry 0-0.7

mgL-1

ε = 2.13×105

450 42

Thionin

Azure B

Spectrophotometry

Spectrophotometry

1.0-12

0.2–16

ε = 2.7×104

ss = 7.9×10–2

ε = 2.06×104

ss = 0.85×10-2

600

644

55

55

Proposed Method

Methylene blue

Rhodamine B

Leuco xylene

cyanol FF

Spectrophotometry

Spectrophotometry

Spectrophotometry

0.5-14

0.5–7.0

0.4 to 14

ε = 1.24×104

ss = 1.41×10-2

ε = 1.406×105

ss = 1.23×10-3

ε = 1.71×104

ss = 1.02×10-2

665.6

553

620

58

2.9 REFERENCES

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7. J. V. Christiansen and L. Carlsen, Riso National Laboratory, Denmark. Riso-M,

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8. G. T. Wong and P. G. Brewer, Geochim. et Cosmochim. Acta, 41 (1977) 151.

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183.

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13. J. E. Mackin, R. C. Allure and W. J. Pullman, Cont. Shelf Res., 8 (1988) 363.

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59

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60

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61

CHAPTER 3

SPECTROPHOTOMETRIC DETERMINATION OF HYPOCHLORITE IN

ENVIRONMENTAL SAMPLES

3.1 INTRODUCTION


3.3 APPARATUS


3.5 PROCEDURE


3.7 APPLICATIONS

3.8 CONCLUSIONS

3.9 REFERENCES

62

3.1 INTRODUCTION

Hypochlorite refers to the salts of hypochlorous acid (HOCl). Hypochlorite is

inherently an unstable compound. It can decompose over the time and results in the

formation of crystalline salts and oxygen gas. Hypochlorite is widely used as

bleaching agent [1], disinfectant in fabrics, wood pulp and food industries. It has

been extensively used for hygienic chemical studies on tap water for treating skin

cancers [2], disinfectant in milk industry [3] and for the determination of blood urea.

It is also used for the preparation of surgically active anticeptic compounds for

controlling and preventing infection in wounds. Sodium hypochlorite is used as a

screening agent for the identification of cocane [4]. The lethal action of calcium

hypochlorite on Bacillus anthracoides spores [5] resulted from the alteration of

structural organization of spores which lead to disturbance of normal permeability

barrier with loss of life – sustaining components and unbalance of metabolic process.

Biological toxins can be extremely hazardous even in minute quantities.

Investigators must ensure that appropriate equipment and safety procedures are in

place for the specific toxin used and the type of experiments performed in their

laboratory. Some toxins are inactivated by autoclaving for one hour at 121°C, while

others are inactivated by exposure to sodium hypochlorite, sodium hypochlorate and

sodium hydroxide [6].

The common method for making sodium hypochlorite is to react chlorine with

a solution of caustic soda. The final concentration of the sodium hypochlorite solution

depends on the initial concentration of the starting caustic soda solution. The

following equation gives the chemical reaction involved, regardless of concentration:

Cl2 + 2NaOH

2O

A more active but less stable sodium hypochlorite can be produced by chlorinating a

solution of soda ash according to the following equation:

Cl2 + 2Na

2CO

3 + H

2O

3

On further chlorination, hypochlorous acid will be produced:

Cl2 + Na

2CO

3 + H

2O

3

Most of the commercial production processes involve the reaction of chlorine with

caustic soda as mentioned in the above equation.

63

Sodium hypochlorite (bleach) manufacturers are now frequently required to

provide high quality sodium hypochlorite with limits on chlorate ion and transition

metal ions. Sodium hypochlorite "decomposes" by two mechanisms. The first is the

2nd

order process that forms chlorate ion

3OCl-

3

-

+ 2 Cl-

In the presence of transition metal ions, decomposing bleach forms oxygen whether

transition metal ion acts as catalyst.

2OCl-

2 + 2Cl

-

Majority of the municipalities require the delivered sodium hypochlorite (9 to 16 wt%

NaOCl) should contain 0.1-0.4 wt% excess caustic, <1,500 mgL-1

ClO3

-

, <0.5

mgL-1

iron and <0.05 mgL-1

nickel and copper. Important considerations for

minimizing ClO3

-

formation include: high pH (i.e. excess caustic), dilution

(decomposition is 2nd

order with respect to OCl-

) and temperature control.

The active ingredient in most of the chlorine bleaches is sodium

hypochlorite(NaOCl). The oxidizing action of hypochlorite ion (OCl-

) kills germs and

also decolorizes many stains and dyes. The quantity of hypochlorite ion in a sample of

bleach can be determined by finding out how much iodine (I2) it can produce by

oxidizing an iodide ion (I-

). The quantity of iodine produced is estimated by titrating

it with sodium thiosulfate, which converts the colored iodine back to colorless iodide

ion.

The equations are:

Oxidation of iodide ion to iodine with bleach:

2H+

+ OCl-

+ 2I-

→ I2 + Cl

-

+ H2O

Titrating iodine with thiosulfate:

I2 + 2S

2O

3

2-

→ 2 I-

+ S4O

6

2-

Sodium hypochlorite is recommended and used by the majority of dentists

because this solution presents several important properties: antimicrobial effect [7,8],

tissue dissolution capacity and acceptable biologic compatibility of less concentrated

solutions. In relation to antimicrobial effect, the studies have shown that sodium

hypochlorite decreases microorganism number during the treatment of teeth with

apical periodontitis.

64

Sodium hypochlorite neutralizes amino acids forming water and salt. With the

reduction in concentration of hydroxyl ions, there is a reduction of pH. Hypochlorous

acid, a substance present in sodium hypochlorite solution, when in contact with

organic tissue acts as solvent, releases chlorine that combined with the protein amino

group and forms chloramines. Hypochlorous acid (HOCl) and hypochlorite ions

(OCl-

) lead to amino acid degradation and hydrolysis.

The antimicrobial effectiveness of sodium hypochlorite based on its high pH

(hydroxyl ions action) is similar to the mechanism of action of calcium hydroxide.

The high pH of sodium hypochlorite interferes in the cytoplasmic membrane integrity

with an irreversible enzymatic inhibition, biosynthetic alterations in cellular

metabolism and phospholipid degradation.

Sodium hypochlorite in higher concentrations is more aggressive while in

lower concentrations (0.5% to 1%), it is biocompatible. For a substance to be

biocompatible, it must present a discrete tissue reaction at all periods and moderate or

intense tissue reaction at 7 days which decreases in intensity with time until reaching

a non-significant tissue reaction [9].


The determination of hypochlorite in environmental and biological samples

such as natural water and tap water can be of interest in biochemical research. Hence

there is a need for a rapid and sensitive method for the determination of hypochlorite.

Iodometric [10], coulometric [11], polarographic [12], chemiluminescence[13-15],

radiolytically-induced redox [16], normal pulse voltametry [17,18], colorimetric [19]

and potentiometric [20] methods are most commonly used. However, colorimetric

methods are often preferred as they involve less expensive instrumentation and

provide better sensitivity when appropriate chromogenic reagents are available.

Anwar et al. reported two simple spectrophotometric procedures for the

quantitative estimation of hypochlorite [21]. One of the method was based directly on

the absorbance of OCl-

in alkaline aqueous media. The other method took the

advantage of the quantitative reaction of OCl-

with NH3 in alkaline solution to form

65

chloramine, which has a higher molar absorptivity. Results of the spectrophotometric

assays are compared with results obtained by the titrimetric procedure of NF XII

(National formulary, 1965).

Bunikiene and Ramanauskas presented an indirect spectrophotometric

detection of trace amounts of OCl-

was based on oxidation of OCl-

with I-

[22].

Subsequent reaction of the oxidized product with brilliant green and measured of

change in the absorbance of brilliant green solution. The detection was conducted in

acid medium (7M HCl) with NaOAc addition or in universal buffer medium and the

absorbance was measured at 628 or 684 nm respectively. The absorbance was

proportional to OCl-

concentration in the range 0.04-1.60 µgmL-1

. The sensitivity of

the method was 0.02-0.05 µgmL-1

of OCl-

.

Fleet and Ho presented a approach to the automated determination of sodium

hypochlorite and hydrogen peroxide [23]. This method was based on the use of a

porous catalytic silver electrode. The principle of both methods involved the

quantitative liberation of oxygen, which was measured colorimetrically by the

electrode.

Isacsson and Wettermark reported a sensitive method for the determination of

hypochlorite in aqueous solution which involved the measurement of the

chemiluminescence, produced during alkaline oxidation of luminol in presence of

hydrogen peroxide. Micromolar and submicromolar quantities could be detected by

this method [24].

Tarasankar et al. described a method for the spectrophotometric determination

of hypochlorite [25]. In this method, AgNO3 was mixed with 0.5 % gelatin at a pH 8,

Ag+

reduced by CO to form a Ag sol solution. Aliquots of the sol solution acidified to

pH <7, were added to water samples containing OCl-

and measured at 415 nm. The

method was used for OCl-

concentrations of 0.04-1.0 mgL-1

.

Bamnolker et al. described a spectrophotometric method for the detection of

hypochlorite traces in solutions containing 4M NaOH based on the reaction of the

66

reagent 3,3'-dimethylnaphthidine (DMN) with chlorine [26]. The reagent containing

DMN, hydrochloric acid, and DMF was used. This common reagent enables the

liberation and detection of chlorine in situ in acidic solution. The effect of factors

such as acidity, temperature, oxidizing agents and metallic impurities on the

absorption spectrum was studied. The chlorine DMN complex has an absorption

maximum at 550 nm and obeys Beer's law for solutions containing up to 3 mgL-1

of

OCl-

.

Gonzalez-Robledo et al. described the reaction of hypochlorite with luminol

by stopped-flow chemiluminescence spectrometry method [27]. The emission was

observed by using a conventional fluorescence detector at 425 nm. The method yields

linear response over three orders of magnitudes with an RSD of about 1 %. The

method was highly selective and rapid (80 samples per hour) and was applied to the

routine determination of hypochlorite in different water samples.

Watanabe et al. presented the simultaneous determination of chlorine dioxide

and hypochlorite by high-performance liquid chromatography [28]. The chromogenic

substance formed by the oxidative condensation reaction using 4-aminoantipyrine

(4-AA) and phenol was determined at 503 nm by a post-column reaction system.

Chlorine dioxide and hypochlorite were separated within 3 minutes by the applied

elution solution (1.7 mM sodium carbonate–1.8 mM sodium carbonate) at 0.5 mL per

minute into a laboratory-prepared PTFE tube column (13 cm×1.0 mm I.D.×2 mm

O.D.) packed with Waters Accell QMA as the anion-exchange material. A linear

correlation between the peak height and concentration was obtained within the range

of 1–20 gmL-1

for chlorine dioxide and 47–200 gmL-1

for hypochlorite with good

reproducibility (relative standard deviations of 4.0 and 2.2%, respectively). The limits

of detection of chlorine dioxide and hypochlorite were approximately 0.2 and

-1

respectively.

Han et al. developed a simple spectrophotometric method for quantitative

detection of hypochlorite (OCl-

) or hypochlorous acid (HOCl) [29]. The OCl-

or

HOCl sample was first incubated with an excess amount of tris(2-

carboxyethyl)phosphine(TCEP). The concentration of the residual TCEP was then

measured as the amount of 2-nitro-5-thiobenzoate produced after reaction with

67

5,5'-dithiobis(2-nitrobenzoic acid). The concentration of OCl-

or HOCl was equivalent

to the amount of decrease in the concentration of TCEP because one mole of TCEP

was rapidly and irreversibly oxidized to TCEP oxide by one mole of OCl-

or HOCl.

This method was more sensitive and convenient than the standard procedure of

NaOCl assay, which involves reaction with KI, followed by titration of the liberated

triiodide with thiosulfate.

Chiswell and O'Halloran reported acid yellow 17 as a spectrophotometric

reagent for the determination of low concentrations of residual free chlorine [30]. The

detection limit was 50 ngmL for free chlorine, and the calibration graph was linear

. Cyclic voltammetry was used to explain the findings of the

spectrophotometric work.

Tian and Dasgupta described a flow injection method for the simultaneous

determination of hydroxide, chloride, hypochlorite and chlorate ions present in

chloralkali cell effluents in concentrations ranging from sub-millimolar to

severaLmolar [31]. For the measurement of hypochlorite and chlorate, colorimetric

iodometry was used.

Icardo et al. reported a flow injection analytical (FIA) procedure for the

detection of free chlorine in industrial formulations and H2O samples [32]. The

manifold was provided with a gas-diffusion unit, which permits the removal of

interfering species and also the preconcentration of chlorine. The detection of chlorine

was performed from the oxidation by o-dianisidine as a chromogenic reagent to a

colored product, which was monitored at 445 nm. The method was linear over the

range 0.04-1.00 mgL-1

of chlorine. The limit of detection was 0.04 mgL-1

, the

reproducibility of the procedure (as relative standard deviation of the slope) was 3.7

% for four independent calibrations, the precision (as relative standard deviation of 30

continuous FIA peaks of 0.56 mgL-1

of chlorine) was 1.4 % and the sample

throughout was 40 per hour.

Narayana et al. presented azure B as a reagent for the facile

spectrophotometric determination of hypochlorite [33]. The method was based on the

reaction of hypochlorite with potassium iodide in an acidic medium to liberate iodine.

68

Bleaching of the blue color of azure B by the liberated iodine was the basis of the

determination and exhibited an absorption maximum at 644 nm. Beer’s law was

obeyed in the range 0.2-1.0 µgmL-1

of hypochlorite in a final volume of 10 mL. The

molar absorptivity and Sandell’s sensitivity for the colored system were found to be

1.49×104

Lmol-1

cm-1

and 3.25×10-4

µgcm-2

respectively.

Kodera et al. reported a electroanalytical method which was based on an

anodic reaction for the hypochlorite ion [34]. The determination was carried out by

linear sweep-voltammetry at a Pt electrode. The peak current of observed oxidation

wave was proportional to the amount of hypochlorite ion. The influences of various

physical and chemical factors (repeatability, supporting electrolyte, pH, sweep rate,

temperature, metal ion, and dissolved oxygen) were investigated. A fairly good

correlation (r=0.987) between this method and iodometric titration was achieved for

standard samples (n=5). This method was useful to analyze the hypochlorite ion.

Ballesta et al. developed a selective and reusable chemiluminescent test strip

to determine hypochlorite [35]. The hypochlorite sensitive test strip contains a 10

mm×9 mm piece of anionic cellulose paper in fluoresceinate cycle, glued to a 10

mm×4cm×0.5 mm polyester strip. The measurement of the chemiluminescence in a

luminometer when 1 mL of sample was injected into a conventional cell containing

the strip makes it possible to determine hypochlorite. The composition of the

membrane and reaction conditions was adjusted to obtain adequate sensitivity and

selectivity. The test strip responded linearly to hypochlorite over two linear ranges,

the first 2.0-10.3 mgL-1

and the second 10.3-51.4 mgL-1

with a detection limit of 0.4

mgL-1

. The reproducibility using the same disposable test strip at a medium level of

the range was 6.6%, as relative standard deviation (R.S.D.), and 12.3% using different

test strips. The procedure was applied to the determination of hypochlorite in different

types of waters.

Narayana et al. described an easy spectrophotometric method for the

determination of hypochlorite using thionin [36]. The method was based on the

reaction of hypochlorite with potassium iodide in acidic medium to liberate iodine.

Bleaching of the violet color of thionin by the liberated iodine was the basis of the

69

determination and was measured at 600 nm. The decrease in absorbance was directly

proportional to the concentration of hypochlorite. Beer’s law was obeyed in the range

0.2–1.2 µgmL-1

of hypochlorite. The molar absorptivity, Sandell’s sensitivity,

detection limit, and quantitation limit are found to be 1.489×104

Lmol-1

cm-1

,

3.25×10 µgcm , 0.1026 µgmL-1

and 0.3112 µgmL-1

respectively.

Antonio et al. described the determination of hypochlorite in bleaching

products with flower extracts to demonstrate the principles of flow injection analysis

[37]. The use of crude flower extracts to the principle of analytical chemistry

automation with the flow injection analysis (FIA) procedure was developed to

determine hypochlorite in household bleaching products. The FIA comprises a group

of techniques based on injection of a liquid sample into a moving, nonsegmented

carrier stream of a suitable fluid.

March and Simonet developed a green method for the determination of

hypochloride in bleaching products [38]. The method was based on a flow injection

system and measurement of the native absorbance of hypochlorite at 292 nm for the

determination of hypochlorite in the range 0.07–0.42 gL of chlorine. A mini-column

containing cobalt oxide was inserted in the flow system catalysed the hypochlorite

decomposition to chloride and oxygen. The method required 20 mg of solid, reusable

catalyst and a NaOH solution of pH 10.4, provided a sample throughput of 12

samples per hour in triplicate injection. The usefulness of the analysis of bleaching

products was demonstrated. Colorimetric methods are often preferred, however, as

they involve less expensive instruments and provide better sensitivity when the

appropriate chromogenic reagents are available.

In this chapter, rhodamine B and methylene blue have been used as rapid,

sensitive and selective reagents for the determination of hypochlorite. The developed

method has been successfully employed for the determination of hypochlorite in tap

water, natural water and milk samples.

70

3.3 APPARATUS

A Secomam Anthelie NUA 022 UV-Visible spectrophotometer with 1 cm

quartz cell was used. A WTW pH 330-pH meter was used.


All chemicals were of analytical reagent grade or chemically pure grade.

Standard hypochlorite solution (1000 µgmL-1

) was prepared by dissolving 1.4446 g of

sodium hypochlorite in 1000 mL water and standardized by the iodometric method

[10]. Rhodamine B (0.05 %), methylene blue (0.05 %), sodium acetate (1 M),

hydrochloric acid (2 M) and potassium iodide (2 %) solutions were used.

3.5 PROCEDURES

3.5.1 Using Rhodamine B as a Reagent


of hypochlorite were

transferred into a series of 10 mL calibrated flasks, 1 mL of 2 M hydrochloric acid and

1 mL of 2% potassium iodide were added and the mixture was gently shaken until the

appearance of yellow color, indicating the liberation of iodine. A 0.5 mL of 0.05 %

rhodamine B solution was then added to it followed by the addition of 2 mL of 1 M

sodium acetate and the reaction mixture was shaken for 2 minutes and the contents

were diluted to 10 mL with distilled water and mixed well. The absorbance of the

resulting solutions were measured at 553 nm against distilled water. A blank was

prepared by replacing the analyte (hypochlorite) solution with distilled water. The

absorbance corresponding to the bleached color which in turn corresponds to the

analyte (hypochlorite) concentration was obtained by subtracting the absorbance of

the blank solution from that of test solution. The amount of the hypochlorite present

in the volume taken was computed from the calibration graph (Figure IIIB1).

3.5.2 Using Methylene Blue as a Reagent


of hypochlorite were

transferred into a series of 10 mL calibrated flasks, 1 mL of 2 M hydrochloric acid and

71

1 mL of 2% potassium iodide were added and the mixture was gently shaken until the

appearance of yellow color, indicating the liberation of iodine. A 0.5 mL of 0.05 %

methylene blue solution was then added to it followed by the addition of 2 mL of 1 M

sodium acetate and the reaction mixture was shaken for 2 minutes, the contents were

diluted to 10 mL with distilled water and mixed well. The absorbance of the resulting

solutions were measured at 665.6 nm against distilled water. A blank was prepared

by replacing the analyte(hypochlorite) solution with distilled water. The absorbance

corresponding to the bleached color which in turn corresponds to the

analyte(hypochlorite) concentration was obtained by subtracting the absorbance of

the blank solution from that of test solution. The amount of the hypochlorite present

in the volume taken was computed from the calibration graph (Figure IIIB2).

3.5.3 Determination of Hypochlorite in Natural / Tap Water Samples

Aliquots of natural / tap water sample containing not more than 6.0 µgmL-1

of

hypochlorite were treated with 0.5 mL of 1M NaOH and 0.5mL of 0.2M EDTA. The

solution was mixed and centrifuged to remove any precipitate formed. The

centrifugate was transferred to a 10 mL calibrated flask and its hypochlorite content

was determined directly according to the general procedure for the determination of

the hypochlorite (Table 3A1 and Table 3A2).

3.5.4 Determination of Hypochlorite in Milk

A known volume of milk (20 mL) was placed in a 50 mL beaker and

coagulated with 8–10 mL of 1 M citric acid. The solution was centrifuged to remove

the precipitate. The centrifugate was transferred to a 100 mL calibrated flask. A

sample solution of suitable aliquot was determined according to the general procedure

for the determination of hypochlorite (Table 3A1 and Table 3A2).



This method involves the liberation of iodine by the reaction of hypochlorite

with potassium iodide in an acidic medium. The liberated iodine selectively bleaches

the color of rhodamine B and is measured at 553 nm. This decrease in absorbance is

72

directly proportional to the hypochlorite concentration and obeys Beer’s law in the

range of 0.1–4.0 µgmL-1

of hypochlorite. The absorption spectrum of rhodamine B

is presented in Figure IIIA1, and the reaction system is represented in Scheme III.

Similarly the liberated iodine selectively bleaches the blue color of methylene

blue and is measured at 665.6 nm. This decrease in absorbance is directly proportional

to the hypochlorite concentration and obeys Beer’s law in the range of 0.1–6.0

µgmL-1

of hypochlorite. The absorption spectrum of methylene blue is presented in

Figure IIIA2, and the reaction system is represented in Scheme III.

3.6.2 Effect of Iodide Concentration and Acidity

The oxidation of iodide to iodine by hypochlorite is effective in the pH range

1.0-1.5, which could be maintained by adding 1 mL of 2M HCl in the final volume of

10 mL. The liberation of iodine from potassium iodide in an acidic medium is

quantitative. The appearance of yellow color indicated the liberation of iodine.

However, concentration of hydrochloric acid should be maintained at the 1.0-1.2 mL

ranges. It is found that 1 mL each of 2 % potassium iodide and 2 M HCl are

sufficient for the liberation of iodine from iodide by hypochlorite and 0.5 mL of each

0.05 % rhodamine B and methylene blue is used for subsequent decolorization. The

bleached reaction system is found to be stable for 3 hours for each rhodamine B and

methylene blue reagents. Effect of pH on color stability is presented in Figure IIIC1

and IIIC2.



The adherence to Beer’s law is studied by measuring the absorbance values of

solutions varying hypochlorite concentration. A straight line graph is obtained by

plotting absorbance against concentration of hypochlorite. Beer’s law obeyed in

the range of 0.1–4.0 µgmL-1

of hypochlorite (Figure IIIB1). The molar absorptivity

and Sandell’s sensitivity for colored system is found to be 2.57×105

Lmol-1

cm-1

,

2.01×10-3

µgcm-2

respectively. Correlation coefficient(n = 10) and slope of the

calibration curve are 0.995 and 0.471 respectively. The detection limit (DL=3.3σ/s)

and quantitaion limit (QL=10σ/s) [where σ is the standard deviation of the reagent

73

blank (n=5) and s is the slope of the calibration-curve] for hypochlorite determination

were found to be 0.070 µgmL-1

and 0.212 µgmL-1

respectively.



solutions varying hypochlorite concentration. A straight line graph is obtained by

plotting absorbance against concentration of hypochlorite. Beer’s law obeyed in the


of hypochlorite (Figure IIIB2). The molar absorptivity and

Sandell’s sensitivity for colored system is found to be 1.12×104

Lmol-1

cm-1

,

4.61×10-3

µgcm-2

respectively. The detection limit (DL=3.3σ/s) and quantitaion limit

(QL=10σ/s) where σ is the standard deviation of the reagent blank (n=5) and s is the

slope of the calibration- curve, for hypochlorite determination were found to be 0.031

µgmL-1

and 0.093 µgmL-1

respectively.

3.6.4 Effect of Diverse Ions

The effect of diverse ions on the determination of hypochlorite by the

proposed procedure is examined. The tolerance limits of interfering species are

established at the concentration required to cause not more than a ± 2% error in the

recovery of hypochlorite at 1.00 µgmL-1

. The tolerance limits of diverse ions are

summarized in Table 3A1. The interference of iron(III) can be masked by the

addition of 1 mL of 2% sodium fluoride while cupric ions were masked by the

addition of 1 mL of 1 % EDTA.

3.7 APPLICATIONS

The proposed method is capable of determining with a high degree of

precision the amount of hypochlorite in samples of tap water, natural water and milk.

The results of analysis of the above samples (Table 3A2 and Table 3A3) compared

favorably with those from a reference method [19,36]. Statistical analysis of the

results by the use of t and F–tests showed that there was no significant difference

between the accuracy and precision of the proposed and reference methods. The

74

precision of the proposed method was evaluated by replicate analysis of samples

containing hypochlorite at different concentrations.

3.8 CONCLUSIONS

1. The reagents provide a simple and rapid method for the spectrophotometric

determination of hypochlorite.

2. The reagents have the advantage of sensitivity and wide range of determinations

without the need for extraction or heating.

3. The method does not involve any stringent reaction conditions and the accuracy of

the method is comparable with most methods reported in the literature.

4. The proposed method has been successfully applied to the determination of traces

of hypochlorite in samples of tap water, natural water and milk. A comparison of

the method reported is made with earlier methods and is given in Table 3A4.

75

FIGURE IIIA1

ABSORPTION SPECTRA OF RHODAMINE B Vs REAGENT BLANK

Wavelength (nm)

0 200 400 600 800 1000

Ab

so

rb

an

ce

0.0

0.5

1.0

1.5

2.0

2.5

FIGURE IIIA2

ABSORPTION SPECTRA OF METHYLENE BLUE Vs REAGENT BLANK

Wavelength (nm)

400 500 600 700 800 900 1000

Ab

so

rb

an

ce

0.0

0.1

0.2

0.3

0.4

76

FIGURE IIIB1

ADHERENCE TO BEER’S LAW FOR HYPOCHLRITE USING RHODAMINE B

AS A REAGENT

H ypochlorite concentration (µgm L

-1

)

0 1 2 3 4 5 6 7

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

FIGURE IIIB2

ADHERENCE TO BEER’S LAW FOR HYPOCHLRITE USING METHYLENE

BLUE AS A REAGENT

Hypochlorite concentration (µgm L

-1

)

0 2 4 6 8 10

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

77

FIGURE IIIC1

EFFECT OF pH ON COLOR INTENSITY USING RHODAMINE B AS A

REAGENT

pH

1 2 3 4 5 6 7 8

Absorbance

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

FIGURE IIIC2

EFFECT OF pH ON COLOR INTENSITY USING METHYLENE BLUE

AS A REAGENT

p H

1 2 3 4 5 6 7 8

Ab

so

rb

an

ce

0 .06

0 .08

0 .10

0 .12

0 .14

0 .16

0 .18

0 .20

0 .22

0 .24

78

SCHEME III

NaOCl + HCl → NaCl + HOCl

KI + HCl → HI + KCl

HOCl + 2HI → I2 + HCl + H

2O

(H3CH

2C)

2N N(CH

2CH

3)2

O

COOH

Cl

I2

(H3CH

2C)

2N N(CH

2CH

3)2

O

COOH

Rhodamine B (colored) Rhodamine B (colorless)

(CH3

)2

N

N

S+ N(CH

3

)2

Cl

-

I2

, H

+

N(CH3

)2

(CH3

)2

N

N

H

S

Methylene blue (colored) Methylene blue (colorless)

79

TABLE 3A1

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF HYPOCHLORITE

USING RHODAMINE B AND METHYLENE BLUE AS REAGENTS (1.0 µgmL-1

)

Foreign ion Tolerance limit

(µg)

Foreign ion Tolerance limit

(µg)

Cd(II)

Zn(II)

Ni(II)

Co(II)

Mg(II)

Ba(II)

Cu(II)*

Fe(III)*

In(III)

V(IV)

Ti(IV)

Al(III)

50

50

75

50

75

25

25

75

50

100

50

50

Mo(VI)

W(VI)

U(VI)

Chloride

Phosphate

Bromide

Nitrate

Acetate

Sulphate

Borate

50

50

100

150

150

250

250

125

100

100

* Masked by masking agents.

80

TABLE 3A2

DETERMINATION OF HYPOCHLORITE IN ENVIRONMENTAL SAMPLES

USING RHODAMINE B AS A REAGENT

Proposed method Reference method [19,36]

Samples

Hypo-

chlorite

added

(µgmL-1

)

Hypo-

chlorite

founda

(µgmL-1

)

Relative

error

Hypo-

chlorite

founda

(µgmL-1

)

Relative

error t-

testb

F-

testc

Tap

water-1

1.00

2.00

3.00

0.98 ± 0.04

2.04 ± 0.02

3.05 ± 0.02

-0.02

+0.02

+0.02

1.04 ± 0.06

2.08 ± 0.02

3.07 ± 0.03

+0.04

+0.02

+0.02

0.67

3.01

0.50

2.25

1.00

2.25

Tap

water-2

1.00

2.00

3.00

1.02 ± 0.01

2.06 ± 0.03

3.07 ± 0.10

+0.02

+0.03

+0.02

1.06 ± 0.01

2.10 ± 0.05

3.09 ± 0.02

+0.06

+0.05

+0.03

4.00

1.33

0.40

1.00

2.72

4.00

Natural

water d

1.00

2.00

3.00

0.97 ± 0.04

1.98 ± 0.06

3.01 ± 0.02

-0.03

-0.01

+0.01

1.06 ± 0.05

2.01 ± 0.10

3.04 ± 0.04

+0.06

+0.01

+0.01

3.00

0.60

1.50

1.56

2.72

4.00

Milk d

1.00

2.00

3.00

1.02 ± 0.03

2.05 ± 0.01

3.04 ± 0.02

+0.02

+0.03

+0.01

1.05 ± 0.04

2.09 ± 0.01

3.06 ± 0.02

+0.05

+0.05

+0.02

1.50

4.00

2.00

1.71

1.00

1.00

a. Hypochlorite concentration (µgmL-1

) ± Standard deviation (n=5)

b. Tabulated t- value for (4,4) degrees of freedom at 95% probability level is 2.306

c. Tabulated F- value for (4,4) degree of freedom at 95% probability level is 6.39

d. Tested and shown to be free from hypochlorite

81

TABLE 3A3

DETERMINATION OF HYPOCHLORITE IN ENVIRONMENTAL SAMPLES

USING METHYLENE BLUE AS A REAGENT

Proposed method Reference method [19,36]

Samples

Hypo-

chlorite

added

(µgmL-1

)

Hypochlorite

founda

(µgmL-1

)

Relativ

e

error

Hypochlorite

founda

(µgmL-1

)

Relativ

e

error

t-testb

F-testc

Tap

water-1

1.00

3.00

5.00

0.994 ± 0.01

3.012 ± 0.01

4.984 ± 0.03

-0.006

+0.004

-0.003

1.014 ± 0.02

3.036 ± 0.01

5.017 ± 0.04

+0.014

+0.012

+0.003

2.00

4.00

1.65

4.00

1.00

1.78

Tap

water-2

1.00

3.00

5.00

1.058 ± 0.02

2.972 ± 0.01

5.028 ± 0.03

+0.018

-0.009

+0.005

1.096 ± 0.02

3.012 ± 0.02

5.032 ± 0.02

+0.096

+0.004

+0.006

2.90

4.00

0.25

1.00

4.00

2.25

Natural

water d

1.00

3.00

5.00

1.022 ± 0.01

3.042 ± 0.02

5.032 ± 0.02

+0.022

+0.014

+0.006

0.986 ± 0.02

3.014 ± 0.015

5.054 ± 0.03

-0.014

+0.005

+0.011

3.60

2.80

1.37

4.00

1.78

2.25

Milk d 1.00

3.00

5.00

1.038 ± 0.02

3.101 ± 0.02

5.028 ± 0.04

+0.038

+0.034

+0.005

1.077 ± 0.03

3.082 ± 0.02

5.032 ± 0.01

+0.077

+0.027

+0.006

2.44

1.46

0.13

2.25

1.00

1.56

a. Hypochlorite concentration (µgmL-1

) ± Standard deviation (n=5)

b. Tabulated t- value for (4,4) degrees of freedom at 95% probability level is 2.306

c. Tabulated F- value for (4,4) degree of freedom at 95% probability level is 6.39

d. Tested and shown to be free from hypochlorite

82

TABLE 3A4


Reagent Method Beer’s law

-1

)

ε (Lmol-1

cm-1

)

-2

)

λmax

(nm)

Ref.

No.

Azure B Spectrophotometry 0.2-1.0 ε = 1.49×104

ss = 3.25×10-4

644 33

Thionin Spectrophotometry 0.2-1.2 ε = 1.489×104

ss = 3.25×10-3

600 36

Proposed

Method

Rhodamine B

Methylene blue

spectrophotometry

spectrophotometry

0.1–4.0

0.1-0.6

ε = 2.57×105

ss = 2.01×10-3

ε = 1.12×104

ss = 4.61×10-3

553

665.6


83

3.9 REFERENCES

1. Asano and Toshio, New food Ind., 18 (1976) 30.

2. I. F. Sporykhin, Otkryt. Izobret, 54 (1977) 204. Chem. Abstr. Vol. 86, p161332a

(1977).

3. S. Hurria, Qum. Ind., 18 (1972) 18.

4. R. W. Samuels, Clin. Toxicol., 12 (1978) 543.

5. L. A. Galamina, L. L Mityushina, V. I. Duda and M. N. Bekhtereva,

Mikrobiologiya, 48 (1979) 470.

6. R. W. Wannemacher, Symposium on Agents of Biological Origin, U.S. Army

Research, Dev. and Engineering Center, Aberdeen proving Ground, MD. (1989)

p.115.

7. A. Bystron and G.Sundqvist, J. Dent. Res, 89 (1981) 321.

8. A. Bystron and G. Sundqvist, Oral Surg. Oral Med. Oral Pathol., 55 (1983) 307.

9. R. Holland, I. J Soares and I. M. Soares, Endod. Dent. Traumatol., 8 (1992) 223.



Edn., (2000).

11. G. Peter and H. Heinz, Talanta, 18 (1971) 147.

12. E. P. Drozdetskaya and K. G. Ilin, Z. Anal. Khim., 27 (1972) 200.

13. M. D. Allan, C. W. Kenneth and N. A. Timothy, Anal. Chem., 51 (1979) 2077.

14. A. V. Terletskaya, N. M. Lukovskaya and N. L. Anatienko, Ukr. Khim. Zh., 45

(1979) 1227.

15. D. F. Marino and J. D. Ingle, J. Anal. Chem., 53 (1981) 455.

16. H. P. Paviet, D. Jacek , H. Thomas, M. Stanislaw, L. Ning-Ping, W. Mark, R.

Andrzej and Z. Zbigniew, (2002) WM’ 02 Conference, February 24-28.

17. H. Iketake, M. Umeda and A. Yamada, J. Tech. Educ. 12 (2005) 9.

18. H. Iketake and A. Yamada, Bunseki Kagaku, 49 (2000) 977.

19. S. Williams, AOAC Official Method of Analysis, 14th

Edn., (1984) 291.

20. L. C. Adam and G. Gordon, Anal. Chem., 67 (1995) 535.

21. H. Anwar, P. Trudell and R. Arnold, J. Pharm. Sci., 59 (1970) 1168.

22. L. Bunikiene and E. Ramanauskas, Chem. Tech., 12 (1970) 57. From: Ref. Zh.,

Khim. 1971, Abstr. No. 9G122

23. B. Fleet and A. Y. W. Ho, Talanta, 19 (1972) 317.

24. U. Isacsson and G. Wettermark, Anal. Chim. Acta, 83 (1976) 227

84

25. P. Tarasankar, G. Ashes and M. S. Durga, Chem. Anal., (Warsaw) 33 (1988) 703.

26. H. Bamnolker, J. Lapid, Y. Givra, Y. Sorek and Z. Gavra, Microchem. J, 40

(1989) 246.

27. D. Gonzalez-Robledo, M. Silva and D. Perez- Bendito, Anal. Chim. Acta, 228

(1990) 123.

28. T. Watanabe, T. Idehara, Y. Yoshimura and H. Nakazawa, J. Chromatogr., 796

(1998) 397.

29. J. Han, C. The-Ching, G. Han, J. Browne, I. Brown and P. Han, Microchem. J, 58

(1998) 218.

30. B. Chiswell and K. R. O'Halloran, Anal. Chim. Acta, 249 (1991) 519.

31. K. Tian and P. K. Dasgupta, Talanta, 52 (2000) 623.

32. M. C. Icardo, J. V. Garcia Mateo and J. M. Calatayud, Analyst, 126 (2001)

2087.

33. B. Narayana, K. Vipin, M. Mathew and N. V. Sreekumar, Indian J. Chem., 43

(2004) 573.

34. F. Kodera, M. Umeda and A. Yamada, Bunseki Kagaku, 53 (2004) 905.

35. C. J. Ballesta, M. C. Valencia Miron and L. F. Capitan Vallvey, Anal. Chim.

Acta, 522 (2004) 267.

36. B. Narayana, M. Mathew, K. Vipin, N. V. Sreekumar and T. Cherian, J. Anal.

Chem., 60 (2005) 706.

37. R. L. Antonio, P. K. Roberta, C. E. Tadeu Gomes and C. C. Cristina Schmitt, J.

Chem. Educ., 82 (2005) 1815.

38. J. G. March and B. M. Simonet, Talanta, 73 (2007) 232.

85

CHAPTER 4

NEW REAGENTS FOR THE SPECTROPHOTOMETRIC

DETERMINATION OF VANADIUM IN ALLOYS, SYNTHETIC AND

PHARMACEUTICAL SAMPLES

4.1 INTRODUCTION


4.3 APPARATUS


4.5 PROCEDURES


4.7 APPLICATIONS

4.8 CONCLUSIONS

4.9 REFERENCES

86

4.1 INTRODUCTION

Vanadium is an essential trace element to man and animals. It was first

discovered by del Rio in 1801 [1-3]. Unfortunately, a French chemist incorrectly

declared Del Rio's new element as only impure chromium and Del Rio thought

himself to be mistaken and accepted the French chemist's statement. The element was

rediscovered in 1830 by Sefstrom, who named the element in honor of the

Scandinavian Goddess Vanadis because of its beautiful multicolored compounds. It

was isolated in nearly pure form by Roscoe in 1867. Vanadium of 99.3 to 99.8 %

purity was not produced until 1922.

Vanadium has abundance in the earth’s crust of about 0.02 %. Vanadium is

found in about 65 different minerals among which carnotite, roscoelite, vanadinite

and patronite are the important sources of the metal [4]. Vanadium is also found in

phosphate rock and certain iron ores, and is present in some crude oils in the form of

organic complexes. It is also found in small percentages in meteorites. High-purity

ductile vanadium can be obtained by the reduction of vanadium trichloride with

magnesium or with magnesium-sodium mixtures. Much of the vanadium being

produced are now made by calcium reduction of V2O5 in a pressure vessel, an

adaptation of a process developed by McKechnie and Seybair. Natural vanadium is a

mixture of two isotopes, 50

V (0.24 %) and 51

V (99.76 %). 50

V is slightly radioactive,

having a half-life of > 3.9×1017

years. Nine other unstable isotopes are recognized.

Pure vanadium is a bright white metal and is soft and ductile. It has good corrosion

resistance to alkalies, sulfuric and hydrochloric acid and salt water, but the metal

oxidizes readily above 660 0

C. The metal has good structural strength and a low

fission neutron cross section, making it useful in nuclear applications. Vanadium is

used in producing rust resistant spring, and high speed tool steels. It is an important

carbide stabilizer in making steels. About 80 % of the vanadium now produced is

used as ferrovanadium or as a steel additive. Vanadium foil is used as a bonding agent

in cladding titanium to steel. Vanadium pentoxide is used in ceramics and as a

catalyst. It is also used in producing a superconductive magnet with a field of 175,000

gauss. Vanadium and its compounds are toxic and should be handled with care.

Ductile vanadium is commercially available and commercial vanadium metal of about

95 % purity.

87

Major sources for the emission of vanadium in the environment include

combustion of fuel oils, dyeing, ceramics, ink, catalyst and steel manufacturing.

Vanadium in trace amounts represents an essential element for normal cell growth,

but it can be toxic when present in higher concentrations. It plays an important role in

physiological systems including normalization of sugar levels and participation in

various enzyme systems as an inhibitor and cofactor of the oxidation of amines [5].

In spite of being a nutritional element, vanadium is not accumulated by the biota. The

only organisms known to bio-accumulate it to any significant degree are some

mushrooms, tunicates and sea squirts. The occurrence of vanadium in sea squirts is

supposed to be one of the main sources of this metal in crude oil and oil shales.

In biology, vanadium ion is an essential component of some enzymes,

particularly the vanadium nitrogenase used by some nitrogen fixing microorganisms.

Vanadium is essential to sea squirts in vanadium chromagen proteins. The

concentration of vanadium in their blood is more than 100 times higher than the

concentration of vanadium in the seawater around them. Rats and chickens are also

known to require vanadium in very small amounts and deficiencies result in reduced

growth and impaired reproduction. Administration of oxovanadium compounds have

been shown to alleviate diabetes mellitus symptoms in certain animal models and

humans. Much like the chromium effect on sugar metabolism, the mechanism of this

effect is unknown.

Vanadium poisoning is an industrial hazard [6]. Environmental scientists have

declared vanadium as a potentially dangerous chemical pollutant that can play havoc

with the productivity of plants, crops and the entire agricultural system. High amounts

of vanadium are said to be present in fossil fuels such as crude petroleum, fuel oils,

coals and lignite. Burning of these fuels release vanadium into the air that then settle

on the soil. Vanadium compounds act chiefly as an irritant to the eyes and respiratory

tract. Exposure may cause conjunctivitis, rhinitis and reversible irritation of the

respiratory tract. More severe cases may cause bronchitis, bronchospasms and asthma

like disease. It may cause polycythemia, red blood cell destruction and anemia,

albuminuria and hematuria, gastrointestinal disorders, nervous complaints and severe

cough [7]. Recently, vanadium has been noticed as the index element in urban

environmental pollution, especially air pollution [8]. Laboratory and epidemiological

88

evidences suggest that vanadium may also play a beneficial role in the prevention of

heart disease [9]. Shamberger has pointed out that human heart disease death rate is

lower in countries where more vanadium occurs in the environment [10]. The

National Institute for Occupational Safety and Health (NIOSH) has recommended that

35 mgm-3

of vanadium be considered immediately dangerous to life and health. This

is the exposure level of a chemical that is likely to cause permanent health problems

or death.

The determination of vanadium provides significant information regarding its

biological effects and the extent of air pollution. Soldi et al. reported that this element

was a useful marker for the potential release of toxic metals from fossil fuels,

especially oils, as it is always present in these materials [11]. Thus, highly and

selective methods are still required for trace vanadium determination in different

kinds of samples.


Several methods have been reported in the literature for the analysis of

vanadium. Various analytical techniques based on fluorescence spectroscopy [12],

atomic absorption spectroscopy [13], inductively coupled plasma-atomic absorption

spectroscopy [14], capillary electrophoresis [15], stripping voltametry [16], neutron

activation analysis [17], high-performance liquid chromatography [18] and ion

exchange separation method [19] are used for its determination.

A survey of literature revealed that a large number of reagents are suitable for

the spectrophotometric determination of vanadium. Telep and Boltz reported

hydrogen peroxide as a reagent for the spectrophotometric determination of vanadium

[20]. The method was based on the reaction of vanadium(V) with H2O

2 in acid

medium to form a reddish-brown colored complex. The complex showed maximum

absorption at 290 nm. Beer's law was valid over the concentration range 0-125 ppm of

vanadium. Eeckhout and Weynants reported diphenylbenzidine as a reagent for the

determination of vanadium [21]. The yellow color resulted from the reaction between

dilute solution of V(V) and diphenylbenzidine was the basis for the

89

spectrophotometric method for the determination of vanadium. Beer's law was valid

over the concentration range 1--1

of vanadium.

Motojima reported oxine as a reagent for the spectrophotometric

determination of vanadium [22]. This method was based on the extraction of

vanadium-oxine complex with chloroform and the complex exhibited an absorption

maximum at 550 nm. Baggett and Huyck reported spectrochemical determination of

vanadium in alkali brines [23]. In this method samples were adjusted to a pH value of

5.0, treated with 8-quinolino1 and quinolates extracted with chloroform. The

chloroform extract was concentrated by evaporation to a known volume and placed on

a graphite electrode previously coated with 20% sodium hydroxide and dried in an

oven with a carbon dioxide atmosphere. Excitation was carried out by a 2300 volt

alternating current arc with photographic recording of the spectra and molybdenum

was used as the internal standard.

Jones and Watkinson described a spectrophotometric method for the

determination of vanadium in plant materials [24]. With minor modifications it was

used to determine vanadium in soils. Priyadarshini and Tandon reported N-benzoyl-

N-phenylhydroxylamine as a reagent for the spectrophotometric determination of

vanadium [25]. Ariel and Manka reported a spectrophotometric method for the

determination of chromium(VI) and vanadium(V) [26]. The presence of iron(III) was

developed by exploiting the color changes which resulted from the oxidation of

o-dianisidine in strong acid medium.

Shibata described a solvent extraction and spectrophotometric determination

of vanadium with 1-(2-pyridylazo)-2-naphthol [27]. Janauer et al. reported a sensitive

and precise spectrophotometric method for the determination of microgram quantities

of vanadium in hydrochloric acid-methanol medium [28]. The photometric reagent

was the azo dyestuff solochrome black-RN, which formed a violet colored complex

with vanadium, which showed maximum extinction at 560 nm. Beer's law obeyed

within the concentration range from 0 to 25 g of vanadium per 10 ml of test solution.

Sailendra Nath and Poddar described spectrophotometric determination of vanadium

with o-hydroxyacetophenone oxime as a reagent [29,30]. Reagents like

p-methoxybenzothiohydroxamic acid [31], 2-(2-thiazolylazo)-5-(diethylamino)phenol

90

[32] and morin [33] were also used as reagents for the spectrophotometric

determination of vanadium. Gagliardi and Ilmaier described a sensitive method for the

spectrophotometric determination of vanadium with 4-(2-pyridylazo)resorcinol(PAR)

[34]. The method was applicable to the analysis of alloys.

Goyal and Tandon described the comparative studies of the reaction of 7-

arylazo-8-hydroxyquinoline-5-sulphonic acid (Azoxine S) dye with vanadium, which

showed that 2:1 yellow, water-soluble complex formed over the pH range 2.5–6.0 and

the phenyl derivative was the most suitable for spectrophotometric determination of

0.2–1.4 ppm of vanadium [35]. The color formed instantaneously and was stable for

about 8 hours. The molar abso max =400 was 1.15×104

equilibrium constant for complex formation was of the order of 102

. These dyes were

used as indicators in the direct complexometric determination of vanadium(IV). The

interference of a number of anions and cations were reported. Tamotsu et al. reported

protocatechuic acid as a spectrophotometric reagent for the determination of

vanadium [36].

Tandon and Bhattacharya used N-aryl hydroxamic acid as a reagent for the

spectrophotometric determination determination of vanadium [37]. Wakamatsu and

Otomo described an extraction and spectrophotometric determination of

vanadium(IV) with Tiron [38]. The vanadium(IV)-Tiron chelate was extracted into a

mixed solvent mixture 1:4, isopentyl alcohol:chloroform in the presence of

1,3-diphenylguanidinium salt. Beer’s law was obeyed upto 36 vanadium per 10

mL of the solvent.

Chakraborti reported 3-hydroxy-1,3-diphenyltriazene and its substituted

derivatives as spectrophotometric reagents for vanadium(V) [39]. Izquierdo and

Lacort described 5,7-dichloro-2-methyl-8-hydroxy-quinoline as a reagent for the

vanadium determination by spectrophotometry [40]. Satyanarayana and Mishra

reported 1,2,3-phenyloxyamidine as a reagent for solvent extraction and

spectrophotometric determination of vanadium(V) [41]. The course of investigations

on the development of organic analytical reagents were able to introduce a type of

functional group for metal ions. 1,2,3-Phenyloxyamidine possessed several useful

properties as an analytical reagent. It was stable and can be readily prepared from

91

common laboratory chemicals. The reagent has great potentialities for the

colorimetric and gravimetric determination of metal ions. Studies carried out in these

laboratories showed that this was an excellent reagent for the spectrophotometric

determination of vanadium(V) by solvent extraction and for the gravimetric

determination of copper and nickel with the functional group.

Naoichi et al. described a spectrophotometric determination of vanadium(V)

with N-benzoyl-N-phenylhydroxylamine [42]. Vojkovic et al. reported the application

of 1-phenyl-2-methyl-3-hydroxy-4-pyridone (HX) for the spectrophotometric

determination of vanadium(V) by extraction into chloroform [43]. The method was

based on the extraction condition and three types of complexes were formed. At pH

1.0-2.2, an orange colored complex of the composition with a maximum absorption at

497 nm was formed. However, at 0.75-1.25 M hydrogen ion concentration and in the

presence of the excess of chloride ions a blue colored complex of the composition

VO2Cl(HX)

2 with a maximum absorption at 625 nm was found. In the presence of an

excess perchlorate ions and at 0.3-0.4 M hydrogen ion concentration a blue colored

complex of composition VO2ClO

4(HX)

3 with a maximum absorption at 605 nm was

established. The latter complex was not recommended for the determination as an

excess of perchlorate influenced the absorption. Procedures for the determination at

497 or 625 nm were very fast and simple. The complexes were also isolated in

crystalline form and identified by elemental analysis and infrared spectroscopy. The

molar absorptivity at 497 nm was 4100 Lmol-1

cm-1

and at 625 nm 5600 Lmol-1

cm-1

.

Uchida et al. reported a spectrophotometric determination of vanadium(V)

with 2-nitroso-5-dimethylaminophenol [44]. Nardillo and Catoggio described a

spectrophotometric determination of vanadium with 3-methyl catechol in alloy steels

[45]. Reagents like 1-(4-tolyl)-2-methyl-3-hydroxy-4-pyridone [46], 4-(4,5-dimethyl-

2-thiazolylazo)-2-methylresorcinol [47] and N-methylaminothioformyl-N'-

phenylhydroxylamine [48] were used for the spectrophotometric determination of

vanadium.

Akama et al. described a spectrophotometric determination of vanadium(V)

using 4-benzoyl-3-methyl-1-phenyl-5-pyrazolone [49]. Bag et al. reported

spectrophotometric method for the determination of vanadium with

92

2:2′-diaminodiphenyldisulphide in strong acidic solution [50]. Vanadium(V) formed a

1:1 cornflower blue colored complex with 2:2′-diaminodiphenyldisulphide in 18 N

sulfuric acid solution which passed onto 1:2 complex with large amount of reagent.

The absorption maxima of the complexes were 590 nm and 700 nm respectively. The

Beer’s law was obeyed in the concentration range 8-36 ppm. The percent relative

error was 2.72. The composition of the complexes were determined by the modified

jobs and molar ratio method. The calculated dissociation constants were 1.6×10–2

and

5×10–9

at 25 °C. The molar extinction coefficient was 1100, while the Sandell’s

sensitivity was 0.046 µgcm-2

.

Reddy and Reddy reported extraction and spectrophotometric determination of

vanadium [51]. In this method vanadium(V) formed a 1:1 yellow colored complex

with salicylaldehyde thiosemicarbazone in n-butanol. The yellow colored complex

was quantitatively extracted from acetic acid medium into n-butanol. Beers law was

obeyed in the range 0.5-6.5 ppm of the metal. Large number of foreign ions did not

interfere.

Montelongo et al. reported a spectrophotometric determination of

vanadium(V) with 4-(1 -1 -triazolyl-3 -azo)-2-methylresorcinol [52]. The

method was based on the reaction of vanadium with the reagent at pH 8.10

(Tris-HClO4 buffer solution), produced a pink-violet, 1:1 complex (λmax=525 nm,

ε=2.55×104

Lmol–1

cm–1

) in a 50% methanol-water medium, which was the basis for

the spectrophotometric determination of 0.1 to 1.51 ppm of vanadium. The method

was applied for the determination of the vanadium content in low alloy steels.

Salinas and Arrabal described a method for the extract and spectrophotometric

determination of vanadium(V) [53]. The violet colored complex formed with

isophthaldihydroxamic acid was extracted into trioctylmethylammonium chloride in

ethylacetate (λmax=380 nm, ε=7.50×103

Lmol–1

cm–1

; λmax=510 nm, ε=5.51×103

Lmol–1

cm–1

) and the range of the determination was 14-80 µg.

Escriche et al. reported a spectrophotometric method for the determination of

vanadium [54]. The method was based on the oxidation of pyrogallol red and

vanadium was determined by the decrease in absorbance of its characteristic band at

93

490 nm and at pH 4. The decrease in absorbance was proportional to the

concentration of vanadium(V) over the range 0–1.83 ppm. The limit of detection of

vanadium was found to be 0.05 ppm. In the presence of potassium bromate the

determination was possible in the ppb levels. The method described the study of the

selectivity of the method with respect to possible interference from 20 species

contained in ferrous and non-ferrous alloys, which was classified according to their

possible mechanism of interference.

Abdullah et al. reported thiophene-2-hydrazide as a reagent for the

spectrophotometric determination of trace amount of vanadium in aqueous solution

[55]. The intense yellow, water-soluble, stable and binary complex formed in acidic

medium was used for the determination of 0.5–5 ppm of vanadium ion with a molar

absorptivity of 12.1×103

Lmol–1

cm–1

at 410 nm. Moreover, the color formation was

very fast. Interferences due to foreign ions were examined.

El-Shahat et al. reported phenylfluorone as a reagent for the

spectrophotometric determination of vanadium [56]. The method was based on the

formation of a 1:1 complex of vanadium - phenylfluorone exhibited an absorption

maximum at 520 nm. The Beer's law was valid over the concentration range of 2-15

µg of vanadium in 10 mL at pH 4. The relative standard deviation was 2 % and the

molar absorptivity of the system was 2.1×104

Lmol-1

cm-1

.

Bhaskar and Surekha reported 2-acetylpyridine thiosemicarbazone as a reagent

for the spectrophotometric determination of vanadium [57]. Vanadium formed a

golden yellow complex at pH 3.5 with 2-acetylpyridine thiosemicarbazone in aqueous

medium. The complex exhibited maximum absorbance at 400 nm, with molar

absorptivity of 5.6×103

Lmol-1

cm-1

. The metal and ligand stoichiometric ratio was 1:1

and the Beer's law was valid over the concentration range 0-8 ppm of vanadium.

Svjetlana and Vladimir reported desferrioxamine-B as a reagent for the

determination of vanadium [58]. A naturally occurring trihydroxamic acid,

desferrioxamine-B, reacted with the vanadium(V) ion in strong acidic aqueous

94

solution and produced a stable 1:1 complex. This red-violet chelate used for the

spectrophotometric determination of trace amounts of vanadium(V). Molar

absorptivity of the system was 3.15×103

Lmol-1

cm-1

and Beer's law was valid over the

vanadium concentration range 0.5-50 ppm.

Krasiejko and Marczenko presented a sensitive and highly selective method

for the spectrophotometric determination of microgram amounts of vanadium(V) [59].

First, vanadium was isolated by extraction with N-benzoyl-N-phenylhydroxylamine

(BPHA) in chloroform from 4 M hydrochloric acid medium. Then, chloroform was

evaporated and the residue mineralized with mixture of concentrated perchloric and

nitric acid. Finally, a color reaction of vanadium(V) separated with 2-(5-bromo-2-

pyridylazo)-5-diethylaminophenol (5-Br-PADAP) in an acetate buffer (pH 4.5). The

molar absorptivity of the method was 5.48×104

Lmol–1

cm–1

at 585 nm. The proposed

method was applied for the determination of traces of vanadium in aluminium

samples. The results obtained showed a good precision and accuracy of the method.

Eshwar and Sharma used 1-(2'-thiazolylazo)-2-naphthol as reagent for the

extractive spectrophotometric determination of vanadium in high speed steel [60].

The sparingly soluble complex formed between vanadium(V) and 4-(2-thiazolylazo)

resorcinol was extracted with chloroform. The complex exhibited an absorption

maximum at 610 nm with molar absorptivity 1.50×104

Lmol-1

cm-1

. Beer's law was

valid over the concentration range 0.08-2.24 -1

of vanadium.

Marczenko and Lobinski described an extraction and spectrophotometric

determination of trace amounts of vanadium with 3,5-dinitrocatechol(DNC) and

brilliant green(BG) [61]. Beer's law was obeyed up to a vanadium concentration of

0.3-1

and the molar absorptivity was 1.7×105

Lmole cm at 630 nm. The

molar ratios of the components and the form of the vanadium(V) cation in the

extracted compound was determined and the formula [VO(OH)(DNC)2

2][BG

+

]2 was

proposed. Titanium, molybdenum, tungsten, EDTA and thiocyanate interfered

seriously. The proposed method has been applied to determination of traces of

vanadium (about 10 %) in alums. Mandelohydroxamic acid [62], alizarine

95

complexone and cetylpyridinium halides [63] were also reported as reagents for the

determination of vanadium in steels.

Kshatriya and Basant developed a selective and sensitive method for the

spectrophotometric determination of vanadium(V) in biological materials with

N-benzylpalmito hydroxamic acid (BPHA) [64]. Vanadium(V) was extracted with

BPHA into chloroform at 3-7 M HCl. The reagent reacted with vanadium to form a

reddish-violet complex with molar absorptivity of 3.79×103

Lmol-1

cm-1

at 500 nm.

Beer's Law was obeyed in the concentraction range of 2-8 ppm vanadium.

Aman et al. reported an improved spectrophotometric determination of

vanadium using benzidine-phosphoric acid [65]. Vanadium reacted with benzidine in

acidic medium, which formed a pink colored complex showed maximum absorbance

at 520 nm. The method was successfully applied to the determination of vanadium in

thermal gas turbine deposits and fuel oil sludge.

Yang et al. reported diantipyryl-(3, 4-dioxymethenyl) phenylmethane as a

reagent for the determination of vanadium in herbal medicine [66]. Molar absorptivity

of the system was 3.21×105

Lmol-1

cm-1

at 470 nm. Beer's law was obeyed in the range

of 0.2-

acids [67] were also reported as reagents for the determination of vanadium in

pharmaceutical and steel samples.

Biao and Rong reported chlorpromazine as a sensitive reagent for the

determination of vanadium [68]. Chlorpromazine reacted with vanadium(V) at room

temperature to form a bright red complex, which exhibited an absorption maximum at

520 nm. Beer's law was obeyed in the concentration range of 2-

10 mL with molar absorptivity of 5.10×103

Lmol-1

cm-1

.

Costa et al. reported a simple and sensitive spectrophotometric method for the

determination of vanadium(IV) using 2-(5-bromo-2-pyridylazo)-5-

diethylaminophenol (Br-PADAP) [69]. The method was based on the oxidation of

vanadium(IV) to vanadium(V) by the addition of iron(III) cation, followed by a

complexation reaction of iron(II) with a spectrophotometric reagent (Br-PADAP).

96

The iron(II) reacted with Br-PADAP which formed a stable complex with a large

molar absorptivity. The vanadium(IV) determination was possible, with a calibration

sensitivity of 0.549 for an analytical curve of 18.8 ngm1-1

,

molar absorptivity of 2.80×104

Lmol-1

cm-1

and a detection limit of 5.5 ngm1-1

. The

proposed method was applied for the vanadium(IV) determination in the presence of

several amounts of vanadium(V

the accuracy obtained were satisfactory (R.S.D. < 2%). Reagents such as N-

phenylcinnamohydroxamic acid and azide [70], 2'-hydroxyacetophenone

benzoylhydrazone [71,72] were also reported for the spectrophotometric

determination of vanadium.

Ahmed and Banoo developed a sensitive, fairly selective direct

spectrophotometric method for the determination of trace amount of vanadium(V)

with 1,5-diphenylcarbohydrazide [73]. The reagent 1,5-diphenylcarbohydrazide

(DPCH) reacted in slightly acidic (0.0001–0.001 M H2SO

4 or pH 4.0–5.5) 50%

acetone media with vanadium(V) to give a red–violet chelate which showed

maximum absorption at 531 nm. The average molar absorption coefficient and

Sandell’s sensitivity were found to be 4.23×104

Lmol cm and 10 ngcm of

vanadium respectively. Linear calibration graph were obtained for 0.1– of

vanadium. The stoichiometric composition of the chelate was 1:3 (V:DPCH). The

reaction was instantaneous and absorbance remain stable for 48 h. The interference

of

vanadium. The method was successfully used in the determination of vanadium in

several standard reference materials (alloys and steels), environmental waters (potable

and polluted), biological samples (human blood and urine), soil samples, solution

containing both vanadium(V) and vanadium(IV) and complex synthetic mixtures. The

).

Agnihotri et al. reported a highly sensitive and selective spectrophotometric

determination of vanadium(V) using 6-chloro-3-hydroxy-7-methyl-2-(2-thienyl)-4H-

chromen-4-one as a complexing agent in a weakly acidified (HCl, pH 0.84–1.09)

medium [74]. The greenish-yellow complex was quantitatively extracted into carbon

tetrachloride and showed maximum absorbance at 417–425 nm. The method obeys

97

of vanadium having molar absorptivity and Sandell’s

sensitivity of 8.26×104

Lmol-1

cm-1

of vanadium respectively. The

method applied to the determination of vanadium in steels, reverberatory flue dust and

water samples. 6-Chloro-3-hydroxy-2-[2'-(5'-methylfuryl)]-4H-chromen-4-one used

as a reagent for the determination of vanadium in various synthetic samples [75].

Vanadium reacted with 6-chloro-3-hydroxy-2-[2'-(5'-methylfuryl)]-4H-chromen-4-

one, which formed a dark yellow (1:1) colored species exhibited an absorption

maximum at 432 nm. Beer's law was valid over the concentration range 0.2-1.4

-1

of vanadium. Molar absorptivity and Sandell's sensitivity of the system was

3.98×104

Lmol-1

cm-1 -2

respectively. The reagents 5,7-dichlorooxine,

rhodamine-6G [76] and isothipendyl hydrochloride [77] were also used and reported

for the determination of vanadium in steels and minerals.

Mohamed and Fawy reported a catalytic spectrophotometric method for the

determination of vanadium in seawater samples [78]. The method was based on the

catalytic effect of vanadium on the bromate oxidative coupling reaction of metol with

2,3,4-trihydroxybenzoic acid (THBA). The optimum reaction conditions are 6.4×10-3

M of metol, 2.0×10-3

M of THBA and 0.16 M of bromate at 35º C and in the presence

of an activator-buffer solution of 1.0×10-2

M of tartarate (pH=3.10). The reagent

phenothiazine derivatives were also reported for the determination of vanadium in

steels, minerals, biological samples and soil samples [79].

Di et al. reported a spectrophotometric method for the determination of

vanadium(V) based on the formation of tungstovanadophosphate-3,3',5,5'-

tetramethylbenzidine-N-propanesulfonic (TMBPS) charge transfer complex [80]. The

spectrophotometric measurements were directly carried out at 450 nm and the

apparent molar absorptivity was 2.74×104

Lmol-1

cm-1

. The linear range of the

determination was 0.02-1.0 1

. The sensitivity was enhanced with a flotation-

extraction preconcentration method and the apparent molar absorptivity was 3.10×105

Lmol-1

cm-1

.

Dian-Wen and Li-Xian reported arsenazo-M as a reagent for the determination

of vanadium in iron ores [81]. This method was based on decolorizing reaction of

arsenazo-M by vanadium(V) in H2SO

4 medium. The decrease in color was directly

proportional to the amounts of vanadium. The maximum absorption was at 547 nm

98

and the molar absorption coefficient was 1.04×103

Lmol-1

cm-1

. Beer’s law was valid

over the concentration range 0- -Hua et al. used

2-(5-carboxy-1,3,4-triazolylazo)-5-diethylamino benzoic acid [82] as a reagent for the

spectrophotometric determination of vanadium in an aluminium alloy sample.

Agnihotri et al. reported 2,4-dihydroxyacetetophenonebenzoylhydrazone

(DABH) and pyridine as reagents for the spectrophotometric determination of

vanadium in variety of synthetic samples [83]. The method was based on the

formation of light brown complex of vanadium with 2,4-dihydroxyacetophenone

benzoylhydrazone and pyridine. The molar absorptivity and relative standard

deviation of the method was 2.83×104

Lmol-1

cm-1

and 0.19 % for vanadium

-1

respectively. Beer's law was valid over the vanadium

concentration range 0--1

. 2-(2-Quinolylazo)-5-diethylaminophenol was also

used as spectrophotometric reagent for the determination of vanadium in water and

biological samples [84].

Cherian and Narayana reported a simple and sensitive spectrophotometric

method for the determination of trace amounts of vanadium using thionin as a

chromogenic reagent [85]. The method was based on the reaction of vanadium(V)

with potassium iodide in acidic medium to liberate iodine. Bleaching of the violet

color of thionin by the liberation of iodine was the basis of the determination and was

measured at 600 nm. Beer's law was obeyed over the range of 0.2--1

of

vanadium. The molar absorptivity, Sandell's sensitivity, detection limit and

quantitation limit of the method were found to be 2.298×104

Lmol-1

cm-1

, 0.520×10-2

-2 -1 -1

respectively. The method was applied to the

analysis of vanadium in synthetic and alloy samples.

Kiran Kumar and Revanasiddappa reported variamine blue as a reagent for the

spectrophotometric determination of trace amounts of vanadium [86]. The method

was based on the oxidation of variamine blue to a violet colored species on reaction

with vanadium(V), having an absorption maximum at 570 nm. Beer’s law was obeyed

in the range of 0.1-2.0 -1

. The molar absorptivity and Sandell’s sensitivity were

found to be 1.65×104

Lmol-1

cm-1 -2

respectively. Optimum reaction

conditions were evaluated in order to delimit the linear range. The effect of interfering

99

ions on the determination was described. The proposed method was successfully

applied to the determination of vanadium in steel, pharmaceutical, environmental and

biological samples.

Mastoi et al. developed a spectrophotometric method for the determination of

vanadium with 2-pyrrolealdehyde phenylsemicarbazone (PPS) [87]. The linear

calibration curve was obtained with 2.5-20 -1

of vanadium. Copper(II),

cobalt(II), iron(II) and palladium(II) were also determined separately using PPS with

linear calibration curves within 2.5-12.5, 5-15, 2.5-15 and 1--1

at 362, 355,

355 and 365 nm, respectively. The vanadium in crude oil was determined with

relative standard deviation of 2.5-5.0%. The method has been applied for the analysis

of copper from copper wires, cobalt from pharmaceutical preparation and palladium

from palladium on barium sulphate with RSD within 2.6-4.5%.

Sao et al. reported a reagent system using rhodamine-B dye for the

determination of vanadium [88]. The method was based on the reaction of vanadium

with acidified potassium iodide to liberate iodine. Bleaching of the pink color of

rhodamine-B by the liberation of iodine was the basis of the determination and was

measured at 553 nm. Beer's law was obeyed over the concentration range of 2-

of vanadium in final solution volume of 25 mL (0.08-0.64 ppm). The apparent molar

absorptivity and Sandell's sensitivity were found to be 1.3×105

Lmol-1

cm-1

and 0.0009

-2

respectively. The method was simple, sensitive and satisfactorily applied to

ppm level for the determination of vanadium in different environmental and

biological samples.

Qi-Li and De-Yun described a simple and highly sensitive spectrophotometric

method for the determination of vanadium(V) [89]. The method was based on

catalytic oxidation of 1,8-dihydroxynaphthalene-3,6-disulfonic acid and

phenylhydrazine with potassium chlorate. The molar absorptivity was 7.8×106

at the

wavelength of 506 nm, detection range was 0.2-5.0 ngmL-1

. It was successfully

applied to the determination of trace amounts of vanadium in spring water, black

bean, corn, tea leaves, and rhodiola schalinensis A leaves.

100

Xianzhong and Yun developed a spectrophotometric determination of

vanadium in carbonaceous shales (stone coal ores) [90]. The method was based on the

reaction of vanadium(V) with the chromophore reagent 2-(5-bromo-2-pyridylazo)-5-

diethylaminophenol (5-Br-PADAP) in the presence of hydrogen peroxide. In a 0.072

M sulfuric acid medium, 5-Br-PADAP reacted with vanadium(V) to form a red-violet

complex with maximum absorption peak at 596 nm with an apparent molar absorption

coefficient of the complex of 8.45×104

Lmol cm . Beer's law was obeyed in the

range 0–

0.9995. Interferences due to various non-target ions were also investigated and high

quantities of other common inorganic ions were tolerable. The method involved the

dissolution of the ore sample by Na2O

2 fusion, followed by filtering of the alkali

solution after which Fe(III), Cu(II), Ni(II) and Co(II) etc. were effectively separated

from the solution by precipitation in a NaOH solution. Selectivity was increased with

the use of EDTA as a masking agent. The vanadium in ore sample was determined

with a relative deviation (RSD) between 0.20 and 0.76 %, and has been successfully

applied to the determination of vanadium-bearing stone coal ores. The results

indicated that the accuracy of 5-Br-PADAP spectrophotometry was comparable with

the ICP-AES method.

Kumar et al. developed a facile, sensitive, selective and rapid

spectrophotometric method for the determination of trace amounts of vanadium(V) in

various samples [91]. The method was based on the interactions of

3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with N-(1-naphthyl)

ethylenediamine dihydrochloride (NEDA) in the presence of vanadium formed a blue

colored derivative or on oxidation of dopamine hydrochloride (DPH) by vanadium in

acidic medium and coupling with MBTH, yielded pink colored derivative. The blue

colored derivative having an absorbance maximum at 595 nm was stable for 9 days

and the pink colored derivative with maximum absorption 526 nm was stable for 5

days. Beer’s law was obeyed for vanadium in the concentration range 0.05–6.0-

1

(blue color derivative) and 0.06–7.0 -1

(pink color derivative), respectively.

The optimum reaction conditions and other important analytical parameters were

established. Interference due to various non-target ions was also investigated. The

proposed methods were applied to the analysis of vanadium(V) in environmental,

biological, pharmaceutical and steel samples.

101

Kumar et al. described a simultaneous second-derivative spectrophotometric

determination of cobalt and vanadium using 2-hydroxy-3-methoxybenzaldehyde

thiosemicarbazone (HMBT) [92]. HMBT reacted with Co(II) and vanadium(V) at pH

6.0 formed green-colored complexes in aqueous dimethyl formamide. The second

derivative spectrum of Co(II) complex showed a zero amplitude at 434.5 nm and a

large amplitude at 409.5 nm, while the V(V) complex showed a sufficient amplitude

at 434.5 nm and a zero amplitude at 409.5 nm. The derivative amplitudes obeyed

Beer’s law at 409.5 and 434.5 nm for Co(II) and V(V) in the range 0.059–3.535 and

0.051–-1

respectively. This enabled the simultaneous determination of

Co(II) and V(V) without separation. Foreign ions did not interfere in the present

method. The method was applied to the simultaneous determination of Co(II) and

V(V) in synthetic mixtures and alloy steel samples. However, most of the reported

methods suffer from a number of limitations, such as interference by a large number

of ions, low sensitivity and need extraction into organic solvents. Therefore a simple

and reliable spectrophotometric method for the determination of vanadium is clearly

recognized.

The present work is to develop a simple spectrophotometric method for the

determination of vanadium using toluidine blue, safranine O and leuco xylene cyanol

FF. The developed method has been successfully applied to the analysis of the

vanadium in alloys, synthetic and pharmaceutical samples.

102

4.3 APPARATUS


quartz cell was used. A WTW pH 330 pH meter was used.



distilled water was used throughout the study. Vanadium stock solution

(1000 µgmL-1

) was prepared by dissolving 0.2395 g of Na3VO

4 in 100 mL of water

and standardized volumetrically [93]. The following reagents were prepared by

dissolving appropriate amounts of reagents in distilled water. Toluidine blue (0.05 %),

safranine O (0.1 %), leuco xylene cyanol FF (0.1 %) (0.1 g of xylene cyanol FF was

dissolved in 25 mL of water containing 30 mg of zinc dust and 2 mL of 1 M acetic

acid, stirred well and kept aside for 20 minutes. The resulting solution was then

diluted to 100 mL with water), hydrochloric acid (2 M), potassium iodide (2 %),

sodium acetate solution (1 M) and sulfuric acid (0.05 M).

4.5 PROCEDURES

4.5.1 Using Toluidine Blue as a Reagent


of vanadium solution

were transferred into a series of 10 mL calibrated flasks. A volume of 1 mL of 2 %

potassium iodide solution was added followed by 1 mL of 2 M hydrochloric acid and

the mixture was gently shaken until the appearance of yellow color, indicating the

liberation of iodine. A 0.5 mL of 0.05 % toluidine blue solution was then added to it

followed by the addition of 2 mL of 1 M sodium acetate solution and the reaction

mixture shaken for 2 minutes. The contents were diluted to 10 mL with distilled water

and mixed well. The absorbance of the resulting solutions were measured at 628 nm

against the corresponding reagent blank. A reagent blank was prepared by replacing

the analyte(vanadium) solution with distilled water. The absorbance corresponding to

the bleached color which in turn corresponds to the analyte(vanadium) concentration

was obtained by subtracting the absorbance of the blank solution from that of test

103

solution. The amount of the vanadium present in the volume taken was computed

from the calibration graph (Figure IVB1).

4.5.2 Using Safranine O as a Reagent


of vanadium were

transferred into a series of 10 mL calibrated flasks. A volume of 1 mL of 2 %



liberation of iodine. A 0.5 mL of 0.1 % safranine O solution was then added to it

followed by the addition of 2 mL of 1 M sodium acetate solution and the reaction




the analyte(vanadium) solution with distilled water. The absorbance corresponding to

the bleached color which in turn corresponds to the analyte(vanadium ) concentration




4.5.3 Using Leuco Xylene Cyanol FF as a Reagent (LXCFF)


of vanadium were

transferred into a series of 10 mL calibrated flasks. Then, volumes of 0.5 mL of the

0.05 M H2SO

4 and 0.7 mL of the 0.1% LXCFF were added and the mixture was kept

on a water bath (≈90°C) for 15 minutes, after being cooled to room temperature

(27 ± 2°C), the contents were diluted to the mark with sodium acetate buffer of pH 4,

and mixed well. The absorbance of the xylene cyanol FF dye formed was then

measured at 614 nm against the reagent blank prepared in the same manner, without

vanadium. The amount of the vanadium present in the volume taken was computed


4.5.4 Determination of Vanadium(V) in Vanadium Steel and Synthetic Mixtures

An accurately weighed amount of vanadium steel (~0.5 g) was treated with 15

mL of concentrated sulfuric acid and 1 mL of concentrated nitric acid and the solution

104

boiled gently to dissolve the sample. The oxides of nitrogen formed were expelled,

the solution was cooled and diluted to 50 mL with double distilled water. Chromium

was extracted with 5 mL of methyl isobutyl ketone [88]. A 0.01 M solution of

potassium permanganate was added dropwise until the solution appeared pink. The

solution was allowed to stand for 5 minutes, warmed and 0.05 M oxalic acid solution

added slowly with stirring until the pink color of the solution was discharged. The

solution was diluted to 100 mL with distilled water. Using the suitable aliquot of the

solution vanadium content was determined using the proposed procedure.

Synthetic mixtures were prepared by mixing exact concentration of different

metal ions keeping the composition of the synthetic mixture as constant and 1 mL of

this sample solution was used for the determination of vanadium(V) according to the

procedure described above. The results are listed in Table 4B1, 4B2, 4B3, 4C1, 4C2

and 4C3.

4.5.5 Determination of Vanadium(V) in Pharmaceutical Sample

A volume of 10 mL of neogadine elixir (Raptakos Brett & Co. Ltd. Mumbai,

India) sample was treated with 10 mL of concentrated HNO3 and the mixture was

then evaporated to dryness. The residue was leached with 5 mL of 0.5 M H2SO4. The

solution was diluted to a known volume with water after neutralizing with dilute

ammonia. An aliquot of the made up solution was analysed for vanadium according to

the general procedure for vanadium determination. The results are listed in Table

4D1.



4.6.1.1 Using toluidine blue as a reagent

This method involves the liberation of iodine by the reaction of vanadate with

potassium iodide in an acidic medium. The liberated iodine bleaches the blue color of

toluidine blue and absorbance of the solution is measured at 628 nm. This decrease in

absorbance is directly proportional to the vanadium concentration. The absorption

spectrum of colored species of toluidine blue is presented in Figure IVA1 and reaction

system is presented in Scheme IV.

105

4.6.1.2 Using safranine O as a reagent

This method involves the liberation of iodine by the reaction of vanadate with

potassium iodide in an acidic medium. The liberated iodine bleaches the pinkish red

color of safranine O and absorbance of the solution is measured at 530 nm. This

decrease in absorbance is directly proportional to the vanadium concentration. The

absorption spectrum of colored species of safranine O is presented in Figure IVA2

and reaction system is presented in Scheme IV.


In this method vanadium quantitatively oxidize leuco xylene cyanol FF into

its blue colored xylene cyanol FF dye in a sulfuric acid medium ( pH 1.4-3.9 ) on a

boiling water bath (∼90°C for 15 minutes); the resulting colored dye shows a

maximum absorbance at 614 nm in an acetate buffer medium ( pH 4.0-4.5 ). The

reagent blank have negligible absorbance at this wavelength. The absorption spectra

of the colored species of LXCFF are presented in Figure IVA3 and reaction system is

presented in Scheme IV.

4.6.2 Effect of the Reagent Concentration and Acidity

4.6.2.1 Using toluidine blue and safranine O as reagents

The effect of iodide concentration and acidity on the reaction system is studied

with 2 µgmL-1

vanadium. The oxidation of iodide to iodine by vanadium is effective

in the pH range 1.0-1.5, which can be maintained by adding 1 mL of 2 M HCl in a

final volume of 10 mL. The liberation of iodine from potassium iodide in an acidic

medium is quantitative. It is found that 1 mL of 2 % KI and 1 mL of 2 M HCl are

sufficient for the liberation of iodine from iodide by vanadium. A 0.5 mL of each

0.05 % toluidine blue and 0.1% safranine O is used for subsequent decolorization.

Constant and maximum absorbance values are obtained in the pH range of

4±0.2. Hence the pH of the reaction system is maintained at 4±0.2 throughout the

study. This can be achieved by the addition of 2 mL of 1 M acetate buffer solution in

a total volume of 10 mL. The maximum absorbance is obtained instantaneously and

requires no heating under the reaction conditions. Under the optimum reaction

106

conditions, toluidine blue and safranine O reaction systems are found to be stable for

a period of 4 hours.


The oxidation of LXCFF by vanadium is studied. Of the various acids

(sulfuric acid, hydrochloric acid and phosphoric acid) studied, sulfuric acid is found to

be the best acid for the system. Constant absorbance readings are obtained in the 0.1-

1.5 mL range of 0.05 M sulfuric acid (or pH 1.4-3.9) at a temperature 90°C for 15

minutes. An increase of the pH above 3.9 markedly affected the stability and

sensitivity of the dye. Color development did not take place below pH 1.4. Hence a

volume of 0.5 mL of 0.05 M sulfuric acid (or maintained pH=2) in a total volume of

10 mL is used in all subsequent work.

The optimum concentration of LXCFF leading to maximum color stability is

found to be 0.7 mL of 0.1 % reagent per 10 mL of the reaction mixture. The

absorbance values are measured in the pH range of 3.5-4.0. This can be achieved by

adding 3 mL of acetate buffer of pH=4. Appreciable results are obtained when the

entire reaction mixture is diluted with the same acetate buffer solution of pH=4. A

change in the pH of the final reaction mixture is affected by the intensity of the

colored dye. The formed colored dye is stable for more than 24 hours.




solutions varying vanadium concentration. A straight line graph is obtained by

plotting absorbance against concentration of vanadium. Beer’s law is obeyed in the

range of 0.4–8.0 µgmL–1

of vanadium (Figure IVB1). The molar absorptivity and

Sandell’s sensitivity of the system is found to be 2.141×104

Lmol-1

cm-1

and 2.36×10-3

µgcm-2

respectively. Correlation coefficient (n=10) and slope of the calibration curve

are 0.995 and 0.141 respectively. The detection limit (DL=3.3σ/s) and quantitation

limit (QL=10σ/s) [where σ is the standard deviation of the reagent blank (n=5) and s is

the slope of the calibration curve] of vanadium determination are found to be 0.234

µgmL-1

and 0.709 µgmL-1

respectively.

107


Adherence to Beer’s law is studied by measuring the absorbance values of






Lmol-1

cm-1

, 1.66×10-3

µgcm-2

respectively. Correlation coefficient (n = 10) and slope of the calibration

curve are 0.997 and 0.144 respectively. The detection limit (DL=3.3σ/s) and

quantitation limit (QL=10σ/s) [where σ is the standard deviation of the reagent blank

(n=5) and s is the slope of the calibration- curve] for vanadium determination are

found to be 0.635 µgmL-1

and 1.920 µgmL-1

respectively.







Sandell’s sensitivity of the colored system is found to be 1.16×104

Lmol-1

cm –1

and

4.38×10-3 -2

respectively. The detection limit (DL=3.3 σ/s) and quantitaion limit

(QL=10 σ/s) [where σ is the standard deviation of the reagent blank (n=5) and s is

the slope of the calibration- curve] for vanadium determination are found to be 0.027

µgmL-1

and 0.08 µgmL-1

respectively.


The effect of various ions at microgram levels on the determination of

vanadium is examined. The tolerance limits of the interfering species are established

at those concentrations, which caused not more than ±2.0 % changes in the

absorbance value during the determination of a fixed amount of vanadium (2 µgmL-1

).

The tolerance limits of the foreign ions are given in Table 4A1 and 4A2. In this

reaction system, various oxidants such as Cu2+

, Cr6+

, Fe3+

, iodate and periodate

interfered. Interference of chromium can be removed by extracting with 5 mL methyl

isobutyl ketone [94]. Iron and copper can be masked with sodium fluoride and 2-

108

mercaptoethanol respectively. However, the tolerance level of other ions may be

increased by the addition of 1 mL of 1 % EDTA.

4.7 APPLICATIONS

The developed method is applied to the quantitative determination of

vanadium in alloys, synthetic and pharmaceutical samples, the results are summarized

in Table 4B1, 4B2, 4B3, 4C1, 4C2, 4C3 and 4D1 respectively. The precision of the

proposed method is evaluated by replicate analysis of samples containing vanadium at

five different concentrations.

4.8 CONCLUSIONS

1. The reagents provide simple method for the spectrophotometric determination of

vanadium.

2. The developed method does not involve any extraction step and hence the use of

organic solvents, which are generally toxic are avoided.


offers the advantages of high stability of the reaction system for toluidine blue

(more than 4 hours), safranne O (more than 5 hours) and leuco xylene cyanol FF

(more than 24 hours).

5. The developed method has been successfully applied to the analysis of the

vanadium in alloy samples, synthetic mixtures and pharmaceutical samples. A

comparison of the method reported is made with earlier methods and is given in

Table 4D2.

109

FIGURE IVA1

ABSORPTION SPECTRUM OF COLORED SPECIES OF TOLUIDINE BLUE

Wavelength (nm)

200 300 400 500 600 700 800 900

Ab

so

rb

an

ce

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FIGURE IVA2

ABSORPTION SPECTRUM OF COLORED SPECIES OF SAFRANINE O

Wavelength (nm)

200 300 400 500 600 700 800

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

110

FIGURE IVA3

ABSORPTION SPECTRA OF COLORED SPECIES OF LEUCO XYLENE

CYANOL FF Vs REAGENT BLANK (a) AND REAGENT BLANK Vs DISTILLED

WATER (b)

Wavelength (nm)

300 400 500 600 700 800 900

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

a

b

FIGURE IVB1

ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF VANADIUM

USING TOLUIDINE BLUE AS A REAGENT

C oncen tra tion o f vanad ium (µgm L

-1

)

0 2 4 6 8 10 12

Ab

so

rb

an

ce

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

111

FIGURE IVB2


USING SAFRANINE O AS A REAGENT

Concentration of vanadium (µgm L

-1

)

0 2 4 6 8 10 12 14 16

Ab

so

rb

an

ce

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FIGURE IVB3


USING LEUCO XYLENE CYANOL FF AS A REAGENT

C oncentration of Vanadium (µgm L

-1

)

0 2 4 6 8 10 12 14

Ab

so

rb

an

ce

0.0

0.5

1.0

1.5

2.0

2.5

112

SCHEME IV

2 VO4

3-

+ 12 H+

+ 2 I-

2 VO2+

+ I2 + 6 H

2O

N

S

+

CH3

NH2

(CH3)2N

N

H

S

CH3

NH2

(CH3)2N

I2 , H

+

Toluidine Blue(Colored) Toluidine Blue(Colorless)

N

N

+

CH3

NH2

NH2

CH3

N

H

N

CH3

NH2

NH2

CH3

I2 , H

+

Safranine O(Colored) Safranine O(Colorless)

NH

CH3

CH3

CH3

NH

CH3

SO3H

SO3Na

H

+V

5+

NH

CH3

CH3

CH3

N

CH3

SO3H

SO3Na

+V

4+

Xylene Cyanol FF (Colorless) Xylene Cyanol FF (Colored)

113

TABLE 4A1

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF VANADIUM

(2 µgmL-1

) USING TOLUIDINE BLUE AND SAFRANINE O

Foreign

ions

Tolerance

limit

-1

Foreign

ions

Tolerance

limit

-1

Fe3+ *

Ni2+

Cu2+ *

Cd2+

Bi3+

Al3+

Ca2+

Ba2+

In3+

Gd3+

Ti4+

Mo6+

10

500

15

500

750

500

1000

1000

750

500

500

250

Cr6+ *

Mg2+

F-

PO4

3-

Iodate*

Citrate

Oxalate

Nitrate

02

250

100

750

< 4

1500

1500

500

* Masked with masking agent

TABLE 4A2

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF VANADIUM

(2 µgmL-1

) USING LEUCO XYLENE CYANOL FF

Foreign

ions

Tolerance

limit

-1

Foreign

ions

Tolerance

limit

-1

Fe3+*

Ni2+

Cu2+

Cd2+

Na+

K+

Sm3+

Eu3+

Mg2+

Zn2+

Mn2+

Al3+

Ca2+

Co2+

≤ 1

500

200

650

2000

1500

1000

500

1000

2500

750

650

2000

500

La3+

Cr2O

7

2-*

F-

In3+

PO4

3-

Ti4+

Mo6+

Gd3+

Oxalate

Acetate

Tartarate

citrate

Sulfate

Iodate*

Nitrate

500

≤ 1

100

1500

50

1000

250

1000

1500

1000

20

20

1000

≤ 1

1500

* Masked with masking agent

114

TABLE 4B1

DETERMINATION OF VANADIUM IN VANADIUM STEELS USING

TOLUIDINE BLUE AS A REAGENT

Sample Composition

%

% of

vanadium

present

% of

vanadium

founda

Relative

error

(%)

Recovery

(%)

1 C,0.56; Si, 0.24; Mn,

0.91; Ni, 0.23; Cr, 1.03;

Mo, 0.04; V, 0.12, Cu,

0.19, P,0.022, S, 0.018

0.120

0.119 ±

0.015 -0.83 99.20

2 C,0.17; Si, 0.13; Mn,

0.53; Ni, 0.20; Cr, 0.20;

Mo, 0.85 ; V, 0.28; Cu,

0.10; P,0.015; S, 0.025

0.280

0.278 ±

0.02 -0.71 99.30

a. Mean ± standard deviation (n = 5)

Cu is masked using 2-mercaptoethanol.

TABLE 4B2

DETERMINATION OF VANADIUM IN VANADIUM STEELS USING

SAFRANINE O AS A REAGENT

Sample Composition

%

% of

vanadium

present

% of

vanadium

founda

Relative

error

(%)

Recovery

(%)

1 C,0.56; Si, 0.24; Mn,

0.91; Ni, 0.23; Cr, 1.03;

Mo, 0.04; V, 0.12, Cu,

0.19, P,0.022, S, 0.018

0.120

0.118 ±

0.02 -0.83 99.20

2 C,0.17; Si, 0.13; Mn,

0.53; Ni, 0.20; Cr, 0.20;

Mo, 0.85 ; V, 0.28; Cu,

0.10; P,0.015; S, 0.025

0.280

0.279 ±

0.01 -0.36 99.30



115

TABLE 4B3

DETERMINATION OF VANADIUM IN VANADIUM STEELS USING LEUCO


Sample Composition

%

% of

vanadium

present

% of

vanadium

founda

Relative

error

(%)

Recovery

(%)

1 C,0.56; Si, 0.24; Mn,

0.91; Ni, 0.23; Cr, 1.03;

Mo, 0.04; V, 0.12, Cu,

0.19, P,0.022, S, 0.018

0.120

0.122 ±

0.06 +1.66 101.66

2 C,0.17; Si, 0.13; Mn,

0.53; Ni, 0.20; Cr, 0.20;

Mo, 0.85 ; V, 0.28; Cu,

0.10; P,0.015; S, 0.025

0.280

0.274 ±

0.02 -2.14 97.85



TABLE 4C1

DETERMINATION OF VANADIUM IN SOME SYNTHETIC MIXTURES USING

TOLUIDINE BLUE AS A REAGENT

Sample Compositions of m-1

) -1

) Recovery

Added Founda

±SD (% )

1 Zn2+

(25)+Cd2+

(25) 1.00 1.01±0.03 101.0

2 Zn2+

(25)+Cd2+

(25)+CrVI

(5)+Mn2+

(20) 0.50 0.49±0.03 98.0

3. Zn2+

(25)+Cd2+

(25)+CrVI

(5)+Mn2+

(20) 0.50 0.52±0.50 104.0

+Ca2+

(50)

a. Average of five analyses of each samples ± standard deviation (n = 5)

Chromium is masked using methyl isobutyl ketone.

116

TABLE 4C2


SAFRANINE O AS A REAGENT

-1

) -1

)

Recovery

Added Founda

±SD (% )

1 Zn2+

(25)+Cd2+

(25) 1.00 0.98±0.02 98.0

2 Zn2+

(25)+Cd2+

(25)+CrVI

(5)+Mn2+

(20) 0.50 0.48±0.04 96.0

3. Zn2+

(25)+Cd2+

(25)+CrVI

(5)+Mn2+

(20) 0.50 0.50±0.08 100.0

+Ca2+

(50)

a. Average of five analyses of each samples ± standard deviation (n = 5)


TABLE 4C3


LEUCO XYLENE CYANOL FF AS A REAGENT

-1

) -1

)

Recovery

Added Founda

±SD (% )

1 Zn2+

(25)+Cd2+

(25) 1.00 1.02±0.03 102.0

2 Zn2+

(25)+Cd2+

(25)+CrVI

(5)+Mn2+

(20) 0.50 0.48±0.06 96.0

3. Zn2+

(25)+Cd2+

(25)+CrVI

(5)+Mn2+

(20) 0.50 0.47±0.12 94.0

+Ca2+

(50)

a. Average of five analyses of each samples ± standard deviation (n = 5),


117

TABLE 4D1

DETERMINATION OF VANADIUM IN PHARMACEUTICAL SAMPLE USING

TOUIDINE BLUE, SAFRANINE O AND LEUCO XYLENE CYANOL FF AS

REAGENTS

Reagent Samples V V Recovery

used Added found a

(%)

-1 -1

) ± SD

Toluidine Blueb

Neogadine Elixir®

-- 1.82 ± 0.02 98.91

(10mL/100mL) 4.0 5.81 ± 0.04 99.75

6.0 7.78 ± 0.05 99.33

Safranine Ob

Neogadine Elixir®

-- 1.80 ± 0.02 97.8

(10mL/100mL) 5.0 6.82 ± 0.06 99.7

10.0 11.77 ± 0.04 99.4

Leuco Xylene Cyanol FF b

Neogadine Elixir®

-- 1.81 ± 0.05 98.3

(10mL/100mL) 3.0 4.83 ± 0.03 99.8

6.0 7.74 ± 0.07 98.7


b. Raptakos Brett & Co. Ltd. Mumbai 400 030, India. [Each 10 mL contains iodised

peptone-0.64 mg, magnesium chloride-13.34 mg, manganese sulphate-2.66 mg,

sodium metavanadate-0.44 mg, zinc sulphate-21.42 mg, pyridoxine HCl-0.50 mg,

cyanocobalamin-0.33 mg, nicotinamide-6.66 mg, alcohol(95 %)-0.63 mL, total

alcohol 6 %(v/v)], vanadium taken-1.84 -1

.

118

TABLE 4D2



Reagent Method Beer’s

law

-1

)

ε (Lmol-1

cm-1

)

-2

)

λmax

(nm)

Ref.

No.

Arsenazo-M Spectrophotometry 0- ε = 1.04×103

-----

547 81

DABH Spectrophotometry 0-1.5 ε = 2.83×104

---- 83

Thionin Spectrophotometry 0.2-10 ε = 2.298×104

ss = 0.520×10-2

600 85

Variamine blue Spectrophotometry 0.1-2.0 ε = 1.65×104

ss = 3.0×10–3

570 86

Rhodamine-B Spectrophotometry 0.08-0.64 ε = 1.3×105

ss = 9.0×10–4

553 88

Proposed Method

Toluidine blue

Safranine O

Leuco xylene

cyanol FF

Spectrophotometry

Spectrophotometry

Spectrophotometry

0.4–8.0

0.5–12.4

0.05–8.0

ε = 2.141×104

ss = 2.36×10-3

ε = 3.06×104

ss = 1.66×10-3

ε = 1.16×104

ss = 4.38×10-3

628

530

614

119

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84. Q. Hu, G. Yang, Z. Huang and J. Yin, Bull. Korean Chem. Soc., 25 (2004) 263.

85. T. Cherian and B. Narayana, Bull. Chem. Soc. Ethiop., 19 (2005) 155.

86. T. N. Kiran Kumar and H. D. Revanasiddappa, J. Iranian Chem. Soc., 2 (2005)

161.

87. G. M. Mastoi, M. Y. Khuhawar and R. B. Bozdar, J. Quan. Spectr. Rad. Trans.,

102 (2006) 236.

88. A. Sao, A. Pillai and V. K. Gupta, J. Indian Chem. Soc., 83 (2006) 400.

89. F. Qi-Li and W. De-Yun, Chem. Anal. (Warsaw), 51 (2006) 319.

90. C. Xianzhong and K. Yun, Geostand. Geoanal. Res. 31 (2007) 353.

91. K. S. Kumar, S. H. Kang, K. Suvardhan and K. Kiran, Environ. Toxicol.

Pharmacol., 24 (2007) 37.

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Edn., (2000).

94. N. Balasubramanian and V. Maheshwari, J. AOAC. Int., 79 (1996) 989.

123

CHAPTER 5

SPECTROPHOTOMETRIC DETERMINATION OF CHROMIUM USING

TOLUIDINE BLUE AND SAFRANINE O AS NEW REAGENTS

5.1 INTRODUCTION


5.3 APPARATUS


5.5 PROCEDURES


5.7 APPLICATIONS

5.8 CONCLUSIONS

5.9 REFERENCES

124

5.1 INTRODUCTION

Nicolas-Louis Vauquelin discovered chromium in 1797 in Siberian red lead,

the mineral crocoite, PbCrO4. In 1798 he isolated the new metal by reduction of CrO

3

with charcoal at high temperature [1,2]. In the same year he analyzed a Peruvian

emrald and found that its green color is due to the new element. Fourcroy and Huay

suggested the name chromium (from the Greek chroma, color) for the new element

because of its many colored compounds [3]. In 1798 Tobias Lowitz and Martin

Heinrich Klaproth independently found chromium in chromite samples from Russia

and in the following year Tassaert, a German Chemist at the Paris school of Mines

found it in French chromite. This ore, a spinel, Fe(CrO2)2

is the only commercial

sources of chromium. Chromium metal was obtained by Moissan in 1893 by

reduction of chromic oxide with carbon in an electric furnance. In 1894 Goldschmidt

developed the alumino-thermit process for producing chromium by the reduction of

oxide with aluminium powder [4]. The higher grades of ore contain 42-56 % Cr2O

3,

10-26 % Fe and varying amounts of other substances such as magnesia, alumina and

silica.

Chromium is the recently recognized biologically essential trace metal. The

first conclusive evidence demonstrating a metabolic role of chromium was obtained

by Mertz and Schwarz in a series of investigations of which the first report appeared

in 1955 [5]. Chromium is found in the body in very low concentrations, with the total

chromium level in the adult human body being about six grams. The highest levels of

chromium are found in the liver, kidney, spleen and bone. The chromium that our

body requires is called trivalent chromium. Chromium helps to improve the body's

responses to the hormone insulin. Insulin is an important hormone for controlling

blood sugar levels as well as for metabolizing fats and proteins in the body.

Chromium deficiency can cause the body to overproduce insulin but the body is

unable to respond to this insulin. This is called insulin resistance and it is a feature of

type-II diabetes. It was once thought that chromium could assist in fat loss and help to

maintain lean body mass (muscle), but these effects are not seen in humans, only in

experimental animals. The recommended daily intake of chromium is 50 to 100

micrograms (0.05-0.1 mg). Various forms of chromium are available at pharmacies

and health food shops. Chromium picolinate may be the one that is best absorbed into

125

the body [6]. Very little chromium in the form of inorganic compounds, such as

chromic chloride is absorbed. The efficiency of absorption of chromium from chromic

chloride is less than 2 % [7]. The efficiency of absorption of chromium from organic

compounds is higher. For example, approximately 2.8 % of an injected dose of

chromium picolinate is absorbed. Following absorption, chromium is bound to

transferrin and albumin and chromium is transported primarily by transferrin. The

evidence which implicates chromium as a critical cofactor in the action of insulin. It

has been variously suggested that chromium enhances insulin-binding, insulin

receptor number, insulin internalization and β-cell sensitivity [8]. Such findings

establish a link between chromium and diabetes. The evidence comes from the studies

conducted with a patient receiving Total Parenteral Nutrition (TPN), who developed

severe signs of diabetes, including weight loss and hyperglycemia that was refactory

to increasing insulin dosage [9].

The two most impotant functions of chromium in steels are improving the

mechanical properties particularly hardenability and increasing the corrosion

resistance [10]. The magnitude of the effect in each case is roughly proportional to the

percentage of chromium in the steel. Low chromium steels (< 3 % of Cr) produced in

all structural shapes such as bars, tubes, sheets, plates, etc., are used extensively as

engineering materials in every branch of industry. For all but the heaviest duty

applications, chromium content is generally less than 6 %. Steels contain more than

10 % chromium and are designated stainless because of their resistance to corrosion

and oxidation. Non-hardenable grade contain 0.08-0.20 % carbon and 11.25-27.0 %

chromium. Type 430 (AISI) is used in large quantities for trim on buildings,

automobiles, etc., and for nitric acid manufacturing equipment. The austenitic

stainless steels (non-hardenable) contain 16-26 % of chromium and 0.15-1.25 %

carbon.

Chromium deficiency can cause insulin resistance and hyperglycaemia (high

blood glucose levels that cannot be controlled by insulin), cardiovascular disease and

elevated fat levels in the blood. Severe chromium deficiency may cause weight loss,

poor coordination, destruction of the nerves in the extremities of the body (peripheral

neuropathy) and inflammation of the brain. The effects of excessive dietary chromium

126

are not well known. Some forms of chromium that are found in the environment may

be cancer causing, but this type of chromium is different from dietary chromium.

Food with high chromium content are fruits, vegetables, whole grains (e.g. oats and

barley), seeds, nuts, legumes (peas and beans) and brewer's yeast. When these food is

processed, particularly using stainless steel equipment (e.g. when cooking or

canning), their chromium content may increase. Allergy to chromium compounds

carries in men, a worse prognosis than dose sensitization to other allergens. The

reason is not known. Continued contact with unrecognized chromium compounds in

the environment or possibly ingestion of chromium compounds has been considered

as possible explanations [11].

The determination of micro amounts of chromium in soils and other naturally

occurring materials are of considerable interest because of the contrasting biological

effects of its two common oxidation states, chromium(III) and chromium(VI) and also

the growing interest in environmental problems. It is known that an increase in the

content in soils makes them infertile and toxic effect depends to some extent on the

chromium oxidation state. On the other hand, the introduction of chromium salts into

soils has some positive effects due to activation of some biochemical processes [12].

Chromium(III) is an essential nutrient for maintaining normal physiological function,

where as chromium(VI) is toxic [13].


Many methods have been reported for the quantitative determination of

chromium. The analytical technique varies from inductively coupled plasma-atomic

emission spectroscopy [14], atomic absorption spectroscopy [15], neutron activation

analysis [16], X-ray absorption spectroscopy [17], complexometric [18], catalytic-

kinetic [19], sequential injection [20] to flow injection methods [21,22].

A survey of literature revealed that a large number of reagents are suitable for

the spectrophotometric determination of chromium. One of the spectrophotometric

method was based on the color of the [Cr(C2O

4)]

3-

ion [23]. Cahnmann and Ruth

reported a spectrophotometric method for the determination of chromium using

127

1,5-diphenylcarbazide reagent [24]. In this method Beckman DU spectrophotometer

was used with a wave length of 543 nm and the reaction was sensitive to 0.005 ppm.

Selmer-Olsen used Complexon-IV as a reagent for the determination of

chromium [25]. Chromium(III) formed a stable water soluble violet complex with

Complexon-IV. The 1:1 chromium complex exhibited absorption maxima at 395 and

540 nm. Beer’s law was obeyed in the range 2--1

of chromium.

Dwight and John used diphenylcarbazide as a spectrophotometric reagent for

the determination of chromium in human plasma and red blood cells [26]. Traces of

chromium in human blood was determined by a method which utilized the red-violet

complex formed by the reaction of Cr2O

7

2-

with diphenylcarbazide. The concentration

of chromium in normal human plasma ranged from 0.017 to 0.052 ppm, which was in

good agreement with previously reported values. The concentration of chromium in

red cells ranged from 0.014-0.038 ppm. Standard deviation by the method was 1.6 %.

8-Hydroxyquinaldine [27] was also used as a spectrophotometric reagent for the

determination of chromium in uranium.

Sumio and Koji used 1-phenylthiosemicarbazide as a reagent for the

spectrophotometric determination of chromium [28]. Chromium formed a brown

color with 1-phenylthiosemicarbazide. Den Boef and Poeder summarized the

interferences in the spectrophotometric determination of chromium(III) with pyridine-

2,6-dicarbonic acid [29,30]. Results were obtained in the determination of Cr(III) in

presence of a 200-fold excess of Cu, Ni, Al, Mn, Zn or Fe(III) and a 100-fold excess

of cobalt. A selective spectrophotometric determinations of chromium(III) with

complexans was described [30]. The method was based on the fact that the

chromium(IIl) complexes were formed rapidly at boiling temperature, but very slow

at room temperature, while the formation of some interfering complexes took place

instantaneously. Determinations with EDTA were more sensitive, but the combined

presence of cobalt and other metals interfered. The combined presence of a l00-fold

amount of copper, nickel, cobalt and iron generally has no effect on the results.

Takao et al. described a extraction-spectrophotometric determination of

chromium(III) with 4-(2-pyridylazo)-resorcinol (PAR) [31]. PAR(H2R) formed a 1:3

128

complex with chromium(III) in a acetate buffer solution at pH 5. The complex formed

an ion-associated compound with tetradecyldimethylbenzylammonium ion which was

extracted into chloroform, the molar absorptivity was 4.7×104

at 540 nm. EDTA was

added as a masking agent after the Cr(HR)3 was formed. Iron, cobalt and nickel

interfered seriously and the method was made specific for chromium by a preliminary

extraction of these metals with cupferron. The sensitivity of the method was seven

times higher than that of the diphenylcarbazide method. Koichi et al. reported xylenol

orange as a reagent for the spectrophotometric determination of chromium [32].

Einar and Walter described a spectrophotormetric determination of chromium

with thioglycollic acid [33]. Cheng reported a simple and sensitive method for the

spectrophotometric determination of chromium with xylenol orange and

methylthymol blue [34]. Xylenol orange and chromium(III) formed a red colored

complex at pH 3 on heating for 20 minutes. The molar absorptivity was 19.0×103

Lmol cm . Methylthymol blue was a less sensitive reagent for chromium; the molar

absorptivity was 11.5×103

Lmol cm .

Johnston and Holland reported a visible spectrophotometric method for the

determination of chromium(III) with 3-thianaphthenoyltrifluoroacetone at a pH of 4.0

to 4.5 [35]. The effects due to pH, time, solvents, reagent concentration and diverse

ions were reported. Beer's law was obeyed and the molar extinction coefficient is

1.3×103

Lmol cm . Katsuya and Yukiteru reportd 2-hydroxy-1-(2-hydroxy-4-sulfo-

1-naphthylazo)naphthalein reagent for the spectrophotometric determination of

chromium [36].

Ferng and Parker described the ternary complex of chromium- peroxo-4-(2-

pyridylazo)resorcinol which exhibited an apparent molar absorptivity of 6.280×103

Lmol–1

cm–1

when extracted into ethyl acetate from 0.1 M sulfuric acid solution for the

determination of chromium [37]. Beer's law was valid up to 6.0 µgmL–1

of chromium.

Conditions for optimum color formation, complex composition, effect of diverse ions

and application to the determination of chromium in steels were described. Li and

Hercules described a method for the determination of chromium in biological samples

by chemiluminescence [38].

129

Kumpulainen et al. reported a method for the determination of chromium in

selected United States Diets [39]. Rizvi used tropolone as a reagent for the

spectrophotometric determination of chromium [40]. Chromium formed a golden

yellow colored complex with tropolone on heating on a water bath; the colored

moiety was extracted to CHCl3. The complex exhibited maximum absorbance at 400

nm.

Reddy and Reddy used cyclohexane-1,3-dionebisthiosemicarbazone

monohydrochloride for the rapid spectrophotometric determination of chromium(VI)

[41]. Cyclohexane-1,3-dionebisthiosemicarbazone monohydrochloride produced

yellow colored solutions with Cr(VI) in NaOAc-HCl medium. Molar absorptivity

value of the system was 1.21×104

Lmol-1

cm-1

at 370 nm.

Gowda and Raj reported fluphenazine hydrochloride as a reagent for the

determination of chromium [42]. Fluphenazine hydrochloride formed a red colored

species with chromium instantaneously at room temperature in 2.0-5.5 M H3PO

4

medium. The red colored species exhibited maximum absorbance at 500 nm with

molar absorptivity of 2.616×104

Lmol-1

cm-1

. Beer's law was valid over the

concentration range 0.05-1.85 ppm of chromium.

Falian and Tao used dibromoalizarin violet for the determination of chromium

in waste water [43]. Chromium reacted with dibromoalizarin violet at pH 5 (using

hexamine-HCl buffer) in the presence of cetyltrimethylammonium bromide at

85-90°C in 30 minutes, formed a blue-green complex. The molar absorptivity of the

complex was 4.54×104

Lmol-1

cm-1

at 620 nm. Beer's law was obeyed at 0--1

of chromium in 25 mL.

Bin reported a spectrophotometric determination of chromium using

salicylfluorone in phosphoric acid medium [44]. Chromium reacted with

salicylfluorone reagent to form a yellow colored complex with molar absorptivity of

2.86×104

Lmol-1

cm-1

at 490 nm. Beer’s law was obeyed in the range 0-50

chromium in 25 mL.

130

Fanqian reported acid chrome blue-K as a new reagent for the

spectrophotometric determination of chromium [45]. The reagent formed a colored

complex with chromium(III). The molar absorptivity was 2.0×104

Lmol-1

cm-1

and

Sandell’s sensitivity 2.2×10-3 -2

at 597 nm. Beer's law was obeyed in the range

0--1

of chromium in 50 mL. Strubel and Rzepka-Glinder described a method

for the analysis of chromium in saliva by atomic absorption spectrometry [46].

Lisheng and Zhaoping used aluminon for the spectrophotometric

determination of micro amounts of chromium in steels [47]. Chromium was

determined in steel by measuring the absorbance at 545 nm of the complex formed by

the reaction of Cr(VI) with aluminon at pH 3.3-4.2 in HOAc-NaOAc buffer solution.

The molar absorptivity of the complex was 1.9×104

Lmol-1

cm-1

. Beer's law was

obeyed in the range 5--1

of chromium in 50 mL. The complex was stable for

about 4 hours. Sodium diethyldithiocarbamate [48], benzyl-tributylammonium [49]

were also reported as spectrophotometric reagents for the determination of chromium

in waste water and steels.

Ram et al. used malachite green [50] for the spectrophotometric determination

of chromium in waste water. The reagent formed a green colored complex with

chromium in acetic acid at pH 2.5. The molar absorptivity of the system was 8.0×104

Lmol-1

cm-1

at 560 nm.

Kamburova described a spectrophotometric determination of chromium with

iodonitrotetrazolium chloride and tetrazolium violet [51-53]. The method was based

on the interaction of iodonitrotetrazolium chloride and tetrazolium violet with

chromium(VI) and the formation of ion-associates with a 1:1 composition in

hydrochloric acid medium. Spectrophotometric determination of chromium(VI) with

methylene blue was developed [52]. In this method the interaction of Cr(VI) and the

methylene blue was examined. The ion-associate formed was extracted into

1,2-dichlorethane. The optimum conditions was established and the values obtained

for the conditional extraction constant Kex

, distribution constant KD and association

Cr(VI) in soils and alloys. Kamburova described triphenyltetrazolium chloride as a

reagent for the spectrophotometric determination of chromium(VI) [53]. Bokic et al.

131

reported a spectrophotometric determination of chromium with diantipyryl(3,4-

dioxomethenyl) phenylmethane [54].

Arya and Bansal described a spectrophotometric determination of

chromium(VI) with ferron [55]. Chromium(VI) formed a pink coloured solution in

chloroform in the presence of ferron when extracted from slightly acidic medium. The

reaction was measured at 510 nm. Beers law was obeyed in the range of 5–70 µgmL-1

.

Most of the important metal ions do not interfered. The relative standard deviation

was 2.78 %. Baluja-Santos et al. described spectrophotometric determination of

chromium(VI) with N-(2-Acetamido)iminodiacetic acid (ADA) [56].

Sasaki et al. reported extraction spectrophotometric determination of

chromium(VI) [57]. The method was based on the reaction of chromium(VI) with

o,o'-dibutyl dithiophosphate ion, which formed tris(dibutyl

dithiophosphato)chromium(III) and tetrabutyl thiophosphoryl disulfide in an acidic

solution (pH 1.2-1.7). Both the products were extracted into hexane and the

absorbance was measured at 278 nm. The chromium(III) complex corresponding to

two-thirds of the initial chromium(VI) concentration and the disulfide corresponding

to three-halves of the initial chromium(VI) concentration were extracted under the

optimum conditions.

El-Sayed and Abd-Elmottaleb described a fourth derivative

spectrophotometric determination of chromium(III) with eriochrome cyanine R (ECR)

[58]. The method was based on the fourth derivative value (D4) at 545 nm. The

experimental and instrumental variables (wavelength range, scan speed, band width

and order of derivative) were optimized. The molar absorptivity was 3.75×105

with a

relative standard deviation of 1.19 %. The method was valid for concentrations

between 20 ngmL-1

and 80 ngmL-1

of chromium(III). The molar ratio of the formed

complex was 1:2 (M:L). The proposed method was successfully applied to the

determination of chromium in steels.

Raj and Gowda reported thioridazine hydrochloride as a sensitive and

selective reagent for the spectrophotometric determination of chromium [59]. The

132

reagent formed a blue coloured radical cation with chromium(VI) instantaneously at

room temperature in 1–4 M orthophosphoric acid medium. The blue colored species

exhibited an absorption maximum at 640 nm with a molar absorption coefficient of

2.577×104

Lmol–1

cm–1

. A 27-fold molar excess of the reagent was necessary for the

development of the maximum colour intensity. Beer's law was obeyed over the

concentration range 0.05–2 ppm of chromium(VI) with an optimum concentration

range of 0.2–1.6 ppm. The effects of acidity, time, temperature, order of addition of

reagents, reagent concentration and the tolerance limit of the method towards various

cations and anions associated with chromium were reported. The method was used

successfully for the determination of chromium in chromium steels.

Rosa et al. reported a sensitive spectrophotometric determination of chromium

with 2-(5-chloro-2-pyridylazo)-5-dimethylaminophenol [60]. A mixture of

hydroethanolic solution of the reagent and an aqueous solution of Cr(III) were heated.

Three different complex species were formed depending on the composition and pH

of the medium (ε=1.7×105 Lmol-1

cm-1

). The stoichiometry of the different complexes

was determined, the formation mechanisms were elucidated and the respective

constants were calculated. A useful spectrophotometric method was proposed for

Cr(III) in concentrations ranged from 15 to 400 ppb. The proper ways to reduce

interferences produced by Fe(III), Nb(V), Ta(V), Ti(IV), V(V), Co(II), Zr(IV), Sn(II),

Al(III), Mn(II) and Cu(II) were described. The methods were applied to chromium

determination in water samples with satisfactory results.

Burns and Dunford described a spectrophotometric determination of

chromium(VI) by extraction of protriptylinium dichromate [61]. In this method

chromium(VI) was determined spectrophotometrically at 365 nm after its extraction

as protriptylinium dichromate into acetone-chloroform (1:1, v/v). The effects of

acidity, diverse ions and masking studies were reported. The relative standard

applied to the spectrophotometric determination of chromium in steels. Maheswari

and Balasubramanian reported rhodamine-6G as a spectrophotometric reagent for the

determination of chromium [62].

133

Wrobel et al. repored enhanced spectrophotometric determination of

chromium(VI) with diphenylcarbazide using internal standard and derivative

spectrophotometry [63]. The following procedure was used: (1) addition of internal

standard and formation of ion pairs of Cr(VI) with benzyltributylammonium bromide

(BTAB) (sample volume 100 mL), (2) extraction to 10 mL of methylene chloride, (3)

evaporation in nitrogen stream, and (4) redissolution in a micro-volume with addition

of diphenylcarbazide for color developm

preconcentration factor achieved was about 400 and it was shown that using internal

standard, the analytical errors due to sample treatment were reduced. The analytical

signals for chromium and internal standard were obtained at 591.30 and 653.50 nm.

,

3.2%, correlation coefficient of linear regression was 0.9985. The proposed procedure

was applied to determination of chromium(VI) in tap water. Total chromium was

determined by electrothermal atomic absorption spectrometry, the recovery of

hexavalent chromium added was then evaluated and compared with the results of the

proposed procedure. In this experiment, good agreement was obtained between results

obtained by the two methods.

Hoshi et al. described a simple and rapid spectrophotometric determination of

trace amounts of chromium(VI) after preconcentration as its colored complex on

chitin [64]. The chromium(VI) was collected as its 1,5-diphenylcarbazide(DPC)

complex on a column of chitin in the presence of dodecyl sulfate as counter-ion. The

Cr-DPC complex retained on the chitin was eluted with a mixture of methanol:acetic

acid(1M) (7:3, v/v) and the absorbance of the eluent was measured at 541 nm. Beer’s

law was obeyed over the concentration range of 0.05–

1 mL of the eluent. The apparent molar absorptivity was 3.5×104

Lmol cm . The

tolerance limits for Fe(III) was low, i.e. ten times that of chromium(VI), but some

metal ions and common inorganic anions did not interfere in the concentration range

of 100–10000 times that of chromium(VI). The method was applied to the

determination of chromium(VI) in natural water samples.

Ressalan et al. reported 3-hydroxy-3-phenyl-1-o-hydroxyphenyltriazene,

3-hydroxy-3-p-tolyl-1-o-nitrophenyl-triazene and 3-hydroxy-3-phenyl-1-o-

134

carboxyphenyl-triazene as reagents for the spectrophotometric determination of

chromium [65-67].

Balogh et al. reported a spectrophotometric study of the complexation and

extraction of chromium(VI) with Cyanine dyes [68]. The molar absorptivities of the

ion associates were in the range of 2.5–3.6×105

Lmol cm . The absorbance of the

coloured extracts obeyed the Beer's law in the range 0.01–2.1 mgL . The extraction

of chromium was the highest during extraction from the sulfuric acid medium

(0.05– 3 M H2SO

4). The method extended to the determination of various types of

soils and sewage doped with chromium(VI).

Abdollahi reported a simultaneous spectrophotometric determination of

chromium(VI) and iron(III) with mixed reagents by H-point standard addition method

[69]. Mixed reagents of diphenylcarbazide and 1,10-phenanthroline in a non-ionic

micellar solution of Triton X-100 was used as a selective chromogenic system for

determination of Cr(VI) and Fe(III). The presence of a micellar system allowed to

eliminate the previous solvent extraction step that was necessary for the determination

of slightly soluble metal complexes in the absence of micelles. This reduced the cost

and toxicity of the method. The total relative standard error for 15 synthetic samples

were in the range 0.20–6.00 of Cr(VI) and 0.20–8.001

of Fe(III) were

1.5 %. The methods were applied to the determination of Cr and Fe in several

synthetic alloy solutions.

Melwanki and Seetharamappa reported propericiazine as a spectrophotometric

reagent for the determination of chromium in environmental samples [70].

Propericiazine formed a red colored radical cation, exhibited maximum absorption at

510 nm in H3PO4 medium. Beer's law was valid over the concentration range of

0.15- 2.25 mgL-1

. The Sandell's sensitivity of the reaction was found to be

3.42 ngcm-2

.

Mohamed and El-Shahat reported a spectrophotometric determination of

chromium and vanadium [71]. The method was based on the reactions with

perphenazine which instantaneously formed a red colored product and exhibited a

maximum absorbance at 526 nm.

135

Melwanki and Seetharamappa described a spectrophotometric determination

of chromium(VI) in soil and steel samples using methdilazine hydrochloride [72]. The

reagent formed a red colored species instantaneously on reaction with chromium(VI)

in phosphoric acid medium and exhibited maximum absorbance at 512 nm with

Sandells’s sensitivity of 3.1 ngcm-2

. Beer's law was obeyed over the concentration

range of 0.1-1.8 ppm.

Llobat-Estelles et al. reported a method for the determination of chromium in

presence of V(V), Mo(VI) and iron(III) [73]. The effects of interferences were

evaluated by using the apparent content curves method and their separation was

performed by solid-phase extraction with an anionic exchanger. The sample treatment

conditions and the influence of the sample conductivity were studied. Tolerance limits

were established and the proposed procedure was used to determine chromium in

certified samples and for speciation of chromium in waste water.

Revanasiddappa and Kiran Kumar reported citrazinic acid as new coupling

agent for the indirect spectrophotometric determination of chromium by the oxidation

of hydroxylamine in acetate buffer of pH 4.0- 0.5 to nitrite [74]. Molar absorptivity

and Sandell's sensitivity of the reaction system were found to be 2.12×104

Lmol-1

cm-1

-2

at 470 nm. Beer's law was obeyed over the concentration range of

2-

hours.

Abdel-Aaty developed a simple and sensitive method for the

spectrophotometric determination of chromium(VI) based on its reaction with

chlorpromazine-HCl, which formed a red product [75]. Beer’s law was obeyed in the

range 0--1

of Cr(VI) and the molar absorptivity value of the system was

3.28×104

Lmol-1

cm-1

.

Xin et al. reported chlorophosphonazo as a reagent for the spectrophotometric

determination of chromium in drinking water and alloy steel [76]. Proposed method

was based on the decolorization of chlorophosphonazo-pA in the presence of

perchlorate and molar absorptivity value of the system was 3.33×105

Lmol-1

cm-1

.

136

Revanasiddappa and Kiran Kumar used trifluoperazine hydrochloride as a

reagent for the rapid spectrophotometric determination of chromium [77] . The method

was based on the oxidation of trifluoperazine hydrochloride by Cr(VI) in the presence

of H3PO

4. The red colored species exhibited an absorption maximum at 505 nm. The

system was obeyed Beer's law at 2-

molar absorptivity of the color system was 2.08×104

Lmol-1

cm-1

and the developed

color was stable for 2 hours. Leuco xylene cyanol-FF a sensitive reagent was used for

the selective spectrophotometric determination of trace amounts of chromium in

steels, industrial effluents and pharmaceutical samples [78]. The method was based on

the oxidation of leuco xylene cyanol-FF to its blue form of xylene cyanol-FF by

Cr(VI) in H2SO

4 medium (pH 1.2-2.4), the absorbance of the formed dye was

measured in an acetate buffer medium (pH 3.0-4.6) at 615 nm. The method was

obeyed Beer's law in the concentration range of 0.05--1

of chromium.

Molar absorptivity and Sandell’s sensitivity of the system was 8.23×104

Lmol-1

cm-1

-2

respectively.

Melwanki and Seetharamappa used isothipendyl hydrochloride as a sensitive

and selective reagent for the simple spectrophotometric determination of

chromium(VI) [79]. The reagent reacted with chromium, which formed a red colored

species in H3PO

4 medium, which exhibited maximum absorbance at 510 nm with

Sandell’s sensitivity of 2.28 ngcm-2

. Beer's law was valid over the concentration range

of 0.1-1.9 mgL-1

of chromium.

Carvalho et al. reported 4-(2-thiazolylazo)-resorcinol (TAR) as a reagent for

the spectrophotometric determination of chromium [80]. Chromium reacted with

TAR, which formed a red complex at pH 5.7. Beer’s law was obeyed in the

concentration range 0.05--1

of Cr(VI) and molar absorptivity of the system

was 2.72×104

Lmol-1

cm-1

at 545 nm. The selectivity was improved by using EDTA

and citrate as masking agents.

Stoyanova reported a catalytic spectrophotometric method for the

determination of chromium(VI) [81]. The method was based on the catalytic effect of

chromium(VI) on the oxidation of sulphanilic acid (SA) by hydrogen peroxide in the

presence of p-aminobenzoic acid (PABA) as an activator. The reaction was followed

137

spectrophotometrically by tracing the formation of the reaction product at 360 nm

after 15 minutes of mixing the reagents. The optimum reaction conditions are

4.0×10-3

M sulphanilic acid, 0.57 M H2O

2 , 1×10

-3

M p-aminobenzoic acid and 0.04

M acetic acid - boric acid - orthophosphoric acid buffer solution (pH 6.6) at 50 °C.

The linear range of the calibration graph was up to 140 ngmL-1

and the detection limit

was 10 ngmL-1

. Interferences of Cu(II) and Cr(III) ions were masked. The method

was applied to the analysis of Cr(VI) in industrial water with recoveries of 95.2–

104.5% and RSD of 2.9–5.8%.

Narayana and Cherian described a rapid spectrophotometric determination of

trace amounts of chromium using variamine blue as a chromogenic reagent [82]. The

method was based on the reaction of chromium(VI) with potassium iodide in acid

medium to liberate iodine. The liberated iodine which oxidized variamine blue to

violet colored species with an absorption maximum 556 nm. Beer’s law was obeyed

in the range 2-12 µgmL-1

of chromium(VI). The molar absorptivity, Sandell’s

sensitivity, detection limit and quantitation limit of the method were found to be

0.911×104

Lmol-1

cm-1

, 1.14×10-2

µgcm-2

, 0.02 µgmL-1

and 0.07 µgmL-1

respectively. The chromium(III) was determined after its oxidation with bromine

water in alkaline medium to chromium(VI).

Stoyanova developed a catalytic spectrophotometric determination of

chromium [83]. The method was based on the catalytic effect of chromium(III) and

chromium(VI) on the oxidation of sulfanilic acid by hydrogen peroxide. The reaction

was followed spectrophotometrically by measuring the absorbance of the reaction

product at 360 nm. Two calibration graphs (for chromium(III) up to 100 ngmL-1

, and

for chromium(VI) up to 200 ngmL-1

) were obtained using the fixed time method with

detection limits of 4.9 ngmL-1

and 3.8 ngmL-1

respectively.

Zaitoun described a spectrophotometric determination of chromium(VI) using

cyclam (1,4,8,11-tetraazacyclotetradecane) as a reagent [84]. This method was based

on the absorbance of its complex with cyclam and the complex showed a molar

absorbtivity of 1.5×104

Lmol-1

cm-1

at 379 nm. Under optimum experimental

conditions at a pH of 4.5 and 1.960×103

mgL-1

cyclam were selected, and all

measurements were performed 10 minutes after mixing. Major cations and anions did

138

not show any interference. Beer’s law was applicable in the concentration range

0.2–20 mgL-1

with a detection limit of 0.001mgL-1

. The standard deviation in the

determination was ± 0.5 mgL-1

for a 15.0 mgL-1

solution (n=7). The described method

provides a simple and reliable for the determination of chromium in real samples.

Cherian and Narayana reported a spectrophotometric determination of

chromium(VI) using saccharin as a reagent [85]. In this method chromium(VI)

oxidized hydroxylamine in acetate buffer of pH 4.0 to nitrite, which then diazotized

p-nitroaniline or sulphanilamide to a diazonium salt. These diazonium salts were then

coupled with saccharin in an alkaline medium, which formed azo dyes with

absorption maximum at 372 and 390 nm for p-nitroaniline and sulphanilamide

respectively. The method obeyed Beer’s law in the concentration range of 1-16

µgmL-1

for chromium with p-nitroaniline-saccharin and 0.6-14 µgmL-1

for chromium

with sulphanilamide-saccharin couples. The molar absorptivity and Sandel’s

sensitivity of the systems with p-nitroaniline-saccharin and sulphanilamide-saccharin

couples were found to be to be 5.41×103

Lmol-1

cm-1

and 1.93×10-3

µgcm-2

and

2.63×104

Lmol-1

cm-1

and 3.90×10-3

µgcm-2

respectively.

Rao et al. described a spectrophotometric determination of chromium(III) after

extraction of its n-methylaniline carbodithioate complex into molten naphthalene [86].

Maximum extraction was obtained in the pH range of 2.0–3.5. Naphthalene

containing the metal complex was dissolved in DMF and the resulting solution

obeyed Beer’s law at 340 nm in the concentration range of 2.6–31.

in 10 mL of the final solution. The molar absorptivity and Sandell’s sensitivity are

8.2×103

Lmol-1

cm-1 -2

respectively. Interference of other metal ions

was studied and the method can be used for the determination of chromium in alloys

and industrial effluents.

Fabiyi and Donnio used variamine blue as a chromogenic reagent for rapid

spectrophotometric determination of nano amount of chromium [87]. The proposed

method was based on the reaction of chromium(VI) with potassium lodide in acid

medium to liberate iodine. The liberated iodine that oxidized variamine blue to violet

colored substances exhibited an absorption maxmium at 615 nm. The molar

139

absorptivity and Sandel’s sensitivity were found to be 8.12×103

Lmol-1

cm-1

and

2.36×10-3

µgcm-2

, respectively. The determination of chromium was found to be

0.0003–15 mgmL-1

. Chromium(III) was determined after its oxidation to Cr(lV) with

bromine water in alkaline medium. The optimum reaction conditions, other analytical

parameters and interference effect of several ions were reported. The reagents

mentioned above are reported to be carcinogenic, while few others are less selective

and are time consuming. The need for a sensitive and simple method for the

determination of chromium is therefore clearly recognized.

The aim of the present work is to provide a simple and sensitive method for

the determination of chromium using toluidine blue and safranine O as new reagents.

The proposed method has been employed to the determination of chromium in steel,

soil, natural water and pharmaceutical samples.

140

5.3 APPARATUS

A Systronics 2201 UV-VIS Double Beam Spectrophotometer with 1 cm




distilled water was used throughout the study. Chromium(VI) stock solution (1000

µgmL-1

) was prepared by dissolving 0.2830 g of K2Cr

2O

7 in 100 mL standard flask

with distilled water and standardized by titrimetric method [18]. The stock solution

was further diluted as needed. Chromium(III) stock solution (1000 µgmL-1

) was

prepared by dissolving 0.2830 g of K2Cr

2O

7 in 50 mL of water, added 1 mL

saturated sodium sulfite solution, acidified with 1 mL 2.5 M sulfuric acid, and then

boil for 2 minutes to remove excess SO2 and diluted with water to 100 mL. Suitable

volume of this solution was diluted to obtain the working standard. The following

reagents were prepared by dissolving appropriate amounts of the reagents in distilled

water: toluidine blue (0.01 %), safranine O (0.02 %), potassium iodide (2 %), acetate

buffer (pH = 4), bromine water (saturated), sulfosalicylic acid (5 %), potassium

hydroxide (4.0 M), sulfuric acid (2.5 M) and hydrochloric acid (2.0 M).

5.5 PROCEDURES


5.5.1.1 Determination of chromium(VI)

Aliquots of sample solution containing 0.5-12.4 µgmL-1

of chromium(VI)

were transferred into a series of 10 mL calibrated flasks. Potassium iodide (2 %,

1 mL) and hydrochloric acid (2 M, 1 mL) were added and mixture was gently shaken

until the appearance of yellow color indicating the liberation of iodine. Toluidine blue

(0.01 %, 0.5 mL) was then added and the reaction mixture was shaken for 2 minutes

for maintaining pH = 4, 2 mL of acetate buffer was added. The contents were diluted

to 10 mL with distilled water and mixed well. The absorbance of the resulting

solutions were measured at 628.5 nm against reagent blank. Reagent blank was

141

prepared by replacing the analyte (chromium) solution with distilled water. The

absorbance corresponding to the bleached color, which in turn corresponds to the

analyte (chromium) concentration was obtained by subtracting the absorbance of the

blank solution from that of the test solution. The amount of the chromium present in

the volume taken was computed from the calibration graph (Figure VA1).

5.5.1.2 Determination of chromium(III)


of chromium(III)

were transferred in to a series of 10 mL calibrated flasks. A volume of 0.5 mL

saturated bromine water and 0.5 mL of 4 M KOH solution were added to each flask

and allowed to stand for 5 minutes. Then 0.5 mL of 2.5 M sulfuric acid and 0.5 mL of

5 % sulfosalicylic were added and then above procedure for chromium(VI) was

followed. The absorbance of the resulting solution was measured at 628.5 nm against

reagent blank.


5.5.2.1 Determination of chromium(VI)


of chromium(VI)

were transferred into a series of 10 mL calibrated flasks. Potassium iodide (2 %,1 mL)

and hydrochloric acid (2 M, 1 mL) were added and mixture was gently shaken until

the appearance of yellow color indicating the liberation of iodine. Safranine O (0.02

%, 0.5 mL) was then added and the reaction mixture was shaken for 2 minutes, for

maintaining pH = 4, 2 mL of acetate buffer was added. The contents were diluted to

10 mL with distilled water and mixed well. The absorbance of the resulting solutions

were measured at 532 nm against reagent blank. Reagent blank was prepared by

replacing the analyte (chromium) solution with distilled water. The absorbance

corresponding to the bleached color, which in turn corresponds to the analyte

(chromium) concentration, was obtained by subtracting the absorbance of the blank

solution from that of the test solution. The amount of the chromium present in the

volume taken was computed from the calibration graph (Figure VA2).

142

5.5.2.2 Determination of chromium(III)

Aliquots of a sample solution containing 0.4-13.8 µgmL-1

of chromium(III)

was transferred in to a series of 10 mL calibrated flasks. A volume of 0.5 mL

saturated bromine water and 0.5 mL of 4 M KOH solution were added to each flask

and allowed to stand for 5 minutes. Then 0.5 mL of 2.5 M sulfuric acid and 0.5 mL of

5 % sulfosalicylic were added and then above procedure for chromium(VI) was

followed. The absorbance of the resulting solution was measured at 532 nm against

reagent blank.

5.5.3 Analysis of Chromium Steel

Steel (0.05 g per 100 mL) was dissolved in approximately 8 mL of aqua regia.

It was evaporated nearly to dryness on a sand bath, sulfuric acid (1-2 mL, 1:1 ) was

added and evaporated until salts crystallized, to this 10 mL of water was added. The

solution was warmed, filtered. The interference of vanadium(V) can be overcome by

extraction of chromium(VI) as chromyl chloride in 5 mL of methyl isobutyl ketone

(MIBK) after the addition of 5 mL of 5 M HCl to provide an overall acidity of

0.3-0.5 M [88]. chromium(VI) in organic layer was stripped by equilibration with 5

mL of water for determination. Suitable aliquots of sample solutions were analyzed

according to the procedure for chromium(III).

5.5.4 Determination of Chromium in Natural Water Samples

Each filtered environmental water samples (100 mL) were analyzed for

chromium. They tested negative. To these samples known amounts (not more than

-1

) of chromium(VI) were spiked and analyzed for chromium by the

proposed procedure. Solutions were also analyzed according to the standard

diphenylcarbazide method [89].

5.5.5 Determination of Chromium in Soil Samples

A known amount of (1 g) air dried soil samples, spiked with known amounts

of chromium(VI) was taken and then fused with 5 g anhydrous sodium carbonate in a

silica crucible and evaporated to dryness after the addition of 25 mL of water. The

dried material was dissolved in water, filtered through whatman No. 40 filter paper in

to 25 mL calibrated flask and neutralized with dilute ammonia. It was then diluted to

143

a known volume with water. An aliquot of this sample solution was analyzed for

chromium(VI). Solutions were also analyzed according to the standard

diphenylcarbazide method [89].

5.5.6 Analysis of Pharmaceutical Samples

Samples of the finely ground multivitamin–multimineral tablets containing

chromium(III) were treated with 5 mL of nitric acid and the mixtures were evaporated

to dryness. The residue was leached with 5 mL of 0.5 M H2SO

4. The solution was

diluted to a known volume with water. Suitable aliquots of the sample solution were

analyzed according to the procedure for chromium(III).




This method is based on the reaction of chromium(VI) with potassium iodide

in acid medium to liberate iodine. This liberated iodine bleaches the blue color of the

toluidine blue. The decrease in absorbance at 628.5 nm is directly proportional to the

chromium(VI) concentration. The absorption spectra of the colored species of

toluidine blue is presented in Figure VA and reaction system is also presented in

Scheme V.


Similarly this method is also based on the reaction of chromium(VI) with

potassium iodide in acid medium to liberate iodine. This liberated iodine bleaches the

pinkish red color of the safranine O. The decrease in absorbance at 532 nm is directly

proportional to the chromium(VI) concentration. The absorption spectra of the

colored species of safranine O is presented in Figure VA and reaction system is also

presented in Scheme V.


The oxidation of iodide to iodine is effective in the pH range 1.0 to 1.5, which

can be maintained by adding 1 mL of 2 M HCl in a final volume of 10 mL. The

liberation of iodine from KI in an acid medium is quantitative. The appearance of

144

yellow color indicates the liberation of iodine. Although any excess of iodine in the

solution will not interfere. It is found that 1 mL each of 2 % KI and 2 M HCl are

sufficient for the liberation of iodine from iodide by chromium(VI) and 0.5 mL of

0.01 % toluidine blue and 0.5 mL of 0.02 % safranine O are sufficient for the

decolorization reaction. The bleached reaction system is found to be stable for about

4 hours for each toluidine blue and safranine O reagents.

Constant and maximum absorbance values are obtained in the pH=4±0.2.

Hence the pH of the reaction system was maintained at 4±0.2 throughout the study.

This could be achieved by the addition of 2 mL of 1 M sodium acetate solution in a

total volume of 10 mL.


5.6.3.1 Using touidine blue as a reagent


solutions varying chromium concentration. A straight line graph is obtained by

plotting absorbance against concentration of chromium. Beer’s law is obeyed in the

concentration range of 0.5-12.4 µgmL-1

of chromium (Figure VA1). The molar

absorptivity and Sandell’s sensitivity of system is found to be 1.457×104

Lmol-1

cm-1

and 5.141×10-3 -2

respectively. The detection limit (DL=3.3σ/S) and quantitation

limit (QL=10σ/S) (where σ is the standard deviation of the reagent blank (n=5) and S

is the slope of the calibration curve) of the chromium determination are found to be

-1 -1

respectively.



solutions varying chromium concentration. A straight line graph is obtained by

plotting absorbance against concentration of chromium. Beer’s law is obeyed in the

concentration range 0.4–13.8 µgmL-1

of chromium (Figure VA2). The molar

absorptivity and Sandell’s sensitivity of the system is found to be 1.093×104

Lmol-1

cm-1

and 6.849×10-3 -2

respectively. The detection limit (DL =3.3σ/S) and

quantitation limit (QL=10σ/S) (where σ is the standard deviation of the reagent blank

145

(n=5) and S is the slope of the calibration curve) of the chromium determination are

-1

a-1

respectively.



chromium is examined. The tolerance limits of interfering species are established at

those concentrations that do not cause more than ±2.0 % error in absorbance values of

-1

). The results are given in Table 5A1.

The reaction involving chromium with potassium iodide, various ions such as Cu2+

,

V5+

, Fe3+

, iodate and periodate are interfered. However, the tolerance level of some of

these ions may be increased by the addition of 1 mL of 1 % EDTA solution. Fe3+

can

be masked using sodium fluoride solution.

5.7 APPLICATIONS

The proposed method is applied to the quantitative determination of traces of

chromium in different samples such as alloys, water, soil and pharmaceutical samples.

The results are summarized in Table 5A2, 5A3 and 5A4. Statistical analysis of the

results by t-tests show that, there is no significant difference in accuracy and precision

of the proposed and reference method [89]. The precision of the proposed method is

evaluated by replicate analysis of samples containing chromium at five different

concentrations.

5.8 CONCLUSIONS

1. The reagents provide a simple and sensitive methods for the spectrophotometric

determination of chromium.

2. The reagents have the advantage of high sensitivity and selectivity.

3. The developed methods does not involve any stringent reaction conditions and

offers the advantages of high color stability compared to the standard

diphenylcarbazide method (30 minutes). The color stability of the reaction system

is found to be stable for 4 hours for toluidine blue and safranine O methods.

4. Statistical analysis of the results by the t- tests show that, there is no significant

146

difference in accuracy and precision of the proposed and reference method.

5. The proposed methods can be used for the determination of traces of chromium in

alloys, water, soil and pharmaceutical samples. A comparison of the method

reported is made with earlier methods and is given in Table 5A5.

FIGURE VA

ABSORPTION SPECTRA OF COLORED SPECIES TOLUIDINE BLUE(b) AND

SAFRANINE O(a)

Wavelength (nm)

200 300 400 500 600 700 800 900

Absorbance

0.0

0.5

1.0

1.5

2.0

a

b

147

SCHEME V

SCHEME OF THE REACTIONS

Cr2O

7

2

+ 6 I + 14 H 2 Cr3

+ 3 I2 + 7 H

2O

N

S

+

CH3

NH2

(CH3)2N

N

H

S

CH3

NH2

(CH3)2N

I2 , H

+

Toluidine Blue(Colored) Toluidine Blue(Colorless)

N

N

+

CH3

NH2

NH2

CH3

N

H

N

CH3

NH2

NH2

CH3

I2 , H

+

Safranine O (Colored) Safranine O (Colorless)

148

FIGURE VA1

ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF CHROMIUM


Concentration of chromium (µgmL

-1

)

0 2 4 6 8 10 12 14 16 18

Ab

so

rb

an

ce

0.0

0.5

1.0

1.5

2.0

2.5

3.0

FIGURE VA2

ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF CHROMIUM


Concentration of chromium (µgmL

-1

)

0 2 4 6 8 10 12 14 16 18

Ab

so

rb

an

ce

0.0

0.5

1.0

1.5

2.0

149

TABLE 5A1

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF CHROMIUM(VI) (2 µgmL-1

)

Foreign io-1 -1

)

Toluidine blue Safranine O Toluidine blue Safranine O

* Fe3+

10 10 *V5+

10 10

Ni2+

75 100 Zn2+

500 250

* Cu2+

10 10 MoO4

2-

750 700

Cd2+

500 600 PO4

3-

1000 1200

Ba2+

750 750 Oxalate 1000 750

Bi3+

1000 1000 F-

750 1000

Mn2+

500 500 Sulfate 1000 1000

Al3+

500 400 Chloride 750 1000

Ca2+

50 75 Tartarate 1250 1000

Co2+

75 50 Nitrate 1250 1500

Acetate 1100 1200


150

TABLE 5A2

DETERMINATION OF CHROMIUM IN ALLOY STEELS USING TOLUIDINE

BLUE AND SAFRANINE O REAGENTS

Toluidine blue Safranine O

Samples Cr Cr Recovery t- Cr Recovery t-

certified found*

(%) testd

found*

(%) testd

(%) (%) ± SD (%) ± SD

GKW Steel, 1.02 1.01± 0.015 99.02 0.37 1.00± 0.03 98.03 1.90

India (0.0501g/100 mL);

C 0.54, Mn 0.89, S 0.018,

P 0.308, Si 0.33, V 0.13a

Stainless steel 18.00 17.96 ± 0.02 99.78 1.25 17.97 ± 0.05 99. 38 1.50

no.394(0.0503g/100 mL);

Ni 8.12,Fe 70-71b

Ferrochrome, Fe 35c

65.00 64.78 ± 0.05 99.70 2.57 64.86 ± 0.04 99.78 1.40

*. Mean ± standard deviation( n=5)

a. Diluted to 5 times before analysis;

b. Diluted to 10 times before analysis;

c. Diluted to 15 times before analysis;

d. Tabulated t-value for 8 degree of freedom at P(0.95) is 2.306 .

151

TABLE 5A3

DETERMINATION OF CHROMIUM IN SOIL SAMPLES AND NATURAL

WATER SAMPLES USING TOLUIDINE BLUE AND SAFRANINE O

REAGENTS


Samples Cr(VI) Cr(VI) Relative Recovery t- Cr(VI) Relative Recovery t-

added founda

error (%) testb

founda

error (%) testb

-1 -1

±-1

± SD (%)

Soil 4.0 4.02 ± 0.02 0.25 100.5 1.67 3.99 ± 0.04 -0.25 99.75 1.57

Samples 6.0 6.01 ± 0.01 0.17 100.2 0.36 6.02 ± 0.08 0.33 100.3 0.89

8.0 7.96 ± 0.02 -0.50 99.5 ----- ------- --- ----- ----

Natural 4.0 4.01 ± 0.02 0.49 100.2 0.83 4.01 ± 0.02 0.25 100.2 1.12

water 6.0 6.01 ± 0.01 0.17 100.1 2.00 5.99 ± 0.02 -0.17 99.83 1.11

Samples 8.0 8.02 ± 0.03 0.25 100.2 ---- ------- ---- ----- ----

a. Mean ± standard deviation(n=5)

b. Tabulated t-value for 8 degree of freedom at P(0.95) is 2.306 .

152

TABLE 5A4

DETERMINATION OF CHROMIUM IN PHARMACEUTICALS SAMPLES

USING TOLUIDINE BLUE OR SAFRANINE O REAGENTS


Samples Cr Cr Recovery Cr Recovery

certified found*

(%) found*

(%)

(mg/tablet) (mg/tablet ± SD) (mg/tablet ± SD)

Chromoplexa

0.200 0.199±0.010 99.5 0.196±0.040 98.0

(0.550 g/100 mL)

Fourts Bb

0.150 0.149±0.007 99.3 0.147±0.020 98.0

(0.650 g/100 mL)

Optisulinc

0.500 0.499±0.006 99.8 0.494±0.035 98.8

(0.300 g/100 mL)

a. Chromoplex (Aristo pharmaceuticals Ltd., Chennai-600 096, India)

Composition – Zinc sulphate monohydrate-27.50 mg; vitamin B1-10 mg; vitamin

B12

-15mg; nicotinamide-50 mg; calcium pantothenate-12 mg; folic acid-1 mg;

vitamin C-150 mg;(0.550 mg).

b. Fourts B (Fourts India Laboratories Private Ltd., Kelambakkam-603 103, Tamil

Nadu, India)

Composition –Thiamine mononitrate-10 mg; riboflavin-10 mg; pyridoxine

hydrochloride-3mg; vitamin C-150 mg; zinc sulphate-80 mg; selenium-100 mg;

(0.650).

c. Optisulin (Dr. Reddy’s Laboratories Ltd., Hyderabad-500 016, India)

Composition – Zinc sulphate monohydrate-27.50 mg; vitamin B6-3 mg; vitamin

B12-15 mg; folic acid-1 mg; (0.3 g).

*. Mean ± standard deviation( n=5)

153

TABLE 5A4



-1

)

ε (Lmol-1

cm-1

)

-2

)

λmax

(nm)

Ref.

No.

4-(2-Thiazolylazo)-

resorcinol

Spectrophotometry 0.05-3.0 ε = 2.72×104

--------

545 80

Variamine blue Spectrophotometry 2-12 ε = 0.911×104

ss = 1.14×10-2

556 82

Cyclam Spectrophotometry 0.2-20 ε = 1.5×104

--------

379 84

p-Nitroaniline-saccharin

Sulphanilamide-

saccharin

Spectrophotometry 1-16

0.6-14

ε = 5.41×103

ss = 1.93×10-3

ε = 2.63×104

ss = 3.90×10-3

372

390

85

Variamine blue Spectrophotometry 0.0003-15

mgmL-1

ε = 8.12×103

ss = 2.36×10-3

615 87

Proposed Method

Toluidine blue

Safranine O

Spectrophotometry 0.5-12.4

0.4-13.8

ε = 1.457×104

ss = 5.141×10-3

ε = 1.093×104

ss = 6.849×10-3

628.5

532


154

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158

CHAPTER 6

SPECTROPHOTOMETRIC DETERMINATION OF ARSENIC IN

ENVIRONMENTAL AND BIOLOGICAL SAMPLES

6.1 INTRODUCTION


6.3 APPARATUS


6.5 PROCEDURES


6.7 APPLICATIONS

6.8 CONCLUSIONS

6.9 REFERENCES

159

6.1 INTRODUCTION

Arsenic is a naturally occurring dissolved element in ground and surface

waters throughout the world. Arsenic is an element classed as a semi-metal or

metalloid. This means it has some properties of metal and some properties of non-

metal. Arsenic occurs in two distinct solid forms. One is a brittle gray metal, while the

other is a yellow, non-metallic form, rarely seen outside the laboratory. Arsenic and

its compounds often have a garlic-like odor when crushed or when scratched with a

hard object. Elemental arsenic has very few uses. Nearly all the applications are as

salts or oxides of arsenic. Arsenic compounds can be very toxic and their uses are

strictly controlled by health and environmental regulations.

Arsenic has been found in nature since antiquity. Aristotle made reference to

sandarach (arsenic trisulfide) in the 4th

century B.C. In the 1st

century A.D., Pliny

stated that sandarach is found in gold and silver mines and arsenic (arsenic trioxide) is

composed of the same matter as sandarach. By the 11th

century three species of

arsenic were known, white, yellow and red - since then recognized as arsenic trioxide,

arsenic trisulfide (orpiment) and arsenic disulfide (realgar) respectively [1].

The name arsenic comes from the Greek word arsenikon, which means

orpiment. Orpiment is a bright yellow mineral composed of arsenic sulfide (As2S

3),

and is the most highly visible common arsenic mineral. Historians say that arsenic

was discovered in 1250 A. D. by Albertus Magnus, a German monk who spent his life

studying and classifying natural materials. It is believed that he heated soap and

orpiment together and isolated elemental arsenic. Arsenopyrite (FeAsS) is the most

common mineral from which, the arsenic sublimes leaving ferrous sulfide on heating.

The terrestrial abundance of arsenic is about 5 g/ton being found widely

dispersed in nature. Some samples of arsenic have been found which vary in purity

from about 90 to 98 %. The commonly associated impurities encountered in these

samples are antimony, bismuth, iron, nickel and sulfur. Normally arsenic is found in

nature, combined as sulfides, arsenides, sulfoarsenides, arsenites and occasionally as

oxide and oxychloride. The most commonly encountered minerals of arsenic are

160

arsenopyrite (FeAsS), loellingite (FeAs2), enargite (CuS.As

2S

5), orpiments (As

2S

3)

and realgar (As2S

3).

Arsenic occurs in nature in inorganic as well as organic forms. Erosion of

arsenic containing surface rocks probably accounts for a significant amount of arsenic

in water supplies [2]. The other major sources of environmental arsenic are the

smelting of nonferrous metal ores, especially copper. Arsenic is an essential nutrient

and is a constituent of many food such as meat, fish, poultry grains and cereals [2]. In

excessive amounts, arsenic causes gastrointestinal damage and cardiac damage.

Chronic doses can cause vascular disorders such as blackfoot disease [2]. Arsenic and

its compound are reported to be carcinogenic, mutagenic and teratogenic in nature.

The maximum permissible limit for arsenic in water is 0.05 mgL-1

as recommended

by WHO [2]. The threshold limit proposed by ACGIH for arsenic in air is

0.5 mgm-3

[3].

The most infamous use of arsenic is as a poison. However, arsenic can now

be detected during autopsy, so this use of the element has become a legend of the

past. These days the most important use of arsenic is in the preservation of wood. It

is used in the form of a compound called chromated copper arsenate (CCA) and is

added to wood used to build houses and other wooden structures. CCA prevents

organisms from growing in the wood and causing it to rot. Arsenic is also used as a

weed killer and rat poison. Arsenic has been used to improve the roundness of lead

shot. Trace amounts of arsenic are alloyed with lead in storage batteries. Arsenic is

also used in the manufacture of high efficiency solar cells. Alloys of gallium, arsenic

and phosphorous are used in the semiconductor industry for the production of light-

emitting diodes (LEDs) in watches, clocks, calculators and numerous other instrument

displays.

Arsenic has significant medicinal properties and it has been used as a

therapeutic agent for more than 2,400 years [4]. In the 15th

century, William

Withering, who discovered digitalis, was a strong proponent of arsenic-based

therapies. He argued, "Poisons in small doses are the best medicines and the best

medicines in too large doses are poisonous" [5]. In the 18th

century, Thomas Fowler

compounded a potassium bicarbonate based solution of arsenic trioxide (As2O

3) that

161

would bear his name. Fowler's solution was used empirically to treat a variety of

diseases during the 18th

, 19th

and early 20th

centuries [6]. Pharmacology texts of the

1880’s describe the use of arsenical pastes for cancers of the skin and breast and

arsenous acid was used to treat hypertension, bleeding gastric ulcers, heartburn and

chronic rheumatism [5]. Arsenic's reputation as a therapeutic agent was enhanced in

1910 when Nobel laureate Paul Ehrlich developed salvarsan, an organic arsenial for

treating syphilis and trypanosomiasis. However, as medicine evolved in the

20th

century, enthusiasm for medicinal arsenic waned rapidly [5].

The concentration of arsenic in plants can vary depending on factors like the

species of plant, the type of arsenic in the soil, and the location of a given species.

Arsenic can exist in a variety of oxidation states in inorganic and organic forms in

many environmental matrices such as natural water and soils [7]. Therefore precise

knowledge of the arsenic compounds present in a system is required for an accurate

assessment of the environmental and biological impact of arsenic, which has resulted

in an increasing need of analytical method for their determination at micro trace or

even ultra trace levels.


Various methods for the analysis of arsenic have been reported in the

literature. Many analytical techniques based on flow injection analysis with hydride

generation [8], atomic absorption spectroscopy [9], gas fluorometry-atomic absorption

spectroscopy [10], inductively coupled plasma-atomic absorption spectroscopy [11],

neutron activation analysis [12] and fluorescence spectroscopy [13] are used for the

arsenic determination.

A literature survey revealed that a large number of reagents are suitable for the

spectrophotometric determination of arsenic. Cristau reported a spectrophotometric

determination of arsenic using hypophosphorus acid [14]. The method was influenced

of the polyvinylpyrrolidone and stannous chloride. Powers et al. reported silver

diethylthiocarbamate as a spectrophotometric reagent for the determination of arsenic

[15]. This method was based on the reduction of arsenic by zinc and generated arsine

was absorbed in silver diethylthiocarbamate. Molybdenum blue was also used as a

162

spectrophotometric reagent for the determination of arsenic in tungstun-free

steels [16].

Takashi and Kazuo reported quercetin as a reagent for the spectrophotometric

determination of arsenic in alloys [17]. Beer's law was obeyed in the range 0-

arsenic in 50 mL and exhibited an absorption maximum at 398 nm.

Steckel and Hall described silver diethyldithiocarbamate as a reagent for the

determination of trace quantities of arsenic [18]. Generated hydride reacted with

As(III) formed a stable red As(III)-silver diethyldithiocarbamate complex and the

absorbance was measured at 540 nm. Beer's law was obeyed for 0-

Takashi and Kazuo used rutin as a reagent for the spectrophotometric determination

of arsenic [19] and Kazuo et al. reported a spectrophotometric determination of

arsenic with morin [20].

Pakalns reported a method for the spectrophotometric determination of arsenic in

a wide variety of salts, white metals, Cu alloys and all types of steels [21]. The

method involved the extraction of the yellow molybdoarsenic acid with BuOH and

subsequent reduction to the blue colored complex. Stara and Stary described

8-mercaptoquinoline as a reagent for the determination of arsenic [22].

8-Mercaptoquinoline reacted with arsenic formed a complex exhibited an absorption

maximum at 380 nm. Only Sn(II) interfered with the determination.

Kellen and Jaselskis described a method for the determination of submicro

amounts of arsenic [23]. The method was based on the reduction of silver and

iron(III) ions by arsine in the presence of ferrozine. Beer’s law was obeyed in range

0.1- Haywood and Riley described a

spectrophotometric determination of arsenic in sea water, potable water and effluents

[24].

Silvia and Victoria described the main sources of arsenic emission in Romania are

ore smelters and refineries [25]. Arsenic determinations were carried out by the silver

163

diethyldithiocarbamate spectrophotometric method on hair and urine samples taken

from smelter workers and individuals residing in two polluted areas and three areas

not polluted by arsenic. Arsenic in hair was found to be a more reliable biological test

than tests on urine, obviously reflecting the differences in arsenic concentrations in

workroom air. Arsenic analysis of hair on people living in two locations near an ore

smelter and a refinery indicated high levels compared to those of individuals residing

in nonpolluted areas. Epidemiological studies were necessary in order to ascertain

effects of heavy arsenic exposure in relation with concurrent exposures to respiratory

irritants and metals.

Takatomi et al. reported bismuthiol-II as a reagent for the spectrophotometric

determination of arsenic [26]. Arsenic(V) reacted with bismuthiol-II which formed a

light yellow precipitate in HCl solution (> 2 M), which was extracted into chloroform.

Arsenic was determined by measuring the absorbance of the CHCl3 extracted at

335 nm. Beer's law was obeyed in the range of 0-

molar absorptivity value of the system was 1.62×104

Lmolcm-1

.

Gowda and Thimmaiah reported promazine hydrochloride as a reagent for the

spectrophotometric determination of arsenic(III), cerium(IV) and nitrite [27]. The

reagent formed a red-colored radical instantaneously in 0.5 M sulfuric acid or

0.5

at 505 nm. Beer's law was valid over the concentration range of 0.5–15 ppm in

sulfuric acid and 0.5–21 ppm in phosphoric acid. The sensitivities of the reaction in

-2

respectively. The

effects of acidity, time, temperature, reagent concentration, and diverse ions were

reported. Arsenic(III) and nitrite were indirectly determined. The proposed method

offered the advantages of good sensitivity, simplicity, rapidity, selectivity and a wider

range of determination without the need for extraction. Hiroshi and Kamihiko

described determination of arsenic using cinchonidine by spectrophotometric method

[28].

Agrawal and Patke reported a simple, sensitive and selective method for the

determination of microamounts of arsenic(III) in the environment [29]. Arsenic

164

formed a yellow colored complex with N-phenylbenzo-hydroxamic acid (PBHA) at

pH 4.5-5.2 which was extracted into chloroform. The effective molar absorptivity of

As-PBHA extract was 1.1×104

Lmol-1

cm-1

at 410 nm. Many common ions associated

with arsenic did not interfere. The effect of pH, reagent concentration and solvent was

described. The arsenic in trace quantities were estimated in the industrial effluents,

soil and grass samples.

Merry and Zarcinas reported sodium tetrahydroborate(III) reagent for the

hydride generation in the spectrophotometric determination of arsenic and antimony

by the silver diethyldithiocarbamate method [30]. The use of oxidising acids for the

digestion of sediment, soil and plant material for arsenic and other metals was not

suitable for antimony. This was overcome by the addition of a reducing agent in the

later stages of digestion which allowed the determination of both arsenic and

antimony simultaneously.

Howard and Arbab-Zavar described the silver diethyldithiocarbamate

spectrophotometric procedure for the determination of arsenic to perform the

differential determination of inorganic arsenic(III) and arsenic(V) species [31]. The

method was based on pH control of the reduction characteristics of the borohydride

ion. Arsine was generated at pH 6 from arsenic(III) following acidification, arsine

was then generated and trapped from arsenic(V) species. The procedure was

appropriate to the determination of 2–40 µg of each arsenic species. Antimony(III),

bismuth(III), chromium(VI), copper(II), gold(III), nickel(II), platinum(IV), silver(I),

tellurium(IV) and tin(II) interfered in the determination of arsenic(III) and

methylarsenic and dimethylarsenic species, platinum(IV) and silver(I) interfered in

the determination of arsenic(V). Interference effected due to platinum was masked by

the addition of 1,10-phenanthroline to the reductant mixture. Interference due to

bismuth, copper, gold, nickel, silver, tellurium and tin was overcome by the

preliminary extraction of the interferents from the sample as their dithizonates.

Elzbieta and Wieckowska used rhodamine-6G as a reagent for the spectrophotometric

determination of arsenic [32].

165

Ali Naki et al. described a method for the spectrophotometric determination of

arsenic using 2-mercaptoethanol as a reagent [33]. Qian-Feng and Peng-Fei reported a

highly sensitive spectrophotometric method for determination of arsenic based on the

formation of an ion-association complex between arsenoantimonomolybdenum blue

and malachite green [34]. The ion-association complex was soluble in the presence of

triton X-305 and arsenic was determined directly in aqueous solution. The molar

absorptivity was found to be 1.13×105

Lmol cm at 640 nm. Beer's law was obeyed

for 0– ation (absorbance = 0.01) was 4

ngmL-1

in the final solution.

Tianze and Ming described a simple and sensitive procedure for the

spectrophotometric determination of traces of arsenic [35]. In this method arsine

generated at pH 5.3 reacted with silver acetate in the aqueous solution in the presence

of tween-80, which formed a yellow silver sol with an absorption maximum at 420

nm. The molar absorptivity was found to be 4.8×104

Lmol-1

cm-1

. Beer's law was

obeyed in the range 0.3-

the determination of arsenic in water and waste water samples.

Maher presented a procedure for the spectrophotometric determination of

arsenic in environmental extracts [36]. In this method arsenic was converted into

arsine using a zinc reductor column, the evolved arsine trapped in a potassium iodide

- iodine solution and the arsenic determined spectrophotometrically as an

arsenomolybdenum blue colored complex. The detection limit was 0.024 µg and the

coefficient of variation was 5.1% at the 0.1 µg level. The method was free from

interferences by other elements at levels normally found in environmental samples.

Kavlentis developed a spectrophotometric determination of arsenic(III) and

antimony(III) using isonicotinoylhydrazones of 4-dimethylaminobezaldehyde

(4-DBIH) and 2-hydroxynaphthaldehyde (2-HNIH) [37]. In this method 4-DBIH and

2-HNIH reacted with As(III) and Sb(III) respectively in CH3COOH medium which

formed colored complexes stable in presence of EDTA. As(III) and Sb(III) did not

166

react with 2-HNIH and 4-DBIH. The Sb(III)-2-HNIH complex was extracted into

isoamyl alcohol.

Lata et al. reported a reducing agent succinyldihydroxamic acid for the extractive

spectrophotometric determination of arsenic in polluted water and environmental

samples [38]. This method was based on the reduction of yellow molybdoarsenic

heteropoly acid with succinyldihydroxamic acid into molybdoarsenic blue. The blue

colored dye exhibited an absorption maximum at 780 nm in n-butanol. Beer's law was

obeyed in the range of 0.02-0.14 ppm of arsenic.

Palanivelu et al. reported a chemical enhancement method for the

spectrophotometric determination of trace amounts of arsenic [39]. Jiayu et al.

reported a spectrophotometric method for the determination of arsenic using

dithioantipyrylmethane (DTPM) as a reagent [40]. The method was based on the

reaction of arsenic with DTPM, which formed 1:2 arsenic-DTPM complex in acidic

medium and the complex was measured at 336 nm with molar absorptivity 3.05×104

Lmol-1

cm-1

. Beer’s law was obeyed in the concentration range of 0–30 µg of arsenic.

Dianwen and Jianping reported methyl orange as a spectrophotometric reagent

for the determination of arsenic [41]. In 0.18-1.08 M H2SO

4 medium, As(III) was

oxidized by KBrO3 in the presence of KBr and methyl orange was bleached by the

excessive KBrO3, and the decrease in color was inversely proportional to the amounts

of arsenic and was used for the determination of arsenic in sludge. The absorption

maximum of methyl orange was 510 nm. The molar absorptivity value of the system

was 4.81×104

Lmol-1

cm-1

.

Yubiao reported gibberellin as a reagent for the spectrophotometric

determination of arsenic [42]. The method was systematically studied on arsenium

molybdenum acid reduction in 0.84 M HCl medium, the As-Mo heteropoly acid was

reduced into very stable As-Mo heteropoly blue by gibberellin. The maximum

absorption wavelength was 838 nm. The molar absorptivity of the system was

167

2.64×104

Lmol-1

cm-1

. Beer’s law was valid over the concentration range 0-1.20

mgmL-1

of arsenic.

Shao-Min et al. used chlorpromazine reagent for the sensitive

spectrophotometric determination of arsenic [43]. The method was based on the

formation of heteropoly arsenomolybdic chlorpromazine complex in aqueous phase

and exhibited maximum absorption at 320 and 350 nm respectively.

Pillai et al. described a sensitive method for the determination of traces of

arsenic(III) [44]. The method involved bleaching of the pinkish red colored dye,

rhodamine B, by the action of iodine which was released by the reaction between

potassium iodate and arsenic in slightly acidic medium. The color of the dye was

measured at 553 nm. Beer’s law was obeyed in the concentration range of

0.04–0.4 mgL of arsenic. The molar absorptivity was found to be

3.24×105

Lmol cm . The proposed method was successfully applied for the

determination of arsenic in environmental and biological samples. Gudzenko et al.

described a spectrophotometric determination of trace amounts of arsenic using

michler's thioketone as a reagent [45]. The molar absorptivity value was

1.02×105

Lmol-1

cm-1

at 640 nm. Beer's law was obeyed in the concentration range

0.008-0.12 mgL-1

of arsenic.

Abd El-Hafeez and El-Syed described a simple, rapid and selective procedure

for the indirect spectrophotometric determination of arsenic(V) [46]. The method was

based on the reduction of As(V) to As(III) with hydroiodic acid (KI + HCl). The

liberated iodine, equivalent to each analyte which was quantitatively extracted with

oleic acid surfactant. The iodine-HOL system exhibited maximum absorbance at

435 nm. The calibration graphs were found to be linear over the range 0.25-20 µgmL-1

of As(V) with lower detection limits 0.15 µgmL-1

. The molar absorptivity and

Sandell’s sensitivity were found to be 0.5×104

Lmol-1

cm-1

and 0.0149 µgcm-2

respectively. The relative standard deviation for five replicate analyses of 4 µgmL-1

of

arsenic was 0.9%. The proposed procedure in the presence of EDTA as a masking

agent for foreign ions was successfully applied to the determination of arsenic in

copper metal, in addition to their determination in spiked and polluted water samples.

168

Kundu et al. reported a spectrophotometric method for the determination of

arsenic in ppm level. The method was based on the color bleaching of methylene blue

in anionic micellar medium [47]. Arsine gas was formed by borohydride reduction of

arsenite/arsenate. Arsine generation and color bleaching (quantification of arsenic)

was done in one-pot. The presence of silver or gold nanoparticles made the

determination faster. Different calibration graphs at the three different ranges of

arsenic concentration such as 0-8.63, 0-1.11 and 0-0.11 ppm were constructed and

limit of detection (LODs) were found to be 1.3, 0.53 and 0.03 ppm respectively. The

method was simple, rapid, reproducible (relative standard deviations lies within ±5%)

and eco-friendly. The method was free from phosphate and silicate interferences and

applied for real sample analysis. Sano et al. reported ammonium

pyrrolidinedithiocarbamate as spectrophotometric reagent for the determination of

arsenic [48].

Taniai et al. described an automated on-line solvent extraction system for the

determination of arsenic or tin in steel by electrothermal atomic absorption

spectrometry (ET-AAS) [49]. The method was based on the formation of AsI3 and

SnI4 in concentrated hydrochloric acid and sulfuric acid media respectively. They are

extracted into benzene and back extracted into water and 0.25 M sulfuric acid,

respectively. An improved gravity phase separator was developed for the recycling of

organic solvent used in the automated on-line solvent extraction system. Using the

proposed system, arsenic or tin contained in the acid dissolved steel sample solution

was automatically extracted and back-extracted. Then, the back-extracted solutions

were used for the determination of arsenic or tin by ET-AAS. In the determination of

arsenic, 800 mgL-1

of cobalt solution had to be used as the matrix modifier to exclude

the effect of coexisting substances such as iodide ion. In the determination of tin,

1000 mgL-1

of palladium solution had to be used in the same manner. By this method,

detection limits of As and Sn we

the 0.05 g of Fe.

Cherian and Narayana reported a spectrophotometric method for the

determination of arsenic in environmental and biological samples using azure B as a

chromogenic reagent [50]. The method was based on the reaction of arsenic(III) with

169

potassium iodate in acid medium to liberate iodine. Bleaching of the violet color of

azur B by the liberated iodine was the basis of the determination and was measured at

644 nm. Beer’s law was obeyed in the range 0.2-10 µgmL-1

of As(III). The molar

absorptivity, Sandell’s sensitivity, detection limit and quantitation limit of the method

were found to be 1.12×104

Lmol-1

cm-1

, 6.71×10-3

µgcm-2

, 0.02 µgmL-1

and 0.08

µgmL-1

respectively.

Behpour et al. described a simple, sensitive, rapid and reliable

preconcentration method for the spectrophotometric determination of trace amounts

of arsenic [51]. Arsenic was retained on a minicolumn of adsorbent naphthalene, as an

ion associate of arsenomolybdate and methyltrioctylammonium ions. The contents of

column was dissolved in a small volume of N,N-dimethylformamide having stannous

chloride as a solvent. The absorbance was measured at 715 nm at room temperature.

The method allowed determination of arsenic in the range of 1–8 ngmL-1

in the initial

solution with r=0.999 (n=6). The relative standard deviation for 15 replicate

measurements of 6.0 ngmL-1

of arsenic was 1.3 % and the detection limit was 0.067

ngmL-1

. The preconcentration factors of 100 and 167 could be achieved when using a

5 and 3 mL DMF for dissolving adsorbent respectively. The optimized method was

successfully applied to determination of arsenic in natural water, synthetic sample and

fish.

Narayana et al. described a spectrophotometric determination of arsenic using

variamine blue reagent [52]. The method was based on the reaction of arsenic(III)

with potassium iodate in acid medium which liberated iodine, oxidized variamine blue

to a violet colored species having an absorption maximum at 556 nm. Beer’s law was

obeyed in the range 0.2-14 µgmL-1

of As(III) in a final volume of 10 mL. The molar

absorptivity and Sandell’s sensitivity for the colored system were found to be

1.43×104

Lmol-1

cm-1

and 5.26×10-2

µg cm-2

respectively.

Graham described a spectrophotometric determination of arsenic in solutions

containing nitric acid necessitates the removal of nitrate ions without the loss of

170

arsenic [53]. A convenient and effective method for its removal was achieved by

treatment with formic acid.

Kunihiro et al. reported a spectrophotometric determination of arsenic in steels

by flow injection analysis using a teflon (PTFE) filter tube concentration method [54].

In this method arsenic coprecipitated with beryllium hydroxide at pH 10, which was

collected with a filter tube for 5 minutes. The precipitate was eluted with 1 M nitric

acid at 0.6 mL per minute and the eluate was reacted with ammonium molybdate.

The molybdoarsenate complex was reduced with ascorbic acid. The obtained

molybdenum blue complex was determined by spectrophotometry at 840 nm. The

calibration curve for arsenic was linear over the range of 0 to 100 ppb. The limits of

detection and determination for arsenic were 0.7 and 2 ppb respectively. The relative

standard deviation for 20 ppb of arsenic was 1.3% (n=8). The iron as a matrix did not

interfere with the determination of arsenic up to a million-fold to arsenic amount.

Keisuke and Emiko described a simple and sensitive spectrophotometric

method for the determination of arsenic in water samples [55]. The method was

based on the formation of micro particles of ethyl violet and molybdoarsenate, which

gave an apparently homogeneous blue color to the solution. The absorption of the

excess dye gradually decreased due to its conversion to a colorless carbinol species

under strongly acidic conditions. Consequently, the sufficiently low reagent blank

enables the spectrophotometric determination of arsenic with the detection limit of 4

µgL-1

. The coefficient of variation for the spectrophotometry at 50 µgL-1

was

3.5% (n = 5).

Revanasiddappa et al. developed a sensitive spectrophotometric method for

the determination of arsenic in environmental samples [56]. Hashemi and Modasser

described a simple spectrophotometric method for the sequential determination of

inorganic arsenic species in a sample [57]. The method was based on the sequential

arsine generation from As(III) and As(V) using selective medium reactions, collection

of the arsine generated in an absorbing solution containing permanganate and ethanol

at 5°C and subsequent reduction of permanganate by arsine. The decrease in

171

permanganate absorbance at 524.2 nm was monitored for arsenic determination. The

acetic acid/sodium acetate and HCl media were used for selective arsine generation

from As(III) and remaining As(V) in one solution, respectively. The effect of

interferences and their possible mechanisms were discussed. Interferences from

transition metal ions were removed by using a Chelex 100 resin. Under optimized

conditions, the established method was applicable to the determination of 3–30

each arsenic species. Good recoveries (96–102%) of spiked artificial sea water, tap

water and standard mixtures of As(III) and As(V) were also found. However, most of

these methods suffer from certain limitations such as; interference by a large number

of ions, low sensitivity and need extraction into organic solvents or heating. Thus

there is need to develop an entirely new method, which would overcome the existing

inadequacies in the determination of arsenic.

The aim of the present work described in this chapter is to provide a simple

and sensitive method for the determination of arsenic using toluidine blue and

safranine O as new reagents. The proposed method has been successfully applied for

the determination of arsenic in various environmental and biological samples.

172

6.3 APPARATUS

A Systronics 2201 UV-Visible Double Beam Spectrophotometer with 1 cm




distilled water was used throughout the study. Arsenic(III) stock solution

(1000 µgmL-1

) was prepared by dissolving 0.1734 g of NaAsO2 in 100 mL of water.

Working standard was prepared by appropriate dilution of stock. Toluidine blue

(0.01 %), safranine O (0.02 %), hydrochloric acid (1 M), potassium iodate (2 %) and

sodium acetate (1 M) were used.

6.5 PROCEDURES



of arsenic(III) were

transferred in to a series of 10 mL calibrated flasks. Potassium iodate (2 %, 1 mL)

then hydrochloric acid (1 M, 1 mL) were added and mixture was gently shaken until

the appearance of yellow color indicating the liberation of iodine. This was followed

by addition of toluidine blue (0.01 %, 0.5 mL) and 2 mL of sodium acetate solution.

The solution was kept for 2 minutes and made up to the mark with distilled water. The

absorbance was measured at 628 nm against the corresponding reagent blank. Reagent

blank was prepared by replacing the analyte (arsenic) solution with distilled water.

The absorbance corresponding to the bleached color, which in turn corresponds to the

analyte (arsenic) concentration, was obtained by subtracting the absorbance of the

blank solution from that of the test solution. The amount of the arsenic present in the

volume taken was computed from the calibration graph (Figure VIA2).



of arsenic(III) were

transferred in to a series of 10 mL calibrated flasks. Potassium iodate (2 %, 1 mL)

173

then hydrochloric acid (1 M, 1 mL) were added and mixture was gently shaken until

the appearance of yellow color indicating the liberation of iodine. This was followed

by addition of safranine O (0.02 %, 0.5 mL) and 2 mL of sodium acetate solution.

The solution was kept for 2 minutes and made up to the mark with distilled water. The

absorbance was measured at 532 nm against the corresponding reagent blank. Reagent

blank was prepared by replacing the analyte (arsenic) solution with distilled water.

The absorbance corresponding to the bleached color, which in turn corresponds to the

analyte (arsenic) concentration, was obtained by subtracting the absorbance of the

blank solution from that of the test solution. The amount of the arsenic present in the

volume taken was computed from the calibration graph (Figure VIA3).

6.5.3 Determination of Arsenic in Polluted Water Samples

Water samples from a river receiving effluent of steel plant and fertilizer

factory were collected in polyethylene bottles, which was filtered through whatman

41 filter paper. A few drops of 10 % KI was added to convert any arsenic(V) to

arsenic(III). Arsenic content was determined directly according to the proposed

method and also by the reference method [44].

6.5.4 Determination of Arsenic in Soil Samples

A known weight (1.0012 g) of a soil sludge sample was placed in a 50 mL

beaker and extracted 4 times with a 5 mL portion of concentrated HCl. The extract

was boiled for about 30 minutes. Arsenic(V) if any was reduced to As(III) by the

process described above. The solution was cooled and diluted to 25 mL volumetric

flask with distilled water. Suitable aliquot of the sample was analyzed by the proposed

method and also by the reference method [44].

6.5.5 Determination of Arsenic in Plant Material

A sample of plant material (grass–5g) was digested with 10 mL of HNO3 for

about 25 minutes. After cooling, 1 mL of perchloric acid was added and heating was

continued for about another 10 minutes. Arsenic(V) if any was reduced to As(III) by

the process described. The solution was transferred to a 25 mL volumetric flask and

diluted to volume with water. Suitable aliquot of the sample was analyzed by the

proposed method and also by the reference method [44]. The results are listed in

Table 6A2.

174




This method involves the liberation of iodine by the reaction of arsenic(III)

with potassium iodate in an acidic medium. The liberated iodine bleaches the blue

color of toluidine blue and measured at 628 nm. This decrease in absorbance is

directly proportional to the As(III) concentration. The absorption spectra of colored

species of toluidine blue are presented in Figure VIA1 and reaction system is

presented in Scheme VI.


In this method is also involves the liberation of iodine by the reaction of

arsenic(III) with potassium iodate in an acidic medium. The liberated iodine bleaches

the pinkish red color of safranine O and measured at 532 nm. This decrease in

absorbance is directly proportional to the As(III) concentration. The absorption

spectra of colored species of safranine O are presented in Figure VIA1 and reaction

system is presented in Scheme VI.


The effect of iodide concentration and acidity on the decolorization is studied

with 2 µgmL-1

of arsenic solution. The oxidation of iodate to iodine was effective in

the pH range 1.0 to 1.5, which could be maintained by adding 1 mL of 1 M HCl in a

final volume of 10 mL. The liberation of iodine from potassium iodate in an acidic

medium is quantitative. The appearance of yellow color indicates the liberation of

iodine. Although any excess of iodate in the solution will not interfere. It is found that

1 mL of 2 % potassium iodate and 1 mL of 1 M HCl are sufficient for the liberation of

iodine from iodate by arsenic and 0.5 mL of 0.01 % toluidine blue or 0.02 % safranine

O was used for subsequent decolorization.

Constant and maximum absorbance values are obtained in the pH=4±0.2. Hence

the pH of the reaction system is maintained at 4±0.2 throughout the study. This could

175

be achieved by the addition of 2 mL of 1 M sodium acetate solution in a total volume

of 10 mL. Effect of pH on color stability is presented in Figure VIA4.




solutions varying arsenic concentration. A straight line graph is obtained by plotting

absorbance against concentration of arsenic. Beer’s law is obeyed in the range of

1.2-10.5 µgmL–1

of arsenic. The molar absorptivity and Sandell’s sensitivity of the

system is found to be 1.076×104

Lmol-1

cm-1

and 9.66×10-3

µgcm-2

respectively.

Correlation coefficient (n = 10) and slope of the calibration curve are 0.9996 and

0.107 respectively. The detection limit (DL=3.3 σ/s) and quantitation limit

(QL=10 σ/s) [where σ is the standard deviation of the reagent blank (n=5) and s is the

slope of the calibration curve] of arsenic determination are found to be 0.308 µgmL-1

and 0.934 µgmL-1

respectively. Adherence to Beer’s law graph for the determination

of arsenic using toluidine blue is presented in Figure VIA2.



solutions varying arsenic concentration. A straight line graph is obtained by plotting

absorbance against concentration of arsenic. Beer’s law is obeyed in the range of

0.4–11.5 µgmL-1

of arsenic. The molar absorptivity and Sandell’s sensitivity of the

system is found to be 1.388×104

Lmol-1

cm-1

, 7.490×10-3

µgcm-2

respectively.

Correlation coefficient (n=10) and slope of the calibration curve are 0.9998 and 0.132

respectively. The detection limit (DL=3.3 σ/s) and quantitation limit (Q

L=10 σ/s)

[where σ is the standard deviation of the reagent blank (n=5) and s is the slope of the

calibration- curve] for arsenic determination are found to be 0.250 µgmL-1

and

0.759 µgmL-1

respectively. Adherence to Beer’s law graph for the determination of

arsenic using safranine O is presented in Figure VIA3.

176


The effect of various foreign ions at microgram levels on the determination of

arsenic using toluidine blue and safranine O is studied. The tolerance limits of

interfering species were established at those concentrations that do not cause more

th-1

, The tolerance

limits of foreign ions are listed in Table 6A1. The results indicated that most of the

common ions did not interfere. Urea, uric acid, glucose, citrate, tartarate and anions

like sulphate and phosphate did not interfere.

6.7 APPLICATIONS

The proposed method is applied to the quantitative determination of arsenic in

various environmental and biological samples. The results of the analysis are

presented in Table 6A2, compare favorably with those from a reference method [44].

The precision and accuracy of the proposed is evaluated by replicate analysis of

samples containing arsenic at four different concentrations.

6.8 CONCLUSIONS

1. The new reagents provide a simple, rapid, sensitive and highly specific method for

the spectrophotometric determination of arsenic.

2. The reagents have the advantage of high sensitivity and selectivity.

3. Proposed method has less interference from the common metal ions and anions.


offers the advantages of high stability of the reaction system (more than 8 hours).

5. The proposed method has been successfully applied for the determination of

arsenic in various environmental and biological samples. A comparison of the

method reported is made with earlier methods and is given in Table 6A3.

177

FIGURE VIA1

ABSORPTION SPECTRA OF COLORED SPECIES OF TOLUIDINE BLUE (a)

AND SAFRANINE O (b)

Wavelength (nm)

200 300 400 500 600 700 800 900

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

a

b

FIGURE VIA2

ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF ARSENIC


C oncentra tion o f arsen ic (µgm L

-1

)

0 2 4 6 8 10 12 14

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

178

FIGURE VIA3

ADHERANCE TO BEER’S LAW FOR THE DETERMINATION OF ARSENIC


Concentration of arsenic (µgmL

-1

)

0 2 4 6 8 10 12 14

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

FIGURE VIA4

EFFECT OF pH ON COLOR INTENSITY

pH

0 1 2 3 4 5 6 7 8

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Toluidine blue

Safranine O

179

SCHEME VI

SCHEME OF THE REACTIONS

2 AsO2

-

+ 2 IO3

-

+ 8 H+

I2 + 2 AsO

2

- +

4 H2O

N

S

+

CH3

NH2

(CH3)2N

N

H

S

CH3

NH2

(CH3)2N

I2 , H

+

Toluidine Blue (Colored) Toluidine Blue (Colorless)

N

N

+

CH3

NH2

NH2

CH3

N

H

N

CH3

NH2

NH2

CH3

I2 , H

+


180

TABLE 6A1

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF ARSENIC

(2 µgmL-1

)

-1 -1

)

Toluidine blue Safranine O Toluidine blue Safranine O

Fe3+

150 100 V5+

125 100

Ni2+

75 100 Zn2+

1000 1000

Cd2+

500 600 PO4

3-

1000 1000

Ba2+

1000 750 Tartarate 1250 1000

Bi3+

1000 1250 Oxalate 1000 1250

Al3+

500 400 Sulfate 750 1000

Ca2+

400 300 Nitrate 750 1000

Co2+

175 150 Glucose 1000 1200

181

TABLE 6A2

DETERMINATION OF ARSENIC IN ENVIRONMENTAL SAMPLES USING

TOLUIDINE BLUE AND SAFRANINE O AS REAGENTS


Samples As3+

added As3+

found Recovery RSD As3+

found Recovery RSD

(µgmL-1

) (µgmL-1

) (%) (%) (µgmL-1

) (%) (%)

a

Ground Water 2.00 2.01 100.50 1.99 1.98 99.00 2.02

Samples 4.00 3.96 99.00 3.03 3.98 99.50 1.51

6.00 5.99 99.83 0.84 5.96 99.33 0.67

8.00 7.92 99.00 0.25 7.95 99.38 0.88

a

Tap Water 2.00 1.94 97.00 1.03 1.96 98.00 3.57

Samples 4.00 3.98 99.50 3.50 3.97 99.25 1.51

6.00 5.94 99.00 0.50 5.96 99.33 0.16

8.00 7.96 99.50 0.75 7.98 99.75 1.25

a

Industrial Water 2.00 1.99 99.50 1.51 1.96 98.00 0.76

Samples 4.00 3.97 99.25 1.51 3.95 98.75 0.51

(Collected from the Indus- 6.00 5.96 99.33 1.34 5.94 99.00 1.34

trial zone of Mangalore city) 8.00 7.95 99.37 1.76 7.97 99.63 0.75

a

River Water 2.00 1.96 98.00 1.67 2.00 100.00 2.62

Samples 4.00 3.95 98.75 1.40 3.95 98.75 1.66

6.00 5.86 98.00 0.72 5.94 99.00 0.57

8.00 7.89 98.62 0.89 7.96 98.50 0.66

a

Soil Samples 2.00 1.98 99.00 1.20 2.00 100.00 1.81

4.00 4.01 100.25 1.31 3.96 99.00 0.37

6.00 5.94 99.00 0.27 5.99 99.83 0.96

8.00 7.96 99.50 0.32 7.95 99.37 0.97

a

Plant Material 2.00 2.01 100.50 1.32 1.98 99.00 1.00

(Grass Samples) 4.00 3.96 99.00 0.60 3.96 99.00 0.80

6.00 6.01 100.17 0.36 5.95 100.17 0.86

8.00 7.96 99.50 0.55 7.92 99.00 0.89

a. Arsenic was not detected.

182

TABLE 6A3




-1

)

ε (Lmol-1

cm-1

)

-2

)

λmax

(nm)

Ref.

No.

Tween-80 Spectrophotometry 0.3- ε = 4.80×104

---------

420 35

Rhodamine B Spectrophotometry 0.04-0.4

mgL-1

ε = 3.24×105

---------

553 44

Oleic acid Spectrophotometry 0.25-20 ε = 0.50×104

ss = 1.49×10-2

435 46

Azure B Spectrophotometry 0.2-10 ε = 1.12×104

ss = 6.71×10-3

644 50

Variamine blue Spectrophotometry 0.2-14 ε = 1.43×104

ss = 5.26×10-2

556 52

Proposed Method

Toluidine blue

Safranine O

Spectrophotometry

Spectrophotometry

1.2-10.5

0.4-11.5

ε = 1.076×104

ss = 9.66×10-3

ε = 1.388×104

ss = 7.490×10-3

628

532

183

6.9 REFERENCES

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9. N. Ybanez, M. L. Cervera and R. Montoro, Anal. Chim. Acta, 258 (1992) 61.

10. G. Samanta, A. Chatterjee, P. P. Chowdhary, C. R. Chanda and D. Chakraborthi,

Environ. Tech., 16 (1995) 223.

11. D. Das, A. Chatterjee, G. Samanta and D. Chakraborthi, Chem. Environ. Res.,

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13. Y. Madrid, D. Chakraborti and C. Camara, Mikrochim. Acta, 120 (1995) 63.

14. B. Cristau, Ann. Pharm. Fr., 16 (1958) 26.

15. G. W. Powers, R. L. Martin, F. J. Piehl and J. M. Griffin, Anal. Chem.,

31(1959)1589

16. M. Elisabeth and M. Jean, Chim. Anal., (Paris), 43 (1961) 276.

17. T. Takashi and H. Kazuo, Bunseki Kagaku, 11 (1962) 1180.

18. L. M. Steckel and J. R. Hall, U.S. At. Energy Comm., Y-1406 (1962) 18.

19. T. Takashi and H. Kazuo, Bunseki Kagaku, 12 (1963) 914.

20. H. Kazuo, T. Takashi and W. Shizuko, Bunseki Kagaku, 12 (1963) 918.

21. P. Pakalns, Anal. Chim. Acta, 47 (1969) 225.

22. V. Stara and J. Stary, Talanta, 17 (1970) 341.

23. G. J. Kellen and B. Jaselskis, Anal. Chem., 48 (1976) 1538.

24. M. G. Haywood and J. P. Riley, Anal. Chim. Acta, 85 (1976) 219.

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25. G. Silvia and C. Victoria, Environ. Health Pers., 19 (1977) 107.

26. M. Takatomi, Y. Yoshikazu and U. Shunzo, Bunseki Kagaku, 27 (1978) 419.

27. S. H. Gowda and K. N. Thimmaiah, Microchem. J., 23 (1978) 291.

28. F. Hiroshi and I. Kamihiko, Bunseki Kagaku, 28 (1979) 627.

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186

CHAPTER 7

SPECTROPHOTOMETRIC DETERMINATION OF SELENIUM IN

ENVIRONMENTAL, BIOLOGICAL AND PHARMACEUTICAL SAMPLES

7.1 INTRODUCTION


7.3 APPARATUS


7.5 PROCEDURES


7.7 APPLICATIONS

7.8 CONCLUSIONS

7.9 REFERENCES

187

7.1 INTRODUCTION

Selenium was discovered by Berzelius and Gahn in 1817. It is widely present

in nature in relatively small concentrations in rocks, plants, coal and other fossil fuels.

Selenium is comparatively rare and its abundance [1] in the lithosphere is 9×10-6

%.

The important minerals containing selenium are clausthalite PbS , crookesite

(Cu,Tl,Ag)2Se, eucairite (Cu,Ag)

2Se, naumannite (Ag,Pb)Se. Selenium is also present

in the soil in certain areas of the U.S.A (the dry plains of Dakota, Wyoming and

Kansas) and is taken up by vegetation which then becomes poisonous to animals;

their meat is then rendered unfit for human consumption. Selenium is however, an

essential trace element in some animal diets.

Selenium is one of the trace element which plays an active role in many

biological systems [2] as it has toxicological and physiological effects [3,4]. Selenium

compounds are extensively used in paints, dyes, glass, electricals, rubber, insecticides,

industries [5] and photocell devices [6] in which variations in the frequencies of the

incident light cause a corresponding variation in the electric current. Grey crystalline

selenium is the only allotrope suitable for this purpose, its conductivity increases

approximately thousand fold when illuminated. Gray selenium is used to a greater

extent for rectification, utilizing the property of asymmetric conduction exhibited by

thin layers of this allotrope [7]. Less important uses of selenium are in the

manufacture of colored (red or reddish-yellow) glasses or ceramic and enamel

pigments. Both selenium and tellurium have been used as secondary vulcanizing

agents for natural rubber in the form of organo-compounds and as oxidation inhibitors

in lubricating oils [8].

Very pure selenium is obtained by heating the crude material in hydrogen at

650o

to form hydrogen selenide, which is then passed through a silica tube at 1000o

to

decompose [9]. Hydrogen sulfide is more stable to heat than the selenide and it

passes out of the system unchanged. The hydrides of elements which are less stable

to heat than hydrogen selenide, are not formed at 650o

C. It is apparent from vapour

density determinations that Se8 molecules are present below 550o

C. The vapour is

188

yellow at the boiling (685o

C) and it dissociates into Se6, Se

2 (above 900

o

C) molecules

and to atomic selenium occurs with the increase in the temperature. A mass

spectrometric study [10] of selenium vapour has provided evidence for the existence

of Se4 and Se

7 molecules in the vapour and the enthalpies of vaporization have been

measured for each of these species.

The determination of selenium is of considerable interest because of its

contrasting biological effects. Selenium is a toxic element as well as a trace element

present in animals and humans. High concentration of selenium causes pulmonary

edema, abdominal pain, jaundice, chronic gastrointestinal diseases, hair loss and

fatigue in humans [11] and its deficiency causes Keshan and Kaschin Beck diseases in

humans, which are frequently reported in China [12]. It also plays a major role in the

life cycle of plants (Cruceferae family), which absorb organoselenium compounds

accumulated in the soils of semiarid areas and may poison livestock that graze on

them. Selenium enters into natural water through seepage from seleniferrous soil and

industrial waste. Water drained from such soil may cause severe environmental

pollution and wild life toxicity. Selenium is also reported to be present in cigarette

paper, tobacco [13] and various cosmetic samples [14].

Selenium is a trace mineral that is essential to good health but required only in

small amounts [15,16]. Selenium is incorporated into proteins to make selenoproteins,

which are important antioxidant enzymes. The anti-oxidant properties of

selenoproteins help to prevent cellular damage from free radicals. Free radicals are

natural by-products of oxygen metabolism that may contribute to the development of

chronic diseases such as cancer and heart disease [16,17]. Other selenoproteins help

to regulate thyroid function and play a role in the immune system [18-20]. Because of

its ant-ioxidant role, selenium has been studied for its potential to protect the body

from many degenerative diseases, including Parkinson’s and cancer. Selenium is

thought to protect cells against cancer because a form of selenium from yeast was

found to have caused cancer cells in test tubes and in animals to undergo apoptosis or

programmed cell death. Selenium is found in some meats and seafood. Animals that

eat grains or plants those were grown in selenium-rich soil have higher levels of

189

selenium in their muscle. In U.S., meats and bread are common sources of dietary

selenium [21,22]. Some nuts are also sources of selenium.

Some industrial and agricultural processes release selenium as a byproduct and

selenium from such sources has caused environmental disaster [23]. Selenium is also

a semiconductor and is used in some types of solid-state electronics as well as in

rectifiers [24], it is an essential nutrient at trace level but toxic in excess [25]. The

threshold limit value for selenium compounds in air is 0.1- 0.2 mgL-1

and in water it is

4.0 ppm [26].

The toxicity, availability and environmental mobility of selenium are very

much dependent on its chemical forms [27]. Selenium can occur in different oxidation

states in organic and inorganic forms. In many environmental matrixes, e.g. natural

water, soils, etc. the predominant oxidation states of selenium are Se(IV) and Se(VI).

Precise knowledge of the amounts of selenium and its compounds present in a system

is therefore required for accurate assessment of the environmental and biological

impact of selenium. This has resulted in an increasing need for analytical methods

suitable for their determination at trace levels.


Selenium is widely spread in relatively small concentrations in rocks, plants,

coal and other fossil fuels. Owing to the importance of selenium, several analytical

techniques have been reported for the determination of selenium [28-31].

Russell et al. described the determination of selenium and tellurium in crude

silver chloride produced as a by-product of the refining of gold and in high-purity

uranium oxide [32]. Selenium and tellurium were separated from one another as well

as from the numerous other substances in the sample before they were extracted into

organic solvents and determined spectrophotometrically. For the determination of

selenium 3,3'-diaminobenzidine was used and for tellurium, diphenylthiourea was

190

used. A minimum of 2 ppm of each element was determined in silver chloride and

0.3 ppm in uranium oxide.

Langmyhr and Omang described a spectrophotometric determination of

selenium(IV) with 1,1 -dianthrimide [33] The method was based on the reaction of

selenium with 1,1 -dianthrimide in concentrated sulfuric acid, which formed a

complex formation was utilized for spectrophotometric determination of up to 0.2 mg

of Se in 25 mL. Boron, germanium, tellurium, bromide and fluoride were interfered.

Langmyhr and Myhrstad described the complex formation in concentrated

sulfuric acid between selenium(IV) and 1,1'-dianthrimide (DA) by spectrophotometry,

infrared spectroscopy and chemical analysis [34]. The system was found to contain

two species, a Se2DA complex and a selenium-1,2,7,8-diphthaloylcarbazole complex.

Langmyhr and Dahl reported an investigation of the applicability of

2,2'-dianthrimide in spectrophotometry and the determination of selenium(IV) [35].

2,2'-Dianthrimide was studied as an analytical reagent and compared with the

properties of 1,1'-dianthrimide, while 1,1'-dianthrimide reacted with B, Ge, Se and Te,

2,2'-dianthrimide was found to react only with selenium(IV). A straight line

but the value of 2,2'-dianthrimide as a reagent for selenium(IV) was reduced by the

high absorption of the reagent.

Kawashima and Ueno reported a spectrophotometric determination of trace

amounts of selenium in iron and steel with 4-methyl-o-phenylenediamine [36]. Brown

reported a spectrophotometric determination of selenium(IV) with diaminochrysazine

[37]. Kasterka reported N- -hydroxypropyl)–o-phenylenediamine and

N-methyl-o-phenylenediamine as reagents for the spectrophotometric determination

of selenium [38,39].

Neve et al. described three important techniques for decomposition of organic

materials for differential determination of the selenium oxidation states [40]. The

method applied to vegetable and biological samples. The only method that was found

191

suitable for the selective determination in aqueous samples gave unsatisfactory results

for organic materials, the recovery of both native and added selenium was very low.

The methods were critically discussed and a procedure was recommended for the

accurate determination of total selenium in organic samples.

Idriss et al. reported a spectrophotometric and potentiometric studies to the

reaction of selenium(IV) with eosin and 1,10-phenanthroline in aqueous solution [41].

An ion association ternary complex with a stoichiometric ratio

selenium-(phenanthroline)2-(eosin)

2was formed. The stability constant of the

complex was determined and the optimum conditions for the spectrophotometric

determination of Se(IV) was established.

Campbell and Yahaya described a spectrophotometric determination of

selenium with dithizone [42]. Microgram amounts of selenium(IV) were determined

by the decrease in absorbance of dithizone in carbon tetrachloride solution at 620 nm.

were 0.6 % and 0.4 % respec -

Neve et al. described an atomic absorption spectrophotometric determination

of ultramicro amounts of selenium in sulphuric acid medium [43]. Selenium(IV) was

determined after the extraction into toluene with an aromatic o-diamine and the

addition of nickel(II) prior to atomization. In the studied samples, total selenium

(0.003–0.022 µg of selenium in 1 mL sulfuric acid) was present only in the tetravalent

state. The detection limit of the method was 0.003 µg of selenium.

Bhat and Gupta described a reagent system consisting of 4-nitrophenyl

hydrazine and 8-quinolnol for the photometric determination of selenium [44].

4-Nitrophenyl hydrazine was oxidized with selenious acid in 6M hydrochloric acid to

4-nitrophenyldiazonium chloride which was then coupled with 8-quinolnol, which

formed a purple colored azoxine dye in alkaline medium with an absorption

maximum at 550 nm. The molar absorptivity and Sandell’s sensitivity were

3.2×104

Lmol cm-2

respectively. The method applied for the

192

detection and determination of complex materials such as cabbage leaf and cigarette

paper.

Bodini and Alzamora described a spectrophotometric determination of trace

amounts of selenium with 4,5,6-triaminopyrimidine(TAP) [45]. In this method TAP

reacted in acidic aqueous medium with selenium(IV), which formed a piazselenol

with an absorption maximum at 362 nm with a molar absorptivity of 1.72×104

Lmol cm . The compound was stable but not extracted into non-polar solvents. The

calibration graph was linear up to 10 ppm of selenium, with a detection limit of

0.1 ppm in the sample solutions. Of the many different ions tested only iron (III)

(in the presence of chloride) and tin (II) interfered. The method produced good

reproducibility with a relative standard deviation of 1.5 % for pure solutions. The

method applied to the analysis of water and electrolytic copper. Lavale and Dave

described a spectrophotometric determination of selenium with chromotropic acid

[46].

Kasterka described a spectrophotometric determination of selenium with

2-aminodiphenylamine in an acidic medium [47]. The optimum hydrogen ion

concentration ranges from about 0.1 to 5M. The molar absorptivity at λ=352 nm was

1.81×104

Lmol–1

cm–1

. The product was extracted as an ion-association complex with

perchlorate into a mixture of hexanol and chlorobenzene. The kinetics of the reaction

was investigated.

Bodini et al. reported a reagent 5,5-dimethyl-1,3-cyclohexanedione which

reacted in dilute acid solution with selenium(IV), which formed a benzoxaselenol and

showed an absorption maximum at 313 nm [48]. The molar absorptivity of the

method was 4.0×103

Lmol-1

cm-1

. The calibration graph was linear up to 30 ppm of

selenium, with a detection limit of 0.1 ppm in the final solutions.

Kasterka reported the condensation reactions of Se(IV) with

3,4-diaminobenzoic acid and 4-bromo-1,2-phenylenediamine by means of UV spectra

and kinetic investigations [49]. A mechanism for the formation of

1,2,3-benzoselenadiazole in acidic medium was proposed. The influence of

193

substitution at C4 in 1,2-phenylenediamine on the reactivity of the system was

discussed.

Manish et al. reported a simple and sensitive method for the

spectrophotometric determination of selenium(IV) using 6-amino-1-naphthol-3-

sulphonic acid as a reagent [50]. The molar absorptivity and Sandell’s sensitivity of

the method were found to be 18.5×103

Lmol-1

cm-1

and 0.004 µgcm-2

respectively.

Beer’s law was obeyed in the concentration range of 0.03-0.3 µgL-1

of selenium.

Safavi and Afkhami reported a highly sensitive catalytic spectrophotometric

method for the determination of selenium(IV) and selenium(VI) [51]. The method

was based on the catalytic effect of Se(IV) in redox reaction of bromate with

semicarbazide in hydrochloric acid media. The determination range of both analyses

was 50-4000 ngmL-1

. Selenium as low as 4.7 ngmL-1

was determined by this method.

The application of the method to the determination of selenium in Kjeldahl tablets and

in a health-care product was described.

Ramachandran and Kumar described a reaction of selenium with

2,3-diaminonaphthalene which was reinvestigated with bromide ion as a catalyst [52].

In acid medium, selenium reacted with the above reagent, which formed a complex

extracted with cyclohexane and with an absorption maximum at 378 nm. The molar

absorptivity of the complex was 17.5×103

Lmol-1

cm-1

. Beer’s law was obeyed in the

concentration range of 0.5--1

of selenium.

Pyrzynska developed the conditions for a spectrophotometric determination of

selenium with 1-naphthyloamine-7-sulfonic acid (Cleve um(IV)

formed a yellow complex with this ligand in sulfuric acid media with maximum

absorbance at 350 nm. The molar absorptivity was 8.9×104

Lmol-1

cm-1

. The

-2

. The amount of Se in a column of unit cross-sectional area with the

absorbance of 0.001. The interference of various ions was studied. The method was

applied for the determination of selenium in a vitamin supplement.

194

Agrawal et al. reported a reagent system for the spectrophotometric

determination of selenium in environmental and cosmetic samples using leucocrystal

violet(LCV) [54]. The method was based on the reaction of selenium with acidified

potassium iodide to liberate iodine, which oxidized LCV to crystal violet with an

absorption maximum at 593 nm. Beer's law was obeyed over the concentration range

of 0.5-

and Sandell's sensitivity were found to be 3.68×105

Lmol-1

cm-1

a-2

respectively.

Mousavi et al. reported a simple and sensitive flow infection

spectrophotometric method for the determination of selenium [55]. The method was

based on the catalytic effect of Se(IV) on the reduction reaction of thionin with

sulphide ion, monitored spectrophotometrically at 598 nm. Beer’s law obeyed in the

range 0.005-1.5 µgmL-1

of selenium. The detection limit was 5 µgmL-1

. The relative

standard deviation for eight replicate measurements was 1.1% for 1 µgmL-1

of

selenium.

Varadarajan et al. reported bis(ethanedithioamido)-2,4-dioxo-3-

oxyminopentane, bis(EDA)DOP as a sensitive and selective reagent for the

spectrophotometric determination of total selenium traces [56]. The method was

based on the color reaction between selenium(IV) and bis(EDA)DOP on heating the

mixture at 50°C for 2.0 minutes which is extracted in 1-octanol from an acidic

medium with respect to 2-3 M HCl on shaking for 1.5 minutes. The absorbance of

the extracted species was measured at 495.0 nm and the molar absorptivity was

1.268×104

Lmol-1

cm-1

. The complex system obeyed Beer’s law within

0.2-15.0 mgmL-1

in Ringbom’s optimum working range of 4.36-12.02 µgmL-1

with a

sensitivity of 2.21 ngcm-2

for effective spectrophotometric determination of total

selenium. The method was applied to the determination of total selenium in various

synthetic mixtures and other samples.

Melwanki and Seetharamappa described spectrophotometric determination of

selenium(IV) using methdilazine hydrochloride as a reagent [57]. The reagent formed

195

a red radical cation by selenium (IV) acid medium and exhibited an absorption

maximum at 513 nm. Beer’s law was valid over the concentration range 0.1-2.3

mgL-1

of selenium(IV). Sandell’s sensitivity of the reaction was found to be

3.57 ngcm-2

and the molar extinction coefficient was 9.32×104

Lmol-1

cm-1

.

Revanasiddappa and Kiran Kumar reported a direct method for the

spectrophotometric determination of micro amounts of selenium(IV) using variamine

blue as a chromogenic reagent [58]. The method was based on the reaction of

selenium with potassium iodide in an acidic medium to liberate iodine, which

oxidized variamine blue to a violet colored species with an absorption maximum at

546 nm. Beer’s law was obeyed in the range 2-20 µgmL-1

of selenium in a final

volume of 10 mL. The molar absorptivity and Sandell’s sensitivity for the colored

system were found to be 2.6×104

Lmol-1

cm-1

and 0.003 µgcm-2

respectively.

Revanasiddappa and Kiran Kumar reported used thionin as a reagent for the

spectrophotometric determination of selenium(IV) in real samples of water, soil, plant

materials, human hair, synthetic cosmetics and in pharmaceutical preparations [59].

The molar absorptivity and Sandell’s sensitivity of the method were found to be

7.33×104

Lmol-1

cm-1

and 0.0011 µgcm-2

respectively. Beer’s law was obeyed in the

range 1.0-5.0 µgmL-1

of selenium in a final volume of 10 mL.

Gurkan and Akcay developed a simple and sensitive catalytic

spectrophotometric method for the determination of trace amounts of selenium [60].

The method was based on the catalytic effect of Se(IV) on the reduction of maxilon

blue-SG by sodium sulfide. Indicator reaction was followed spectrophotometrically

by measuring an absorption maximum at 654 nm. Selenium could quantitatively be

determined in the range 0.004-0.200 µgmL-1

Se(IV) with a detection limit of

0.205 ngmL-1

selenium(IV).

Narayana et al. described a rapid and sensitive spectrophotometric method for

the determination of trace amounts of selenium using starch and iodine as

chromogenic reagents [61]. The proposed method was based on the reaction of

selenium with potassium iodide in an acidic medium to liberate iodine. This reacted

196

with starch to form a blue colored species with an absorption maximum of 570 nm.

Beer's law was obeyed in the range of 2-

and Sandell's sensitivity were found to be 1.40×104

Lmol-1

cm-1

and 5.45×10-3 -2

respectively. The proposed method was successfully applied to determine selenium in

a sample of natural water, polluted water, soil sludge, biological samples and human

hair. Guo et al. described a new vapor generation technique utilizing UV irradiation

coupled with atomic absorption for the determination of selenium in aqueous

solutions [62].

Ningli reported a spectrophotometric method for the determination of trace of

selenium. The method was based on the bromopyrogallol red oxidized fading reaction

by selenium(IV) in neutral solution [63]. The optimum conditions were studied.

Beer's law was obeyed in the concentration range of 0-3.0 µgmL-1

. The molar

absorptivity of the method was found to be 8.05×103

Lmol-1

cm-1

. The method could

be applied to the detection of selenium in tea and mineral water samples with

satisfactory results.

Ensafi and Lemraski described a sensitive and rapid kinetic

spectrophotometric method for the detection of ultra trace amounts of selenium(IV)

[64]. The method was based on the catalytic effect of Se(IV) on the reduction of

sulfonazo by sodium sulfide. The limit of detection was 0.3 ngmL-1

of Se(IV) at 680

nm. The selectivity of the selenium detection was greatly improved using the cation

exchange resin. The method was used for the detection of Se(IV) in a food sample,

natural water and synthetic samples with satisfactory results.

Khajehsharifi et al. reported a kinetic spectrophotometric method for the

simultaneous determination of selenium(IV) and tellurium(IV) [65]. The method was

based upon the catalytic effect of these cations on the reaction of toluidine blue with

sulfide. Partial least squares calibration method was employed for the data

manipulation and analysis. The concentrations were varied between 0.02–0.24 and

0.01–0.08-1

for Se(IV) and Te(IV) respectively. Cross-validation method was

used to select the optimum number of factors. The root mean square errors of

difference for selenium and tellurium were 1.2 and 1.7-1

respectively.

197

Application of the method to artificial samples and several mixtures of standard

solutions of Se(IV) and Te(IV) were performed and satisfactory results were obtained.

Liang et al. described a method of kinetic spectrophotometry for the detection

of trace amounts of selenium was established by catalytic kinetics [66]. The

sensitivity of the method was 0.905 µgmL-1

. Beer’s law was obeyed in the

concentration range of 0-9.6 µgmL-1

. The method was used for the detection of trace

amounts of Se(IV) in Chinese herbal medicine with satisfactory results.

Feng-Shang and Di reported UV spectrophotometric determination of

selenium in black fungus [67]. The method was sensitive and precise with relative

standard deviation (RSD) of 0.054% and the recovery of 97.74%-100.75% and was

applied for the detection of selenium in blank fungus with satisfactory results.

Gudzenko et al. developed a catalytic spectrophotometric determination of

nanogram amounts of selenium(IV) [68]. The method was based on the reduction of

nitrate with iron(II)-EDTA catalyzed by Se(IV) compounds. The reaction proceeded

in several stages and formed iron(III)-EDTA, the nitrosyl complex of iron, nitrous

acid and other products. Nitrous acid entered into the diazotization reaction with

aromatic amine. The resulting diazo compound was coupled with another aromatic

amine to form the azo compound. 4-Nitroaniline was used as the diazo component

and N-diethyl-N′-(1-naphthyl)ethylenediamine was used as the azo component. The

molar absorptivity of the solution of the azo compound was 4.5×104

at 540 nm. The

detection limit of selenium by the proposed method was 0.1 ngmL-1

. In the

determination of 0.2 and 2 ngmL-1

selenium, the relative standard deviation was 6 and

2 % respectively.

Zhengjun et al. developed a flow injection catalytic kinetic spectrophotometric

method for rapid determination of trace amounts of selenium [69]. The method was

based on the accelerating effect of Se(IV) on the reaction of EDTA and sodium nitrate

with ammonium iron(II) sulfate hexahydrate in acidic medium. The absorbance

intensity was registered in this reaction solution at 440 nm. The calibration graph was

linear in the range of 5×10 –2×10 and 2×10 –2×10 gmL . The detection limit

was 2×10 gmL . The relative standard deviation was 3.4% for 5×10 gmL

198

selenium(IV) (n=11), 2.7% for 5×10 gmL selenium(IV) (n = 11). This method was

very simple, rapid and suitable for automatic and continuous analysis. The method

was applied successfully to determination of Se(IV) of seawater samples. Muramoto

et al. reported a novel method for the determination of trace amounts of selenium in

iron and steel has been demonstrated by a HPLC using 2,3-diaminonaphthalene

(DAN) as a derivatizing reagent [70].

Revanasiddappa and Dayananda reported a highly sensitive

spectrophotometric determination of selenium using a reagent leuco malachite green

[71]. The method was based on the reaction of selenium(IV) with potassium iodide in

an acidic condition to liberate iodine, the liberated iodine oxidized leuco malachite

green to malachite green dye. The green coloration was developed in an acetate buffer

(pH 4.2–4.9) on heating in a water bath (40°C). The formed dye exhibited an

absorption maximum at 615 nm. The method obeys Beer’s law over a concentration

range of 0.04–0.4 µgmL selenium. The molar absorptivity and Sandell’s sensitivity

of the color system were found to be 1.67×105

Lmol cm-1

and 0.5 ngcm

respectively. The method was successfully applied to the determination of selenium in

real samples of water, soil, plant material, human hair and cosmetic samples.

Li et al. described a catalytic spectrophotometric method for the determination

of trace amount of Se(IV) in microemulsion medium [72]. The method was based on

the catalytic effect of traces of selenium(IV) on the oxidation of

2 -dichlorophenylfluorone by potassium bromate with HNO3 as an activator in the

presence of nonionic microemulsion medium. Under optimum conditions, the

calibration graph was linear in the range of 0.4– of Se(IV) at 480 nm. The

detection limit achieved was 9.86×10 . Samples were dissolved and the

obtained trace amounts of Se(IV) was separated and enriched by sulphydryl dextrane

gel. The method was applied for the determination of trace selenium with satisfactory

results.

Cherian and Narayana was reported a system for the spectrophotometric

determination of trace amounts of selenium [73]. The proposed method was based on

the oxidation of phenylhydrazine-p-sulphonic acid and the coupling reaction.

Selenium(IV) oxidized phenylhydrazine-p-sulphonic acid into its diazonium salt in an

199

acidic medium. The diazonium salt was then coupled with acetylacetone or ethyl

acetoacetate in an alkaline medium, which formed azo dyes with absorption

maximum at 490 or 470 nm respectively. The method obeyed Beer’s law in the

concentration range of 0.5-20 µgmL-1

of selenium with phenylhydrazine-p-sulphonic

acid-acetylacetone and 1.0-24 µgmL-1

of selenium with phenylhydrazine-p-sulphonic

acid-ethyl acetoacetate couples. The molar absorptivity and Sandell’s sensitivity for

the colored system with phenylhydrazine-p-sulphonic acid-acetylacetone and

phenylhydrazine-p-sulphonic acid-ethyl acetoacetate couples were found to be

1.02×104

Lmol-1

cm-1

, 7.69×10-3

µgcm-2

and 1.18×104

Lmol-1

cm-1

, 6.67×10-3

µgcm-2

respectively.

Mathew and Narayana used azure B as a chromogenic reagent for the

spectrophotometric determination of selenium [74]. The molar absorptivity and

Sandell’s sensitivity of the method were found to be 0.9473×105

Lmol-1

cm-1

and

8.33×10-4

µgcm-2

respectively. Beer’s law was obeyed in the range 2.0-10.0 µgmL-1

of selenium.

Kumar et al. described a flow injection spectrophotometric method for the

determination of selenium (IV) in pharmaceutical formulations [75]. The method was

based on the oxidation of 4-aminoantipyrine (4-amino-1,2-dihydro-1,5-dimethyl-2-

phenyl-3H-pyrazole-3-one; 4-AAP) by selenium in presence of acidic medium and

the coupling with N-(naphthalen-1-yl)ethane-1,2-diamine dihydrochloride, which

formed a violet color derivative. Beer's law was obeyed for selenium in the

concentration range 0.05-5.0 µgmL-1

and Sandell's sensitivity was found to be

0.00286 µgcm-2

. The reported methods are either not sensitive enough or required

complicated and expensive instruments and are time consuming. The need for a

simple and sensitive spectrophotometric method for the determination of selenium is

therefore clearly recognized.

The aim of the present work is to provide a simple, accurate and sensitive

method for the determination of selenium using toluidine blue and safranine O as new

reagents. The proposed method is well adopted for the determination of selenium in

various environmental and pharmaceutical samples.

200

7.3 APPARATUS





double distilled water was used throughout the study. A standard stock solution of

selenium was prepared by dissolving 1.912 g of NaHSeO3

in 1000 mL of water and

standardized by the dithiozone method [42]. Toluidine blue solution (0.02%),

safranine O solution (0.02%), potassium iodide (2%), hydrochloric acid (1M), acetate

buffer solution (pH=4) were used.

7.5 PROCEDURES



of selenium solution

were transferred into a series of 10 mL calibrated flasks. A volume of 1 mL of 2 %



liberation of iodine. A 0.5 mL of 0.02 % toluidine blue solution was then added to it

followed by the addition of 2 mL of acetate buffer solution of pH=4 and the reaction




the analyte(selenium) solution with distilled water. The absorbance corresponding to

the bleached color which in turn corresponds to the analyte(selenium) concentration



from the calibration graph (Figure VIIA1).

201


Aliquots of solution containing 0.8–15.4 µgmL-1

of selenium were transferred

into a series of 10 mL calibrated flasks. A volume of 1 mL of 2 % potassium iodide

solution was added followed by 1 mL of 1M hydrochloric acid and the mixture was

gently shaken until the appearance of yellow color, indicating the liberation of iodine.

A 0.5 mL of 0.02% safranine O and 2 mL of acetate buffer solution of pH=4 were

added to each flask and the reaction mixture was shaken for 2 minutes. The contents

were diluted to 10 mL with distilled water. The absorbance of the resulting solutions

were measured at 532 nm against a reagent blank. A blank solution was prepared by

replacing the selenium solution with distilled water. The absorbance corresponding to

the bleached color, which in turn corresponds to the selenium concentration, was

obtained by subtracting the absorbance of the blank solution from that of the test

solution. The amount of the selenium present in the volume taken was computed from

the calibration graph (Figure VIIA2).

7.5.3 Determination of Selenium in Water

Aliquots (≤5 mL) of water sample containing not more than 15.0 µgmL-1

of

selenium were treated with 0.5 mL of 1M NaOH and 0.5 mL of 0.2M EDTA. The

solutions were mixed and centrifuged to remove the formed precipitate. The

centrifugate was transferred to a 10 mL calibrated flask. They all tested negative. To

these samples a known amount of the selenium was added. An aliquot of the made up

solutions containing selenium was determined directly according to the proposed

method (using toluidine blue or safranine O) and also by the reference method[48].

The results are listed in Table 7A2.

7.5.4 Determination of Selenium in Soil

A known weight (50.0 g) of a soil sludge sample was placed in a 50 mL

beaker and extracted 4 times with a 5 mL portion of concentrated HCl. The extract

was boiled for 10 minutes to convert any Se(VI) present in the soil to Se(IV) cooled

and neutralized (pH =7) with 10% NaOH. A volume of 5 mL of 5 % EDTA solution

was added and the contents were made up to 25 mL with water. An aliquot (≤5 mL)

of the made up solution containing selenium was determined directly according to the

202

proposed method (using toluidine blue or safranine O) and also by the reference

method[48]. The results are listed in Table 7A2.

7.5.5 Determination of Selenium in Pharmaceutical Samples

A volume of 10 mL of each Fourts B (Fourts India Laboratories Private Ltd.,

Kelambakkam-603 103, Tamil Nadu, India) and Homoxid (Angle French drugs,

India) samples were treated with 10 mL of concentrated HNO3 and the mixture was

then evaporated to dryness. The residue was leached with 5 mL of 0.5 M H2SO

4. The

solution was diluted to a known volume with water after neutralizing with dilute

ammonia. An aliquot of the made up solution was analysed for selenium according to

the general procedure described earlier. The results are listed in Table 7A2.



This method involves the liberation of iodine by the reaction of selenium with

potassium iodide in an acidic medium. The liberated iodine bleaches the blue color of

toluidine blue and absorbance of the solution is measured at 628 nm. This decrease in

absorbance is directly proportional to the selenium concentration. At the same time

the liberated iodine bleaches the pinkish red color of safranine O and the absorbance

of the solution is measured at 532 nm. This decrease in absorbance is directly

proportional to the selenium concentration. The absorption spectra of colored species

of toluidine blue and safranine O is presented in Figure VIIA3 and reaction system is

presented in Scheme VII.


The effect of iodide concentration and acidity on the decolorization is studied

with 5 µgmL-1

of selenium solution. The oxidation of iodide to iodine is effective in

the pH range 1.0 to 1.5, which could be maintained by adding 1 mL of 1 M HCl in a

final volume of 10 mL. The liberation of iodine from KI in an acidic medium is

quantitative. The appearance of yellow color indicates the liberation of iodine.

Although any excess of iodide in the solution did not interfere. It is found that 1 mL of

2 % KI and 1 mL of 1 M HCl were sufficient for the liberation of iodine from iodide

203

by selenium and 0.5 mL of 0.02 % toluidine blue or 0.02 % safranine O is used for

subsequent decolorization.

Constant and maximum absorbance values are obtained in the pH=4±0.2.

Hence the pH of the reaction system was maintained at 4±0.2 throughout the study.

This could be achieved by the addition of 2 mL of 1 M sodium acetate solution in a

total volume of 10 mL. In the case of toluidine blue method, the bleached reaction

system is found to be stable for more than 6 hours and also in safranine O reagent

case, the bleached reaction system is found to be stable for 4 hours.




solutions varying selenium concentration. A straight line graph is obtained by

plotting absorbance against concentration of selenium. Beer’s law obeyed in the

range of 1.0–16.0 µgmL–1

of selenium (Figure VIIA1). The molar absorptivity and


Lmol-1

cm-1

and 6.37×10-3

µgcm-2


are 0.9992 and 0.150 respectively. The detection limit (DL=3.3 σ/s) and quantitation

limit (QL=10 σ/s) [where σ is the standard deviation of the reagent blank (n=5) and

s is the slope of the calibration curve] of selenium determination are found to be 0.220

µgmL-1

and 0.670 µgmL-1

respectively. Adherence to Beer’s law graph for the

determination of selenium using toluidine blue is presented in Figure VIIA1.



solutions varying selenium concentration. A straight line graph is obtained by


range of 0.8 – 15.4 µgmL-1

of selenium (Figure VIIA2). The molar absorptivity and


Lmol-1

cm-1

, 6.63×10-3

µgcm-2


are 0.9995 and 0.154 respectively. The detection limit (DL=3.3 σ/s) and quantitation

limit (QL=10 σ/s) [where σ is the standard deviation of the reagent blank (n=5) and

204

s is the slope of the calibration- curve] for selenium determination are found to be

0.214 µgmL-1

and 0.649 µgmL-1

respectively. Adherence to Beer’s law graph for the

determination of selenium using safranine O is presented in Figure VIIA2.

7.6.4 Effect of Diverse Ions


selenium is examined. The tolerance limits of interfering species are established at

those concentrations that do not cause more than

-1

. Ions such as Ni2+

, Cu2+

, Al3+

, Co2+

, V5+

, Fe3+

, sulfate are

interfered. However, the tolerance level of some of these ions may be increased by the

addition of 1 mL of 1 % EDTA solution and the interference of Fe3+

was masked

using sodium fluoride solution. The tolerance limits of various foreign ions are given

in Table 7A1.

7.7 APPLICATIONS

The developed method is applied to the quantitative determination of selenium

in various environmental and pharmaceutical samples. The results of are presented in

Table 7A2 and the analysis of the above samples are compared with those from a

reference method [48]. The precision and accuracy of the proposed method is

evaluated by replicate analysis of samples containing selenium at two different

concentrations.

7.8 CONCLUSIONS

1. The reagents provide a facile, rapid and accurate method for the spectrophotometric

determination of selenium.

2. The reagents have an advantage of high sensitivity and selectivity.

3. The method needs neither heating for the complete color development nor

extraction into any organic phase.

4. The accuracy of the method is comparable with most methods reported in the

literature.

5. The proposed method is used for the determination of traces of selenium in

various environmental and pharmaceutical samples. A comparison of the method

reported is made with earlier methods and is given in Table 7A3.

205

FIGURE VIIA1

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF SELENIUM

TOLUIDINE BLUE AS REAGENT

Concentration of selenium (µgmL

-1

)

0 2 4 6 8 10 12 14 16 18 20

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

FIGURE VIIA2

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF SELENIUM


Concentration of selenium (µgmL

-1

)

0 2 4 6 8 10 12 14 16 18

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

206

FIGURE VIIA3

ABSORPTION SPECTRA OF COLORED SPECIES OF TOLUIDINE BLUE (A)

AND SAFRANINE O (B)

W avelength (nm)

200 300 400 500 600 700 800 900

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

a

b

SCHEME VII

H2SeO

3 + 4I

-

+ 4H+

Se + 2I2

+ 3 H20

N

S

+

CH3

NH2

(CH3)2N

N

H

S

CH3

NH2

(CH3)2N

I2 , H

+

Toluidine Blue (Colored) Toluidine Blue (Colorless)

N

N

+

CH3

NH2

NH2

CH3

N

H

N

CH3

NH2

NH2

CH3

I2 , H

+


207

TABLE 7A1

EFFECT OF DIVERSE IONS ON THE DETERMINATION OF SELENIUM

(5 µgmL-1

)

Foreign ions

Tolerance limit

(µgmL-1

)

Foreign ions

Tolerance limit

(µgmL-1

)

Ni2+*

Cu2+ *

Cd2+

Ba2+

Fe3+*

Bi3+

Al3+*

Ca2+

75

50

100

200

75

200

50

200

Co2+*

V5+*

Zn2+

Tartarate

Oxalate

PO4

3-

Sulfate*

Glucose

75

75

200

500

500

250

50

200

*Masked by secondary masking agents.

208

TABLE 7A2

DETERMINATION OF SELENIUM IN VARIOUS ENVIRONMENTAL AND

PHARMACEUTICAL SAMPLES USING TOLUIDINE BLUE AND SAFRANINE

O AS REAGENTS


Samples Se(IV) Se(IV) Recovery RSD Se(IV) Recovery RSD

added* found* (%) (%) found* (%) (%)

a

Tap Water 4.00 3.96 99.00 0.65 3.98 99.50 0.51

Samples 8.00 7.92 99.00 0.75 7.95 99.38 0.88

a

Rain Water 4.00 3.98 99.50 2.50 3.97 99.25 1.81

Samples 8.00 7.96 99.50 0.85 7.98 99.75 0.65

a

Industrial Water 4.00 3.97 99.25 1.51 3.95 98.75 0.58

Samples 8.00 7.95 99.37 0.76 7.97 99.63 1.35

(Collected from the industrial

zone of Mangalore city)

Soil Samples ___ 1.35 ___ 0.74 1.32 ___ 2.03

4.00 5.36 100.25 1.34 5.28 99.00 1.37

8.00 9.31 99.50 0.92 9.27 99.37 0.97

ab

Fourts B 4.00 3.99 99.75 0.40 3.96 99.00 0.76

8.00 7.95 99.37 0.75 7.92 99.00 1.04

ac

Homoxid 4.00 3.96 99.00 1.21 3.94 98.50 1.76

8.00 7.91 98.87 1.11 7.93 99.13 0.75

-1

a. Selenium was not detected in ground water, tap water, industrial water and

pharmaceutical samples.

b. Fourts B (Fourts India Laboratories Private Ltd., Kelambakkam-603 103, Tamil

Nadu, India) Composition –thiamine mononitrate-10 mg; riboflavin-10 mg;

pyridoxine hydrochloride-3mg; vitamin C-75 mg; zinc sulphate-55 mg; selenium-

100 µg; folic acid-1mg; niacinamide-50µg; chromium-200µg; L-cysteine HCl-

25mg; glycine-25mg, glutamic acid-25mg; vanadium-100µg;

c. Homoxid (Angle-French drugs, India) Composition – pyridoxine HCl-10mg; folic

acid-1mg; cyanocobalamin-0.4mg; vit c-150mg; β-carotene-10mg; selenium-70µg;

209

TABLE 7A3

COMPARISON OF THE METHOD REPORTED IS MADE WITH EARLIER

METHODS



-1

)

ε (Lmol-1

cm-1

)

-2

)

λmax

Ref.

No.

5,5-dimethyl-1,3-

cyclohexanedione

Spectrophotometry upto 30 ε = 4.00×103

ε = 3.77×103

313

300

48

Variamine Blue Spectrophotometry 2.0-20 ε = 2.60×104

ss = 3.0×10-3

546 58

Starch Spectrophotometry 2.0- ε = 1.40×104

ss = 5.45×10-3

570 61

Leuco malachite

green

Spectrophotometry 0.04-0.4 ε = 1.67×105

ss = 0.50 ngcm-2

615 71

Ethyl acetoacetate Spectrophotometry 1.0-24 ε = 1.18×104

ss = 6.67×10-3

470 73

Acetylacetone Spectrophotometry 0.5-20 ε = 1.02×104

ss = 7.69×10-3

490 73

Azure B Spectrophotometry 2.0-10 ε = 0.947×105

ss = 8.33×10-4

644 74

Proposed Method

Toluidine blue

Safranine O

Spectrophotometry

Spectrophotometry

1.0-16.0

0.8-15.4

ε = 1.240×104

ss = 6.37×10-3

ε = 1.190×104

ss = 6.630×10-3

628

532

210

7.9 REFERENCES

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6. B. Lange, Photoelements and Their Applications, Reinhold Publishing Co. Inc.,

New York, (1938).

7. H. K. Henisch, Metal Rectifiers, Clarendon Press, Oxford (1945).

8. K.W. Bagnall, The Chemistry of Selenium, Tellurium and Polonium, Elsevier,

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9. S. Nielsen and R. J. Heritage, J. Electrochem. Soc., 106 (1959) 39.

10. R. Yamdagni and R. F. Porter, J. Electrochem, Soc., 115 (1968) 601.

11. M. I. Smith, K. W. Franke and B. B. Westfall, U.S. Public Health Rep., 51 (1936)

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13. P. W. West and A. D. Shendrikar, Anal. Chim. Acta, 82 (1977) 403.

14. J. R. Shapira, “Organic Selenium Compounds their Chemistry and Biology”,

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16. S. B. Goldhaber, Regulatory Toxicology and Pharmacology, 38 (2003) 232.

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18. O. A. Levander, J. Nutr., 127 (1997) 948.

19. J. R. Arthur, Can. J. Physiol Pharmacol., 69 (1991) 1648.

20. B. Corvilain, B. Contempre, A. O. Longombe, P. Goyens, C.Gervy-Decoster,

F. Lamy, J. B. Vanderpas and J. E. Dumont, Am. J. Clin. Nutr., 57 (1993) 244S.

21. J. A. Pennington and S. A. Schoen, Int. J. Vitam. Nutr. Res., 66 (1996) 342.

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23. F. A. Patty, Industrial Hygiene and Toxicology, Vol 2, Wiely, New York, (1962)

p.886.

24. H. K. Henisch, Metal Rectifiers, Clarendon Press, Oxford, 1945.

211

25. R. W. Andrews and D. C. Johnson, Anal. Chem., 47 (1975) 249.

26. Spallholz J. E, Martin J. L, Ganther H. E, 2nd

Edn., Selenium in Biology and

Medicine. A VI, West Port, CT., 1981

27. R. L. Tatken and R. J. Lewis, Registry of Toxic Effects of Chemical Substances.

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28. A. H. Jeffrey, G. B. Roger and A. G. Carolyn, J. AOAC Int., 75 (1992) 269.

29. I. I. Stewart and A. Chow, Talanta, 40 (1993) 1345.

30. H. Da-Qing, X. Guo-Hong, Z. Yi-Min, T. Guo-Jun, Talanta, 43 (1996) 595.

31. F. MacLeod, B. A. McGaw and C. A. Shand, Talanta, 43 (1996) 1091.

32. B. G. Russel, W. V. Lubbe, A. Wilson, E. Jones, J. D. Taylor and J. W. Steele,

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33. F. J. Langmyhr and S. H. Omang, Anal. Chim. Acta, 23 (1960) 565.

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37. R. S. Brown, Anal. Chim. Acta, 74 (1975) 441.

38. B. Kasterka, Chem. Anal., (Warsaw), 74 (1975) 441.

39. B. Kasterka, Chem. Anal., (Warsaw), 25 (1980) 215.

40. J. Neve, M. Hanocq and L. Molle, Mikrochim. Acta, 73 (1980) 259.

41. K. A. Idriss, M. M. Seleim and M. S. Abu-Bakr, Mikrochim. Acta, 74 (1980) 179.

42. A. D. Campbell and A. H. Yahaya, Anal. Chim. Acta, 119 (1980) 171.

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213

CHAPTER 8

SPECTROPHOTOMETRIC DETERMINATION OF CEPHALOSPORINS IN

PHARMACEUTICAL SAMPLES

8.1 INTRODUCTION


8.3 APPARATUS


8.5 PROCEDURES


8.7 APPLICATIONS

8.8 CONCLUSIONS

8.9 REFERENCES

214

8.1 INTRODUCTION

Cephalosporins are penicillinase-resistant antibiotics with significant activity

against both gram-positive and gram-negative bacteria. The key intermediate for

semisynthetic production of a large number of cephalosporins is

7-aminocephalosporanic acid (7-ACA) [1]. A few thousand semisynthetic

cephalosporins have been described in the scientific literature, but only a small

number of those have shown clinical importance.

Cephalosporin C is an antibiotic isolated in 1956 from a species of

Cephalosporium, possesses greater acid and penicillinase stability than other β-lactam

containing antibiotics but much weaker antibacterial action [2-4]. The antimicrobial

activity can be enhanced greatly by N-acylation of 7- aminocephalosporanic acid,

which has been obtained in a low yield by mild acid hydrolysis of cephaiosporin C

[5]. However, 7-ACA has not been sufficiently available to evaluate fully this

interesting class of antibiotics.

Cephalosporin C was measured by a cup agar diffusion assay of Salmonella

gallinarum grown on pH 6 nutrient agar medium containing penicillinase

(1250 µmL-1

) to destroy penicillin N [6]. The cephalosporin structure is well

established as a mono-release prodrug nucleus owing to rapid elimination of the

3′-substituent following enzyme-catalyzed scission of the β-lactam ring. Examples are

known where antimicrobial (quinolones) [7] and cytotoxic components (melphalan,

doxorubicin) [8] have been incorporated at this position.

Deacetoxycephalosporin C synthase (DAOCS) is an iron- and α-ketoglutarate-

dependent oxygenase that catalyzes the ring expansion of penicillin N to

deacetoxycephalosporin C (DAOC) in all cephalosporin producing microorganisms

[9-12]. In bacteria, the subsequent hydroxylation of DAOC to deacetylcephalosporin

C (DAC) is catalyzed by a closely related enzyme deacetylcephalosporin C synthase

(DACS), whereas in the fungus Acremonium chrysogenum (previously

Cephalosporium acremonium), the activities of expandase and hydroxylase reside in a

single functional protein [10].

215

Several research groups have reported on the narrow substrate specificity and

lack of detectable activity of expandase on inexpensive and available penicillins such

as penicillin V and G [9,13,14]. Chemical ring expansion of penicillin G plus

enzymatic removal of the phenylacetyl side chain is currently being used in industry

to obtain 7-aminodeacetoxycephalosporanic acid (7-ADCA) that is used for the

manufacture of semisynthetic cephalosporins. However, this chemical process

requires several steps and is expensive and polluting [15]. A biological route requiring

only two enzymatic steps (ring expansion and deacylation) might replace the chemical

process, thereby reducing costs and environmental problems.

Cefotaxime, ceftriaxone, cefadroxil and cephalexin are β-lactam antibiotics

possessing a broad spectrum of antibacterial properties [16,17]. These drugs are found

to be very useful in pre and post operative chemotherapy against infections in

abdominal, pelvic, orthopaedic, cardiac, pulmonary, oesophageal and vascular surgery

[18]. Cefotaxime, ceftriaxone and cefadroxil were also determined in pharmaceutical

preparations [19-23], urine [22,24-27] and human serum [28]. Recently a rapid

development in chromatographic determination methods of pharmaceuticals have

been observed too [29,30].

The hydrolysis of β-lactum ring, which is the common feature for

cephalosporins and penicillins, has been achieved by the sodium hydroxide addition.

Major difficulties in the determination of cephalosporins were encountered at the β-

lactum ring hydrolysis step [31]. A β-lactum enzyme [32] is used for the hydrolysis

of the analyte which reacted with iodate in acid medium and liberates iodine. The

liberated iodine bleaches the violet color species is the basis for the

spectrophotometric determination of the analytes. The reaction mechanism followed

the course similar to the one described for penicillins [33].


Several methods have been reported for the quantitative determination of

cephalosporins. These include fluorimetric [34], polorographic [35], chromatographic

[36-39], isotachophoretic [40] and flow injection chemiluminescence methods [41].

216

Sastry et al. reported spectrophotometric determination of penicillins and

cephalosporins in bulk and in dosage forms [42]. Chloranilic acid formed colored

complexes with penicillins and cephalosporins in dioxane and dioxane-DMF media at

maximum absorption 520 nm.

Issopoulos described cephalosporins, cefaclor, cefazolin, cefotaxime, cefoxitin

and cefamandole nafate in pharmaceuticals and bulk by a spectrophotometric method

[43,44]. The method was based on the reaction with (NH4)6Mo

7O

24 and 0.5 M sulfuric

acid and measured the absorbance of the blue colored solution at 810 nm. A linear

relation between the absorbance and anion was observed for all antibiotics studied.

The recoveries and relative standard deviations were 96.7-104.7 % and 0.60-2.08 %

respectively. In the other method molybdophosphoric acid was used as an oxidising

agent for the spectrophotometric determination of 4 cephalosporin derivatives;

cefadroxil (I), cefapirin (II), ceforanide L-lysine (III) and cefuroxime (IV) in pure

form or in pharmaceutical formulations [44]. Beer's law was obeyed up to 100 µgmL-1

for I, up to 60 µgmL-1

for II and IV and up to 80 µgmL-1

for III. The molar

absorptivities were 4.58×103

, 11.3×103

, 9.8×103

and 10.9×103

Lmol-1

cm-1

and the

Sandell sensitivities were 83.3, 39.3, 53.0 and 41.0 ngcm-2

for I, II, III and IV,

resectively.

Abdel-Razeq described two spectrophotometric procedures for the

determination of three cephalosporins; cefixime trihydrate (I), cefoperazone sodium

(II) and cefotaxime sodium (III) [45]. The first procedure was based on the reduction

of ferric ion into ferrous ion in presence of o-phenanthroline by the mentioned drugs,

which formed a highly stable orange-red ferroin chelate [Fe-(Phen)3]

2+

and was

measured at 513 nm. The second procedure was also based on the reduction of

tetrazolium blue in alkaline medium by the above cephalosporins, which formed

purple colored formazan, which was measured at 526 nm. Beer's law was obeyed in

the ranges of 0.4-2.4 and 4-20 µgmL-1

for I, 0.8 - 3.6 and 4 - 24 µgmL-1

for II or 0.4 -

2.4 and 4-16 µgmL-1

for III by ferric-phenanthroline and tetrazolium blue procedures

respectively. The optimum assay conditions and their applicability to the

217

determination of the cited drugs in pharmaceutical formulations were described. The

recoveries of the drugs were 90.7-96.0% from urine and 71.7-78.5% from serum.

Abd El-Sattar et al. reported three simple, rapid and accurate

spectrophotometric methods for the determination of cephalosporins; cefepime

dihydrochloride and cefprozil monohydrate [46]. The 1st

method was based on the

reaction of the named drugs as n-donors with three acceptors; chloranilic acid (CA),

2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and 7,7,8,8-tetracyanoquinodi-

methane (TCNQ), which yielded highly colored radical anions measured at 527 nm,

460 nm and 841 nm, respectively. The 2nd

method was based on the reaction of each

of the two drugs with ninhydrin in boiling water bath, in presence of pyridine, which

produced a bluish violet product and measured at 566 nm. The 3rd

method was based

on the reduction of Folin Ciocalteu's reagent (FCR) in alkaline medium by the

investigated drugs into blue colored products and measured at 755 nm. Beer's law was

obeyed for cefepime salt at concentration range of 50-450 µgmL-1

, 20-180 µgmL-1

,

10-60 µgmL-1

, 5-25 µgmL-1

and 10-60 µgmL-1

for CA, DDQ, TCNQ, ninhydrin and

FCR resectively. However, for the cefprozil salt, the concentration range were 50-400

µgmL-1

, 20-140 µgmL-1

, 1-7 µgmL-1

, 2-14 µgmL-1

and 2.5-25 µgmL-1

in the same

order of reagents. The methods were successfully applied to the analysis of the

studied cephalosporins, in either pure form and in pharmaceutical formulations.

Walily et al. reported a spectrophotometric and spectrofluorimetric procedures

for the determination of four penicillins [amoxycillin, bacampicillin, piperacillin and

sultamcillin] and ten cephalosporins [cefadroxil, cefamandole nafate, cefuroxime

axetil or sodium, cefaclor, ceftazidime, ceftizoxime, ceftriaxone, cefoperazone,

cefixime and cefpodoxime proxetil] [47]. Both methods were based on the oxidation

of the antibiotics with cerium(IV) at elevated temperature. The effect of acid

concentration and temperature were studied to optimize the reaction conditions. Each

antibiotic was determined at 317 nm or the cerous inherent fluorescence at 256 and

356 nm for excitation and emission wavelengths respectively. The two procedures

were successfully applied to the assay of these antibiotics in their pharmaceutical

dosage forms.

218

Nabi et al. reported a simple, rapid and sensitive method for the determination

of sodium cefazolin, cefaclor and cefadroxil in pharmaceutical preparations [48]. The

drug β-lactam ring was hydrolyzed with sodium hydroxide at 800°C for 10-15

minutes. Cooled samples were acidified with 1 M HCl and CCl4 was added. Aliquots

of each sample were titrated with a standard 0.01 M potassium iodate solution. The

colorless CCl4 layer gradually developed to a deep red colored as the titration end-

point. The absorbance of the CCl4 layer was measured at 520 nm. The cephalosporins

were determined in the concentration range of 1-5 mgmL-1

.

Agbaba et al. described cephalexin, cefixime, ceftriaxone and cefotaxime by

spectrophotometry in bulk and in pharmaceuticals by using the ferrihydroxamate

method [49]. Reaction optimization with respect to reaction time and temperature was

investigated. Using cefotaxime sodium as the model drug with an ester functional

group, it was shown that the method gave equally accurate and precise results even in

the presence of the ester functional group.

Mahrous and Abdel-Khalek described simple, accurate and selective

spectrophotometric method for determination of cephalothin sodium, cefoxitin

sodium and cephaloridine [50]. The methods was based on condensation of

acetaldehyde, vanillin or p-dimethylaminobenzaldehyde with the free thienyl moiety

of these cephalosporins in sulfuric acid medium, which formed colored chromophores

measured at the selected wavelengths. The results obtained are reasonably

reproducible with a coefficient of variation of <0.7 %.

Alwarthan et al. described spectrophotometric assay of certain cephalosporins

based on formation of ethylene blue [51]. The hydrolytic degrdation of antibiotics was

very often used as a preliminary step in the analytical procedures for their

determination. Therefore, a procedure was developed for measuring small amounts of

cefadroxil and cefotaxime in pure samples as well as in formulations. The method was

based on the formation of a vis-absorbing compound with N,N-diethyl-p-

phenylenediamine sulfate (N,N-DPPD) (ethylene blue dye), after the hydrolysis of

cefadroxil and cefotaxime in sodium hydroxide solution, which formed hydrogen

sulfide. The method was selective for cephalosporins, since other β-lactam

219

compounds such as penicillins do not give hydrogen sulfide under alkaline hydrolysis.

Variables such as pH, temp, reagent concentration and stability of the colored

products were evaluated. Beer's law was obeyed over the concentration range 0.5-10

µgmL-1

and 0.5-7 µgmL-1

for cefadroxil and cefotaxime resectively. The detection

limit was 0.1 µgmL-1

and 0.05 µgmL-1

for cefadroxil and cefotaxime respectively.

The method was successfully applied to the analysis of some pharmaceutical

formulations.

Sengun and Fedai described a determination of cephalosporins in

pharmaceutical formulations [52]. Cephalothin, cephacetrile, cefamandole,

cefamandole and cefoperazone were determined in pharmaceuticals by treatment with

a Hg(II)-imidazole reagent and measured the absorbances at 325 nm for cephacetrile

and at 345 nm for the other cephalosporins. The relative standard deviation was 0.53-

1.73%, the limit of detection was 8 µgmL-1

for cephalothin and 25.36 µgmL-1

for the

other cephalosporins.

Morelli and Peluso described a spectrometric determination for cephalosporin

[53]. The procedure applied successfully to a wide variety of cephalosporins, also in

pharmaceutical preparations: cephalothin, cefacetrile, cephapirin, cefotaxime,

ceftizoxime, cephaloridine, cefazolin, cefamandole nafate, cephalexin, cefadroxil,

cefoxitin and cefuroxime. The method employed a reaction with ammonium

molybdate in H2SO

4 medium. The antibiotic was heated at 91.5°C for 15 minutes and

the absorbance of the colored product was measured at 670 nm against a reagent

blank. Beer's law was obeyed up to 125-150 µg of cephalosporin in the 5 mL final

solution. The effects of reagent concentration and reaction conditions were discussed.

Abdalla et al. reported a selective spectrophotometric determination of

cephalosporins by alkaline degradation to hydrogen sulphide and the formation of

methylene blue [54]. The method was selective for cephalosporins in the presence of

penicillins.

Abdel Khalek and Mahrous reported a spectrophotometric method for the

determination of some cephalosporins [55]. The drug was boiled with (NH4)VO

3

220

solution in H2SO

4 for 10 minutes and the absorbance of the color developed was

measured at 750 nm. Beer's law was obeyed in the range 20-100 µgmL–1

.

Mahrous and Abdel Khalek presented ninhydrin as a reagent for the

spectrophotometric determination of certain cephalosporins in H2SO

4 medium [56].

The method was applied successfully to the analysis of injections. Absorbance was

measured at 458 nm against a reagent blank. Beer's law was valid in the concentration

range of 2.5-30 µgmL–1

. The coefficient of variation were <0.88%. Excipients in the

injections did not interfere.

Sengun and Ulas described ceftriaxone in bulk and pharmaceuticals by a

spectrophotometric method based on the reaction with imidazole-HgCl2 reagent and

measurement of the absorbance was at 370 nm [57].

Sastry et al. described haematoxylin-chloramine-T as a reagent for the

spectrophotometric determination of penicillins and cephalosporins in pure samples

and pharmaceutical preparations [58]. The method was based on acid hydrolysis of

penicillins and cephalosporins with 5M HCl and subsequent treatment with oxidized

haematoxylin. The resulting color exhibited maximum absorption at 555 nm.

Abdalla reported a spectrophotometric method for the determination of some

cephalosporins [59]. The method was based on the hydrolysis of the cephalosporins in

NaOH solution to produce H2S and the reaction of the sulfide with N,N-diethyl-p-

phenylenediamine to form ethylene blue. The method was successfully applied to the

pharmaceutical formulations and the results were statistically compared with those

obtained by the official methods and the imidazole and mercury(II) method.

Hosny described a spectrophotometric method for the determination of some

cephalosporins using 2,2'-diphenyl-1-picrylhydrazyl [60]. Beer's law was obeyed in

the range of 5-30, 5-25 and 10-30 µgmL–1

for cephalexin, cefadroxil and cephradine

respectively with a maximum absorbance at 520 nm.

221

Helaleh et al. reported a spectrophotometric determination of ceftriaxone and

cephalexin in pharmaceuticals [61]. In this method cephalosporin were treated with

potassium iodate in a moderately acidic medium after the β-lactam ring had been

hydrolyzed for 10 minutes with sodium hydroxide at 80°C. Beer's law was obeyed in

the concentration range of 20-600 and 20-800 µgmL–1

for ceftriaxone and cephalexin

respectively. The correlation coefficients were 1.0000 and 0.9999 respectively.

Al-Momani developed a spectrophotometric determination of selected

cephalosporins in drug formulations using flow injection analysis [62]. In this method

cephalosporins were hydrolyzed for 15 minutes with 0.1M NaOH at 80°C and then

oxidized with Fe3+

in H2SO

4 medium, which produced Fe

2+

. The produced Fe2+

was

then complexed by o-phenanthroline in citrate buffer at pH 4.2 and the red complex

formed exhibited an absorption maximum at 510 nm. The method was successfully

applied to the analysis of pharmaceutical preparations.

Amin and Shama reported vanadophosphoric acid in acidic medium as a

modified reagent for the spectrophotometric determination of cephalexin, cephaprine

sodium, cefazolin sodium and cefotaxime in pure samples and in pharmaceutical

preparations [63]. The method was based on acid hydrolysis of cephalosporins and

subsequent oxidation with vanadophosphoric acid. The resulted solution exhibited

maximum absorption at 516 nm. The effect of reaction conditions were investigated.

Beer’s law was obeyed over a concentration range of 0.4– . The proposed

method was applied to the determination of the drugs in pharmaceutical formulations.

Buhl and Barbara described a sensitive spectrophotometric method for the

determination of cefotaxime, ceftriaxone and cefradine with leuco crystal violet

presented [64]. The determination was based on the reduction of potassium iodate in

acidic medium, followed by hydrolysis of β-lactam ring of cephalosporins with

sodium hydroxide. The formed iodine oxidized with leuco crystal violet to crystal

violet dye of maximum absorption at 588 nm. Its absorbance was measured within pH

range of 4.0-4.2. Beer's law was obeyed in the concentration range: 0.8-4.8, 0.4-1.6

and 0.2-2.0 µgmL–1

for cefotaxime, ceftriaxone and cefradine respectively. The molar

absorptivity of the colored compound was 8.4×104

Lmol-1

cm-1

for cefotaxime,

222

2.4×105

Lmol-1

cm-1

for ceftriaxone, and 1.6×105

Lmol-1

cm-1

for cefradine. The

analytical parameters were optimized and the method was successfully applied to the

determination of cefotaxime, ceftriaxone and cefradine in pharmaceuticals.

Alaa and Ragab described a spectrophotometric determination of certain

cephalosporins in pure form and in pharmaceutical formulations with metol-

chromium(VI) reagent [65]. Beer's law was obeyed in the range 0.2-28 µgmL–1

at

maximum absorption 520 nm. The molar absorptivity and Sandell sensitivity were

calculated.

Abdel-Ghani et al. developed a spectrophotometric method for determination

of some cephalosporins in presence of each of their acid and alkaline induced

degradation products by two-different spectrophotometric methods [66]. Linear

correlations were obtained in a range 4.0-40.0 µgmL–1

. The proposed method was

successfully applied for the determination of the cephalosporins in pure form and in

laboratory prepared mixtures with their acid and alkaline induced degradation

products and in pharmaceutical preparations.

El-Ansary et al. developed a spectrophotometric determination of some

cephalosporins using palladium(II) chloride [67]. This method was based on the

reaction of cephalosporins with palladium(II) chloride in the pH range 2.5-6.0 and

yellow water-soluble complexes formed with maximum absorbance at 337-350 nm.

Beer's law was obeyed in the concentration range of 1.5-12.6, 2.0-14.4 and 3.0-19.2

µgmL–1

of cefadroxil, cephradine and cefotaxime respectively. The proposed method

was used for the determination of the above mentioned drugs in their pharmaceutical

preparations.

Vadia and Patel described a spectrophotometric determination of cefetamet

pivoxil hydrochloride in bulk and in pharmaceutical formulation [68]. Two simple

and sensitive colorimetric methods were developed for the analysis of cefetamet

pivoxil hydrochloride in bulk and in pharmaceutical formulations. The colored

complex formed was measured at 645 nm and the calibration curve was linear in the

range of 1--1

, while in the second method the measurement was at 524 nm and

223

the calibration curve was linear in the range of 2-18 mgmL-1

. The developed methods

were successfully applied to the pharmaceutical formulations.

Patett and Fischer reported a spectrophotometric assay for quantitative

determination of 7-aminocephalosporanic acids from direct hydrolysis of

cephalosporin C [69]. Nkeoma et al. reported a simple and accurate

spectrophotometric method for the analysis of ceftriaxone, cefotaxime and cefuroxime

in pharmaceutical dosage [70]. The method was based on the formation of Prussian

blue complex. The reaction between the acidic hydrolysis product of the antibiotics

with the mixture of Fe3+

and hexacyanoferrate(III) ions was evaluated. The maximum

was 3.0×104

Lmol-1

cm-1

. The linear range of the calibration graph was 2-20 µgmL-1

for ceftriaxone and cefotaxime and 2-18 µgmL-1

for cefuroxine. The method was

successfully applied to the determination of the selected antibiotics in bulk drugs and

pharmaceutical formulations.

In the present investigation, a facile and sensitive method has been reported

for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin with a

new reagents variamine blue and thionin. The developed method was successfully

applied for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin in

pharmaceuticals.

224

8.3 APPARATUS

A Systronics 2201 UV-VIS Double Beam Spectrophotometer with 1 cm

quartz cell was used for the absorbance measurements and a WTW pH 330, pH meter

was used.


All chemicals used were of analytical grade and double distilled water was

used for dilution of the reagents and samples. Cefotaxime, ceftriaxone, cefadroxil, and

cephalexin stock solutions (1000 µgmL–1

) were prepared by dissolving standard

sodium cefotaxime (Alkem Lab. Ltd. Mumbai) or sodium ceftriaxone (Aristo

Pharmaceuticals Ltd. Mumbai) or standard cefadroxil (Alkem Lab. Ltd. Mumbai) or

standard cephalexin (Ranbaxy, India) in water. These compounds were chosen to

represent cephalosporins. They were prepared freshly, as required, by dissolving an

appropriate amount of each antibiotic in water to provide a 1 µgmL–1

solution. The

standard solution must be protected from light. The structures of the cephalosporins

studied are listed in 8A1. Sodium hydroxide (0.1 M), hydrochloric acid (1 M),

potassium iodate (0.1 M) were used.

Taxim (Alkem Lab. Ltd. Mumbai), Monocef (Aristo Pharmaceuticals Ltd.

Mumbai), Cefadrox (Aristo Pharmaceuticals Ltd. Mumbai) and Sporidex (Ranbaxy,

India) were examined. A 0.05% solution of variamine blue (E-Merck Limited,

Mumbai) in (75:25) water-ethanol mixture was used and stored in an amber bottle.

Thionin (S. D. fine – Chem Limited, Mumbai) 0.1% was prepared by dissolving 0.1g

of thionin in 25 mL of methanol and made up to 100 mL with distilled water.

8.5 PROCEDURES

8.5.1 Using Variamine Blue as a Reagent

Aliquots of sample solution containing 0.5–5.8 µgmL–1

of cefotaxime, 0.2-7.0

µgmL–1

of ceftriaxone, 0.2-5.0 µgmL–1

of cefadroxil and 0.5-8.5 µgmL–1

of

cephalexin were transferred into a series of 25 mL calibrated flasks, 1 mL of 0.1 M

225

sodium hydroxide were added and the mixture was kept on a water bath (80°C) for 10

minutes after being cooled to room temperature (27±2° C), 1.5 mL of 0.1 M

potassium iodate and 2 mL of 1M hydrochloric acid were added The mixture was

gently shaken until the appearance of yellow color, indicating the liberation of iodine,

1 mL of 0.05 % of variamine blue was then added to it followed by the addition 2 mL

of 1 M of acetate buffer of pH 4 and the reaction mixture was shaken for 2 minutes.

The contents were diluted up to 25 mL with distilled water and mixed well. The

absorbance of the oxidized species variamine blue formed was then measured at 556

nm against the reagent blank prepared in the same manner without the analyte. The

amount of the cefotaxime, ceftriaxone, cefadroxil and cephalexin present in the

volume taken was computed from the calibration graph (Figure VIIIC1).

8.5.2 Using Thionin as a Reagent

Aliquots of sample solution containing 0.5–6.4 µgmL–1

of cefotaxime, 0.4–5.2

µgmL–1

of ceftriaxone, 0.8–4.2 µgmL–1

of cefadroxil and 1.0–7.5 µgmL–1

of

cephalexin were transferred into a series of 25 mL calibrated flasks, 1 mL of 0.1 M

sodium hydroxide were added and the mixture was kept in a water bath (80° C) for 10

minutes after being cooled to room temperature (27±2° C), 1.5 mL of 0.1 M

potassium iodate and 2 mL of 1 M hydrochloric acid were added The mixture was

gently shaken until the appearance of yellow color, indicating the liberation of iodine,

1 mL of 0.1 % of thionin was then added to it followed by the addition 2 mL of 1 M

of acetate buffer of pH 4 and the reaction mixture was shaken for 2 minutes. The

contents were diluted up to 25 mL with distilled water and mixed well. The

absorbance of the resulting solution was measured at 600 nm against distilled water.

A blank was prepared by replacing the analyte (cefotaxime, ceftriaxone, cefadroxil

and cephalexin) solution with distilled water. The absorbance corresponding to the

bleached color that in turn corresponds to the analyte concentration was obtained by

subtracting the absorbance of the blank solution from that of test solution. The

amount of the cefotaxime, ceftriaxone, cefadroxil and cephalexin present in the

volume taken was computed from the calibration graph (Figure VIIIC2).

226

8.5.3 Analysis of injection solution

An appropriate amount of each antibiotic was dissolved in water so as to

prepare 1 mgmL–1

solution and then the recommended procedure was followed

without modification. The presence of other substances caused no significant

interference with the determination of antibiotics.

8.5.4 Analysis of Formulations

Weighed an amount of the sample equivalent to about 250 mg (0.2503 g) of

cephalosporin and was dissolved in a sufficient amount of distilled water. The

solution was shaken and filtered through whatman No. 1 filter paper and washed with

water. The filtrate was diluted with distilled water and made upto 100 mL. The

general procedure was applied with no modification and the presence of excipients in

the sample such as glucose, fructose, lactose, sucrose and calcium caused no

interference in the determination and process of separation was not required.



8.6.1.1 Using variamine blue as a reagent

This method is based on the hydrolysis of β-lactum ring of the analytes on

heating with sodium hydroxide and the reaction of the hydrolysed product with

potassium iodate in acidic medium. The liberated iodine oxidizes variamine blue to

violet colored species of maximum absorption at 556 nm. Determination of

cefotaxime, ceftriaxone, cefadroxil, and cephalexin are represented in Scheme

VIIIA1. The absorption spectra of colored species of variamine blue are presented in

Figure VIIIA1, the absorption spectra of colored species of variamine blue with

cefotaxime, ceftriaxone, cefadroxil and cephalexin against reagent blank in the range

300–800 nm are illustrated in Figure VIIIB1. The maximum absorption is at 556 nm

and reaction systems are presented in Scheme VIIIA3.

8.6.1.2 Using thionin as a reagent

This method involves the liberation of iodine by the hydrolysis of β-lactum

ring of the analytes on heating with sodium hydroxide and the reaction of the

227

hydrolysed product with potassium iodate in acidic medium to which liberates iodine.

The liberated iodine bleaches the violet color of thionin and is measured at 600nm.

This decrease in absorbance is directly proportional to the cefotaxime, ceftriaxone,

cefadroxil and cephalexin concentration. Determination of cefotaxime, ceftriaxone,

cefadroxil and cephalexin are represented in Scheme VIIIA2. The absorption

spectrum of colored species of thionin is presented in Figure VIIIA2, the absorption

spectra of colored species of thionin with cefotaxime, ceftriaxone, cefadroxil and

cephalexin against reagent blank in the range 300–800 nm are illustrated in Figure

VIIIB2. The maximum absorption is at 600 nm and reaction systems are presented in

Scheme VIIIA4.

8.6.2 Effect of Sodium Hydroxide Concentration

The effect of sodium hydroxide concentration on the absorbance is studied

with 2 µgmL–1

of cephalosporins. Volumes from 0.5–2.0 mL of 0.1 M NaOH

solutions are examined. The investigation showed that 1.0–1.5 mL of 0.1 M NaOH

solution gave maximum absorbance and 1.0 mL of 0.1 M NaOH solution is chosen

for the procedure.

8.6.3 Effect of Temperature, Time and pH

The effect of different variables such as temperature, time and pH on the

colorization is studied with 2 µgmL–1

of cephalosporins. It is observed that the

optimum reaction temperature is 80°C–90°C, lower or higher temperature gives

inaccurate results and the reaction time for complete hydrolysis of β-lactum ring is

10–15 minutes. Constant and maximum absorbance values are obtained in the

pH=4.0–4.2 hence the pH of the reaction system is maintained at pH=4.0–4.2

throughout the study by adding 2 mL of 1 M sodium acetate solution.


8.6.4.1 Using variamine blue as a reagent


solutions varying cephalosporins(analyte) concentration. A straight line graph is

obtained by plotting absorbance against concentration of analyte. Beer’s law is

obeyed in the range of 0.5–5.8 µgmL–1

, 0.2-7.0 µgmL–1

, 0.2-5.0 µgmL–1

and 0.5-8.5

228

µgmL–1

of cefotaxime, ceftriaxone, cefadroxil and cephalexin respectively (Figure

VIIIC1). The correlation coefficients for cefotaxime, ceftriaxone, cefadroxil and

cephalexin are found to be 0.9980, 0.9992, 0.9996 and 0.9991 respectively. The

following regression coefficients a -0.014,

are obtained: 1.07×105

Lmol-1

cm–1

, 1.02×105

Lmol-1

cm–1

, 2.68×104

Lmol-1

cm–1

and

5.90×104

Lmol-1

cm–1

for cefotaxime, ceftriaxone, cefadroxil and cephalexin

respectively.

8.6.4.2 Using thionin as a reagent


solutions varying cephalosporins(analyte) concentration. A straight line graph is

obtained by plotting absorbance against concentration of analyte. Beer’s law is

obeyed in the range of 0.5–6.4 µgmL–1

, 0.4–5.2 µgmL–1

, 0.8–4.2 µgmL–1

and 1.0–7.5

µgmL–1

of cefotaxime, ceftriaxone, cefadroxil and cephalexin respectively(Figure

VIIIC2). The correlation coefficients for cefotaxime, ceftriaxone, cefadroxil and

cephalexin are found to be 0.9990, 0.9992, 0.9981, and 0.9975 respectively. The

following regression coefficients are calculated: for cefotaxime a=0.1563 b=-0.0016,

for ceftriaxone a=0.2251 b=0.0083, for cefadroxil a=0.1694 b=-0.0013 and for

cephalexin a=0.0966 b=0.0161. The following relative molar absorption coefficients

are obtained: 7.21×104

Lmol-1

cm–1

, 1.23×105

Lmol-1

cm–1

, 6.91×104

Lmol-1

cm–1

and

4.08×104

Lmol-1

cm–1

for cefotaxime, ceftriaxone, cefadroxil and cephalexin

respectively.


The effect of foreign substances is examined for the proposed method. The

maximum tolerance in the determination of 100 µgmL– 1

cephalosporins is 54.0 mg

for glucose, 35.5 mg for fructose, 56.5 mg for lactose, 32.4 mg for sucrose and 22.0

mg for calcium. In case of thionin method the maximum tolerance in the

determination of 100 µgmL–1

cephalosporins is 47.0 mg for glucose, 32.0 mg for

fructose, 54.3 mg for lactose, 34.2 mg for sucrose and 16.0 mg for calcium. The

results are summarized in Table 8A2.

229

8.7 APPLICATIONS

The proposed method is successfully applied to the determination of studied

antibiotics in pharmaceuticals. Cefotaxime was determined in 1g vials of taxim,

ceftriaxone in 250 mg vials of monocef, cefadroxil in 250 mg tablets of cefadrox and

cephalexin in 125 mg tablets of sporidex. Their contents in the investigated drug

samples are calculated from the calibration curves mentioned above are found to be in

a good agreement with the labelled amounts. The results of the analysis are presented

in Table 8A3 and 8A4, compared favorably with those from a reference method [64].

The precision of the proposed method was evaluated by replicate analysis of 3

samples containing cephalosporins at different concentrations.

8.8 CONCLUSIONS

A simple method for the determination of β-lactum antibiotics is described.

The method is based on the reaction of iodate with the hydrolysed product of β-

lactum antibiotics which liberates iodine, subsequently oxidizes variamine blue into

violet colored species and measured at 556nm and also bleaches the violet colour

species of thionin and measured at 600 nm. The developed method does not involve

any stringent reaction conditions and offers the advantages of high stability of the

reaction system (4 hours). The reagents have an advantage of high sensitivity,

selectivity, and low absorbance of the reagent blank. The proposed method was

applied to the determination of cephalosporins in pharmaceuticals. A comparison of

the method reported is made with earlier methods and is given in Table 8A5.

230

FIGURE VIIIA1

ABSORPTION SPECTRA OF COLORED SPECIES OF VARIAMINE BLUE

WITH REAGENT BLANK

Wavelength (nm)

200 300 400 500 600 700 800 900

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

FIGURE VIIIA2

ABSORPTION SPECTRUM OF COLORED SPECIES OF THIONIN

W avelength / nm

200 300 400 500 600 700 800 900

Ab

so

rb

an

ce

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

231

FIGURE VIIIB1

ABSORPTION SPECTRA OF COLORED SPECIES OF VARIAMINE BLUE

WITH CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN

AGAINST REAGENT BLANK: CEPHALOSPORINS = 2 µgmL-1

Wavelength (nm)

200 300 400 500 600 700 800 900

Absorbance

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Cefotaxime

Ceftriaxone

Cefadroxil

cephalexin

Reagent blank

232

FIGURE VIIIB2

ABSORPTION SPECTRA OF COLORED SPECIES OF THIONIN WITH

CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN AGAINST

REAGENT BLANK: CEPHALOSPORINS = 2 µgmL-1

Wavelength (nm)

200 300 400 500 600 700 800 900

Absorbance

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Cephalexin

Cetriaxone

Cefotaxime

Cefadroxil

233

FIGURE VIIIC1

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF

CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN USING

VARIAMINE BLUE AS A REAGENT

0 2 4 6 8 10

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Cefotaxime

Ceftriaxone

Cefadroxil

Cephalexin

Volume of Cefotaxime, Ceftriaxone, Cefadroxil and Cephalexin (µgmL– 1

)

234

FIGURE VIIIC2

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF

CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND CEPHALEXIN USING

THIONIN AS A REAGENT

Volume of Cefotaxime, Cefriaxone, Cefadroxil and Cephalexin (µgmL

- 1

)

0 2 4 6 8

Absorbance

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cefotaxime

Ceftriaxone

Cefadroxil

Cephalexin

235

SCHEME VIIIA1

DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL AND

CEPHALEXIN USING VARIAMINE BLUE AS A REAGENT

To the sample containing

0.5 –5.8 µgmL–1

0.2-7.0 µgmL–1

0.2-5.0 µgmL–1

0.5—8.5 µgmL–1

Cefotaxime Ceftriaxone Cefradroxil Cephalexin

+ 1 mL of 0.1 M NaOH

Kept 10 min on water bath at 80ºC

Cooled to room temperature

+ 1.5 mL of 0.1 M potassium Iodate

+ 2 mL of 1 M HCl

Analyte + IO3

-

+ 6H+

oxid.+ ½I

2 + 3H

2O

+ 1 mL of 0.05% VB

½ I2 + VB

-

(colorless) (colored)

+ 2 mL of 1 M sodium acetate

( pH is 4.0-4.2 )

Quantitatively transfered to the 25 mL volumetric flask,

and diluted to 25 mL with water

Measured the absorbance at 556 nm in the 1cm thick cell against the blank solution

236

SCHEME VIIIA2


CEPHALEXIN USING THIONIN AS A REAGENT

To the sample containing

0.5-6.4 µgmL– 1

0.4-5.2 µgmL– 1

0.8-4.2 µgmL– 1

1.0-7.5 µgmL– 1

Cefotaxime Ceftriaxone Cefadroxil Cephalexin

+ 1 mL of 0.1 M NaOH

Kept 10 min on water bath at 80ºC

Cooled to room temperature

+ 1.5 mL of 0.1 M potassium Iodate

+ 2 mL of 1M HCl

Analyte + IO3

-

+ 6H+

oxid.+ ½I

2 + 3H

2O

+ 1 mL of 0.1% thionin

½ I2 + Thionin

-

(coloured) (colourless)

+ 2 mL of 1M sodium acetate

( pH is 4.0-4.2 )

Quantitatively transfered to the 25 mL volumetric flask,

and diluted to 25 mL with water

Measured the absorbance at 600nm in the 1cm thick cell against the blank solution

237

SCHEME VIIIA3

Analyte + IO3

-

+ 6H+

oxid.+ ½I

2 + 3H

2O

½ I2 + VB -

(colourless) (coloured)

NH

NH2

O

CH3

I2

+

+ I

-

N

NH2

O

CH3

+

1

/2

VARIAMINE BLUE VARIAMINE BLUE

(LEUCOFORM) (VIOLET COLOR)

SCHEME VIIIA4

½ I2 + Thionin

-

(coloured) (colourless)

S

N

NH2

NH2

1

/2 I

2

S

N

H

NH3

+

NH2

+ I

-

I2

H

+

+

THIONIN THIONIN

(VIOLET COLOR) (LEUCOFORM)

238

TABLE 8A1

STRUCTURES OF THE CEPHALOSPORINS STUDIED

S

N

R'

O O

R''

HH

N H

R

O

O

Cephalosporin R R' R"

S

N

N

OCH3

NH2 CH

2

OCOCH3 Na

S

N

N

OCH3

NH2

N

N

N

CH3

O

H2CS OH

Na

CH3

H

1. Cefotaxime

2. Ceftriaxone

4. CephalexinCH

2

NH2

CH3

H

CH

NH2

HO3. Cefadroxil

239

TABLE 8A2

TOLERANCE OF EXCIPIENTS FOR THE DETERMINATION OF

CEPHALOSPORINS USING VARIAMINE BLUE AND THIONIN AS

REAGENTS

Common excipient Tolerance limit (mg) Tolerance limit (mg)

Variamine blue Thionin

Glucose 54.0 47.0

Fructose 35.5 32.0

Lactose 56.5 54.3

Sucrose 32.4 34.2

Calcium 22.0 16.0

240

TABLE 8A3


CEPHALEXIN IN PHARMACEUTICALS PREPARATIONS USING VARIAMINE

BLUE AS A REAGENT

Pharmaceutical Declared Quantity Found in the samplea

(µgmL–1

) (µgmL–1

) ± S.D

TAXIM 1 2.00 1.984 ± 0.03

TAXIM 2 4.00 3.975 ± 0.02

TAXIM 3 5.50 5.454 ± 0.02

MONOCEF 1 1.00 0.986 ± 0.03

MONOCEF 2 3.00 2.992 ± 0.04

MONOCEF 3

MONOCEF 4

5.00

7.00

4.954 ± 0.02

6.966 ± 0.025

CEFADROX 1 2.00 1.994 ± 0.01

CEFADROX 2 4.00 3.986 ± 0.02

SPORIDEX 1

SPORIDEX 2

SPORIDEX 3

SPORIDEX 4

2.00

4.00

6.00

8.00

1.984 ± 0.015

3.896 ± 0.01

5.982 ± 0.04

7.940 ± 0.08

a

Average of three determinations.

241

TABLE 8A4

DETERMINATION OF CEFOTAXIME, CEFTRIAXONE, CEFADROXIL, AND

CEPHALEXIN IN PHARMACEUTICALS PREPARATIONS USING THIONIN AS

A REAGENT

Pharmaceutical Declared Quantity Found in the samplea

(µgmL–1

) (µgmL–1

) ± S.D

TAXIM 1 2.00 1.896 ± 0.04

TAXIM 2 4.00 3.936 ± 0.015

TAXIM 3 6.00 5.898 ± 0.03

MONOCEF 1 1.00 0.982 ± 0.04

MONOCEF 2 3.00 3.010 ± 0.02

MONOCEF 3 5.00 4.924 ± 0.025

CEFADROX 1 2.00 1.962 ± 0.015

CEFADROX 2 4.00 3.891 ± 0.02

SPORIDEX 1

SPORIDEX 2

SPORIDEX 3

2.00

4.00

6.00

1.994 ± 0.05

3.985 ± 0.03

5.954 ± 0.02

a

Average of three determinations.

242

TABLE 8A5



Reagent Method Analyte Beer’s law

-1

)

ε (Lmol-1

cm-1

)

-2

)

λmax

(nm)

Ref.

No.

Etylene blue Spectropho-

tometry

cefotaxime

cefadroxil

0.5-7.0

0.5-10

--------

--------

----- 51

2,2′-Diphenyl-1-

picryl-hydrazyl

Spectropho-

tometry

cephalexin

cefadroxil

cefradine

5.0-30

5.0-25

10-30

--------

--------

--------

520 60

Leuco crystal

violet

Spectropho-

tometry

cefotaxime

ceftriaxone

cefradine

0.8-4.8

0.4-1.6

0.2-2.0

ε = 8.40×104

ε = 2.40×105

ε = 1.60×105

588 64

Proposed

Method

Variamine blue

Thionin

Spectropho-

tometry

Spectropho-

tometry

cefotaxime

ceftriaxone

cefadroxil

cephalexin

cefotaxime

ceftriaxone

cefadroxil

cephalexin

0.5-5.8

0.2-7.0

0.2-5.0

0.5-8.5

0.5-6.4

0.4-5.2

0.8-4.2

1.0-7.5

ε = 1.07×105

ε = 1.02×105

ε = 2.68×104

ε = 5.90×104

ε = 7.21×104

ε = 1.23×105

ε = 6.91×104

ε = 4.08×104

556

600

243

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247

CHAPTER 9

SPECTROPHOTOMETRIC DETERMINATION OF MOSAPRIDE BY

DIAZOTIZATION METHOD

9.1 INTRODUCTION


9.3 APPARATUS


9.5 PROCEDURES


9.7 APPLICATIONS

9.8 CONCLUSIONS

9.9 REFERENCES

248

9.1 INTRODUCTION

Mosapride is a new prokinetic agent widely used in Japan and is structurally

related to cisapride. Mosapride citrate, known as 4-amino-5-chloro-2-ethoxy-N-[[4-

(4-fluorobenzyl)-2-morpholinyl]methyl]benzamide citrate dihydrate is a novel

gastroprokinetic agent and plays an important role in conjunction with life-style

modifications in short and long term management of gastroesophageal reflux disease

and dyspepsia in many of the Asian countries [1]. Unlike the conventional

gastroprokinetic agents, it is free of dopamine D2 receptor antagonist and neither

stimulates colon motor activity nor causes adverse effects such as central nervous

system depression and extra pyramidal syndrome in man [2–4].

O

N

F

NH

O

NH2

O

Cl

CH3

CO2H

CO2H

CO2H. H

2OHO

Mosapride

Mosapride improves insulin sensitivity and glycaemic control in patients with

type-II diabetes mellitus. It behaves as a selective 5-HT4-receptor agonist and

enhances only upper gastroprokinetic motor activity [5,6]. However, it binds

exclusively to serotonin 5-HT4 receptors and has no dopaminergic action [7].

Mosapride accelerates gastric emptying and is used for the treatment of acid reflux [8]

and functional dyspepsia [9]. The 5-HT4 receptor is a member of the seven

transmembrane-spanning G protein-coupled family of receptors [10-12]. Dumuis

characterized a neuronal cells and shown to be linked positively to adenylyl cyclase

[13]. Clarke discovered that the neuronal receptor was similar in the gastrointestinal

system where it was shown to be responsible for stimulating motility [14].

Considerable interest has developed in this receptor because it provided a mechanism

of action for the gastric prokinetic drugs, a number of which were found to be 5-HT4

249

receptor agonists [15]. Many of these compounds are members of the generic

benzamide family, derived from metoclopramide [16]. They are amides or esters of 4-

amino-5-chloro-2-methoxybenzoic acid derivatives such as renzapride, cisapride,

prucalopride and mosapride. The pharmacological characterization of this receptor

was facilitated by the synthesis of potent and selective antagonists such as 5-HT4

receptors are expressed in a wide variety of tissues: brain, heart, bladder, intestine and

kidney and have been implicated in a number of pathological disorders: memory

deficits, irritable bowel syndrome, gastroparesis, urinary incontinence and cardiac

atrial arrhythmias [17,18]. Recently, several groups reported the cloning of the

receptors from different species [10-12]. They are characterized by several splice

variants which differ in the length and sequence of their C termini. They described the

cloning and pharmacological characterization of four splice variants of the human 5-

HT4 receptor: 5-HT4(a), 5-HT4(b), 5-HT4(c) and 5-HT4(d) [11]. More recently

another isoform (5-HT4(e)) has been characterized. Thirteen other 5-HT receptor

subtypes also have splice variants in particular the 5-HT7 receptor, which has four

isoforms [19] produced by alternative splicing in the carboxyl terminus. The

expression of 5-HT4 receptor isoforms depends on the tissue. Mosapride’s

pharmacokinetic profiles in rats [20], dogs, monkeys [21] and in healthy subjects [22]

have been well characterized.


A Few methods have been reported for the determination of mosapride such as

spectrofluorophotometry [23] and liquid chromatography. Spectrophotometric

method for the determination of mosapride by diazotization reaction was also

reported.

Krishnaiah et al. reported the determination of mosapride citrate in bulk drug

samples and pharmaceutical dosage forms using high performance liquid

chromatography [24,25]. The retention time (tR) of mosapride citrate reference

substance and the internal standard risperidone were 6.10 and 4.07 minutes

respectively. The method was validated for its intra and inter day precision. In the

range of 0.1--1

, the coefficient of variation based on the peak area ratio were

found to be between 0.06% and 1.31%. The inter day assay precision was expressed

250

as coefficient of variation and ranged between 1.25% and 0.54% respectively.

Risperidone was used as an internal standard [25]. A HPLC system consisting of

gradient pump, reverse phase C-18 analytical column, a variable UV-visible detector

set at 274 nm and an integrator was used. The mobile phase consisted of acetonitrile.

A 0.02 M potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with o-

phosphoric acid) and was pumped at 1 mL per minute at 40º C. The drug and internal

standard were eluted at 8.10 and 2.27 minutes respectively. The peak drug/ internal

standard area ratio versus drug concentration relationship was linear (r=0.9998). The

method was validated for its linearity, precision and accuracy. The calibration curve

was linear in the range of 0.5 to 30 -1

. The lower detection limit was found to

be 0.23 -1

. The intra and inter day variation was found to be less than 1%

showing high precision of the assay method. The mean recovery of the drug from the

solutions containing 2, 4 or 10 -1

was 101.55±0.97% indicating high accuracy of

the proposed HPLC method.

Rao et al. reported an isocratic reverse-phase high-performance liquid

chromatographic (RP-HPLC) method for determination and evaluation of purity of

mosapride citrate in bulk drugs and pharmaceuticals using water symmetry C18

column with acetonitrile [26]. The method was simple, rapid, selective and capable of

detecting all process related impurities at trace levels in the finished products with

detection limits ranging between 0.2×10 g and 6.4×10 g. The method was validated

with respect to accuracy, precision, linearity, ruggedness and limit of detection and

quantification. The linearity range was 125–1000-1

. The percentage recoveries

from pharmaceutical dosages were ranged from 95.53 to 100.7. The method was

found to be suitable not only for monitoring the reactions during the process

development but also quality assurance of mosapride citrate.

Kuchekar et al. reported a simple colorimetric method for the determination of

mosapride citrate in solid dosage forms [27]. This method was based on diazotization

of mosapride and coupling of the diazonium salt with N-(1-napthyl)ethylenediamine

dihydrochloride to form a stable purple colored chromogen with absorbance

maximum at 540 nm, -1

.

251

Prabhakar et al. described a spectrophotometric method for the determination

of mosapride by diazotization method [28]. The method was based on diazotization of

mosapride with nitrous acid followed by its coupling in situ with N-(1-

naphtyl)ethylene diamine dihydrochloride (Method-a) which formed purple colored

species, N-methyl aniline (Method-b) which formed reddish orange colored species

and N-phenylaniline (Method-c) which formed orange colored species. A simple and

sensitive spectrophotometric method for the determination of mosapride [29] was

described. The method was based on the diazotization of mosapride with nitrous acid

followed by its coupling in situ with phloroglucinol to yield a yellow coloured species

with absorption maximum at 434 nm and obeyed Beer's law in the concentration

range of 2--1

. The proposed method was simple, sensitive, selective and

economical for the quantitative determination of mosapride in bulk drug and

pharmaceutical formulations.

Raju and Shobha reported UV spectrophotometric method for the quantitative

estimation of mosapride citrate in bulk drug and its formulations [30]. Mosapride

citrate in alcohol exhibited absorption maximum at 272 nm and Beer's law was

obeyed in the concentration range of 2--1

. The method was extended to

pharmaceutical preparations. There was no interference from any common

pharmaceutical additives and diluents.

Yokoyama et al. reported a simultaneous enantiomeric determination of

mosapride citrate and its metabolite in plasma using alpha 1-acid glycoprotein HPLC

column [31]. In this study the enantiomeric separation and determination were

successfully achieved using an alpha 1-acid glycoprotein column and gradient elution

with a fluorimetric detection (excitation 314 nm/emission 352 nm). Both enantiomers

of mosapride and M-1 were well separated between 20 and 22 minutes at pH 4.4 and

between 4 and 7 minutes at pH 5.0 respectively. Accurate determinations were

possible in the concentration ranges of 10-5000 ngmL-1

for mosapride enantiomers

and 50-5000 ngmL-1

for M-1 enantiomers. The intra and inter day coefficients of

variation were satisfactory for the pharmacokinetic study of mosapride.

Carlsson et al. developed an electrophysiological characterization of the

prokinetic agents cisapride and mosapride in vivo and in vitro [32]. Mine et al.

252

described a comparison of effect of mosapride citrate and existing 5-HT4 receptor

agonists on gastrointestinal motility in vivo and in vitro [33]. Karlsson and Aspegren

reported the effect of mobile phase, pH and column temperature on the retention

behavior of enantiomers of mosapride citrate using chiral-AGP [34].

Itoh et al. reported the effect of mosapride citrate (mosapride) on plasma

levels of gastrointestinal peptides (motilin, gastrin, somatostatin and secretin) [35]. In

this method after a single oral administration of mosapride (15 mg), the plasma

mosapride level (85.0±13.7 ngmL-1

) was highest in 60 minutes sample after the

administration and then the plasma mosapride level fell. Peak plasma motilin levels

(18.6±1.7 pgmL-1

) were achieved 60 minutes after administration of mosapride and

returned to baseline levels within 120 minutes. Plasma gastrin levels (42.4±3.6 pgmL-

1

) increased 60 minutes after administration of mosapride. Plasma somatostatin and

secretin levels did not change significantly. These results suggest that the

pharmacological effects of mosapride on gastrointestinal functions were closely

related to changes in motilin-immunoreactive substance levels in human plasma.

Asakawa et al. described the study to investigate the effects of mosapride

citrate on feeding behavior in obese mice with decreased gastric emptying [36].

Mosapride citrate (1 mg/kg/day) was orally administered for 7 days. Food and water

intake and body weight were measured daily. Blood glucose, serum insulin and

fructosamine concentrations were measured after 7 days of treatment. Orally

administered mosapride citrate significantly increased food intake in obese mice, with

a tendency to decrease fasting blood glucose and fructosamine concentrations

compared with controls. There were no significant changes in body weight after 7

days of treatment with oral mosapride citrate. These observations suggest that

mosapride citrate may be useful in the treatment of appetite loss and improve the

quality of life in patients with diabetes mellitus.

Revanasiddappa and Veena described two simple spectrophotometric methods

(M1 and M2) for the determination of mosapride in pure and in pharmaceutical

preparations [37]. These methods were based on the interaction of diazotized

mosapride coupling with chromotropic acid [M1] in alkaline medium and

diphenylamine [M2] in acidic medium. The resulting azo dyes exhibited maximum

253

absorption at 560 nm and at 540 nm for methods M1 and M2, respectively. All

variables were studied in order to optimize the reaction conditions. No interference

was observed from excipients.

Aoki et al. described a simple method for determination of mosapride citrate

and its metabolite, des-p-fluorobenzyl mosapride (M-1) in equine muscle, liver,

kidney, adipose tissue and intestine by liquid chromatography–tandem mass

spectrometry [38]. (±)-4-Amino-5-chloro-2-ethoxy-N-[[4-(2-

chlorobenzyl)morpholinyl]methyl]benzamide was used as an internal standard. The

analytes and internal standard were spiked and extracted from tissues by acetonitrile.

The chromatographic separation was performed on a reverse phase TSK-GEL SUPER

ODS column with a mobile phase of acetonitrile–0.05% (v/v) formic acid containing

5 mM nonafluoropentanoic acid (2:3, v/v). The method exhibited a large linear range

from 0.0005 to 0.2-1

for both mosapride citrate and M-1 (r > 0.9976). In the

intra day assay (n = 5), the relative standard deviations ranged from 1.1 to 7.8% for

mosapride citrate and 1.6 to 7.2% for M-1. In the inter-day assay (n = 3), the relative

standard deviations ranged from 1.0 to 13% for mosapride citrate and 0.8 to 11% for

M-1. The extraction recovery at 1.28

ranged from 97 to 107%. The lower limit of quantification for mosapride citrate was

found to be 0.004-1

. Stability studies were carried out at different storage

conditions.

Sandhiya et al. described a role of ion channel modifiers in reversal of

morphine–induced gastrointestinal inertia by prokinetic agents in mice [39]. Patel et

al. described a simple, sensitive high performance liquid chromatographic (HPLC)

and thin-layer chromatographic (TLC) methods for the quantitative estimation of

rabeprazole and mosapride in their combined pharmaceutical dosage forms [40]. In

HPLC, rabeprazole and mosapride are chromatographed using ammonium acetate

buffer–methanol–acetonitrile (40:20:40, v/v, pH 5.70±0.02) as the mobile phase at a

flow rate of 1.0 mL per minute. In TLC, the mobile phase was ethyl acetate–

methanol–benzene (2:0.5:2.5, v/v). Both the drugs were scanned at 276 nm. The

retention times of rabeprazole and mosapride were found to be 4.93±0.01 and

9.79±0.02 respectively. The Rf values of rabeprazole and mosapride were found to be

0.42±0.02 and 0.61±0.02 respectively. The linearities of rabeprazole and mosapride

254

were in the range of 400–2000 ngmL-1

and 300–1500 ngmL-1

respectively for HPLC.

In TLC, the linearities of rabeprazole and mosapride were in the range of 400–1200

ng/spot and 300–900 ng/spot respectively. The limit of detection was found to be 97.7

ngmL-1

for rabeprazole and 97.6 ngmL-1

for mosapride in HPLC. In TLC, the limit of

detection was found to be 132.29 ng/spot for rabeprazole and 98.25 ng/spot for

mosapride. The method was applied to the determination of rabeprazole and

mosapride in combined pharmaceutical products. The reagents mentioned above,

some have been reported to be carcinogenic, while few others are not too sensitive

and are time consuming. The need for a sensitive and simple method for the

determination of mosapride is therefore clearly recognized.

In the present work a facile and selective method for the determination of

mosapride in pharmaceutical samples by spectrophotometric method is described. The

amino group in mosapride is diazotized with nitrite in acidic medium at room

temperature and the diazonium salt thus formed is coupled with acetylacetone or ethyl

acetoacetate to give water-soluble colored dye in alkaline conditions is the basis for

the determination of mosapride. The method has been successfully applied to the

determination of mosapride in pharmaceutical samples.

255

9.3 APPARATUS




All chemicals used were of analytical reagent grade or chemically pure grade

and double distilled water was used for the dilution of reagents and samples. A

standard solution of mosapride (Torrent Pharmaceuticals Ltd. India) was prepared by

dissolving 0.1004 g of mosapride in absolute alcohol and diluted to 100 mL with

distilled water. Working standard solutions were prepared by appropriate dilution.

Sodium nitrite solution (0.05 %), acetylacetone (2 %), ethyl acetoacetate (2 %),

hydrochloric acid (0.2 M), sodium hydroxide (2 M), EDTA (0.2 M) and sodium

carbonate (1 %) were used.

Somapride (Alkem Lab. Ltd. Mumbai), Mozax (Sun Pharmaceuticals

Industries Ltd. Mumbai), Mosid (Torrent Pharmaceuticals Ltd. India), Moza (Intas

Pharmaceuticals Ltd. Ahamedabad) and Mozasef (Sun Pharmaceuticals Industries

Ltd. Mumbai) were examined.

9.5 PROCEDURES

9.5.1 Using Sodium Nitrite and Acetylacetone


of mosapride were

transferred into a series of 10 mL calibrated flasks. To this 1 mL of 0.05 % solution of

sodium nitrite and 0.5 mL of 0.2 M hydrochloric acid were added and the solution

was shaken thoroughly for 2 minutes and kept aside for completion of diazotization

reaction. Then, volumes of 1 mL of 2% acetylacetone and 2 mL of 2 M sodium

hydroxide solutions were added and the contents were diluted to 10 mL using double

distilled water and mixed well. After 5 minutes, the absorbance of the colored azo dye

formed was measured at 402 nm against the reagent blank (Figure IXA2).

256

9.5.2 Using Sodium Nitrite and Ethyl acetoacetate


of mosapride were

transferred into a series of 10 mL calibrated flasks. To this 1 mL of 0.05 % solution of

sodium nitrite and 0.5 mL of 0.2 M hydrochloric acid were added and the solution

was shaken thoroughly for 2 minutes and kept aside for completion of diazotization

reaction. Then, volumes of 1 mL of 2% ethyl acetoacetate and 2 mL of 2 M sodium

hydroxide solutions were added and the contents were diluted to 10 mL using double

distilled water and mixed well. After 5 minutes, the absorbance of the colored azo dye

formed was measured at 422 nm against the reagent blank (Figure IXA3).

9.5.3 Analysis of Formulations

Weighed an amount of the sample equivalent to about 5 mg mosapride and

was dissolved in a sufficient amount of distilled water. The solution was shaken and

filtered through whatman No. 1 filter paper and washed with water. The filtrate was

diluted up to the mark with distilled water and made upto 100 mL. The general

procedure was applied with no modification and process of separation was not

required. The results are listed in Table 9A2 and 9A3.



9.6.1.1 Using sodium nitrite and acetylacetone

The method involves the diazotization of sodium nitrite in acid medium

followed by the coupling with acetylacetone in alkaline medium to give a colored dye.

The azo dye formed by sodium nitrite and acetylacetone has an absorption maximum

at 402 nm and the reagent blank has low absorbance at this wavelength. The

absorption spectrum of the azo dye is presented in Figure IXA1 and reaction system is

presented in Scheme IX.

9.6.1.2 Using sodium nitrite and ethyl acetoacetate

The method involves the diazotization of sodium nitrite in acid medium

followed by the coupling with ethyl acetoacetate in alkaline medium to give a colored

257

dye. The azo dye formed by sodium nitrite and ethyl acetoacetate has an absorption

maximum at 422 nm and reagent blank having low absorbance at this wavelength.

The absorption spectrum of the azo dye is presented in Figure. IXA1 and reaction

system is presented in Scheme IX.

9.6.2 Effect of Acid Concentration and Temperature on Diazotization

Diazotization is carried out at room temperature (25±50

C). The effect of

acidity on the diazotization reaction is studied in the range of 0.1-0.5 M hydrochloric

acid and constant absorbance is observed in this range. Above this range, a decrease

in the absorbance is observed. The optimum acidity for the diazotization is fixed to be

0.2 M hydrochloric acid and minimum time for the complete diazotization is found to

be 2 minutes.

9.6.3 Effect of Sodium Hydroxide Concentration

The effect of sodium hydroxide concentration on the absorbance is studied.

Volumes from 0.5-2.0 mL of 2 M sodium hydroxide solutions are examined. The

investigations shows that 1.0-1.2 mL of NaOH give maximum absorbance and 2.0

mL of 2 M NaOH solutions is chosen for the procedure. Other alkaline solutions are

tried, but best results are obtained by using sodium hydroxide.


9.6.4.1 Using sodium nitrite and acetylacetone


solutions varying mosapride concentration. A straight line graph is obtained by

plotting absorbance against concentration of mosapride. This method obeys Beer’s

law in the concentration range of 0.4-13.5 µgmL-1

for mosapride with sodium nitrite-

acetylacetone couple (Figure IXA2). The molar absorptivity, Sandell’s sensitivity of

the colored system is found to be 1.026×104

Lmol-1

cm-1

, 4.484×10-3

µgcm-2

respectively. The detection limit (DL = 3.3 σ/S), quantitation limit (Q

L= 10 σ/S)

[where σ is the standard deviation of the reagent blank (n=5) and S is the slope of the

calibration curve] and correlation coefficient for the mosapride determination with

nitrite-acetylacetone diazocouple is found to be 0.137 µgmL-1

, 0.417 µgmL-1

and

0.9996 respectively.

258

9.6.4.2 Using sodium nitrite and ethyl acetoacetate


solutions varying mosapride concentration. A straight line graph is obtained by

plotting absorbance against concentration of mosapride. This method obeys Beer’s

law in the concentration range of 0.5-17.0 µgmL-1

for mosapride with sodium nitrite-

ethyl acetoacetate couple (Figure IXA3). The molar absorptivity, Sandell’s sensitivity

of the colored system is found to be 1.097×104

Lmol-1

cm-1

, 4.193×10-3

µgcm-2

respectively. The detection limit (DL=3.3σ/S), quantitation limit (Q

L=10σ/S) [where

σ is the standard deviation of the reagent blank (n=5) and S is the slope of the

calibration curve] and correlation coefficient for the mosapride determination with

nitrite-ethyl acetoacetate diazocouple is found to be 0.141 µgmL-1

, 0.427 µgmL-1

and 0.9994 respectively.


The extent of interference by common ions is determined for the proposed

method in the determination of 10 µgmL–1

of mosapride and the results are given in

Table 9A1. Majority of the common ions did not interfere. An error of 2% in the

absorbance readings was considered tolerable. Some of the common excipients, which

often accompany the pharmaceutical preparations did not interfere in the present

method.

9.7 APPLICATIONS

The proposed method was applied to the determination of mosapride in

pharmaceuticals samples. The results of assay compare favorably with the reference

method. (Table 9A2 & 9A3). Statistical analysis of the results by t and F-tests showed

no significant difference in accuracy and precision of the proposed and reference

methods [24]. The other active ingradients and excipients usually present in

pharmaceutical sample forms did not interfere. The reliability of the method to

analyze pharmaceutical samples were checked by recovery experiments, which gave

quantitative results with good reproducibility.

259

9.8 CONCLUSIONS

The proposed method for the determination of mosapride is simple, sensitive

and reliable and has a wide analytical range without the need for extraction or heating.

The reagents proposed have the advantage of high sensitivity and low absorbance of

reagent blank. The developed method does not involve any stringent reaction

conditions and offers the advantages of high color stability (8 hrs). The proposed

method has been successfully applied to the determination of mosapride in various

pharmaceutical samples. A comparison of the method reported is made with earlier

methods and is given in Table 9A4.

260

FIGURE IXA1

ABSORPTION SPECTRA OF MOSAPRIDE WITH DIAZOCOUPLE OF NITRITE-

ACETYLACETONE AGAINST REAGENT BLANK (a), MOSAPRIDE WITH

DIAZOCOUPLE OF NITRITE-ETHYL ACETOACETATE AGAINST REAGENT

BLANK (b) AND REAGENT BLANK AGAINST DISTILLED WATER (c)

Wavelength (nm)

250 300 350 400 450 500 550 600 650

Ab

so

rb

an

ce

0.0

0.2

0.4

0.6

0.8

1.0

a

b

c

261

FIGURE IXA2

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF MOSAPRIDE

USING ACETYLACETONE

C oncentra tion of m osapride (µgm L

-1

)

0 2 4 6 8 10 12 14 16

Ab

so

rb

an

ce

0

1

2

3

4

FIGURE IXA3

ADHERENCE TO BEER’S LAW FOR THE DETERMINATION OF MOSAPRIDE

USING ETHYL ACETOACETATE

C oncen tra tion o f m osapride (µgm L

-1

)

0 2 4 6 8 10 12 14 16 18 20

Ab

so

rb

an

ce

0

1

2

3

4

5

262

SCHEME IX

NH

O

N

+

O

N

Cl

CH3

Cl

-

+

CH3

O

CH3

O

OH

-

O

O

C2H

5

OCH3

OH

-

O

N

F

O

N

F

NH

O

N

O

N

CH3

O

O

CH3

Cl

CH3

O

N

F

NH

O

N

O

N

C2H

5

O

O

OCH3

Cl

CH3

(Ethyl acetoacetate)(Acetylacetone)

Coloured azo dyeColoured azo dye

H

+

NO2

-

O

N

F

NH

O

NH2

O

Cl

CH3

+

Mosapride

263

TABLE 9A1

TOLERANCE OF EXCIPIENTS FOR THE DETERMINATION OF MOSAPRIDE

USING ACETYLACETONE AND ETHYL ACETOACETATE

Excipient Tolerance limit (mg) Tolerance limit (mg)

Acetylacetone Ethyl acetoacetate

Stearate 30 40

Cellulose 20 30

Lactose 20 30

Glucose 30 20

Dextrose 30 30

Sodium alginate 20 40

Talc 40 50

Starch 20 30

264

TABLE 9A2

DETERMINATION OF MOSAPRIDE IN DIFFERENT PHARMACEUTICAL

SAMPLES USING ACETYLACETONE


Samples

SMP

taken

-1

)

SMP found ±

SDa -1

)

Recovery

%

SMP found ±

SDa -1

)

Recovery

% t- testb

F-testc

Somapride

(5 mg/tab)

4.0

8.0

12.0

4.01 ± 0.04

7.98 ± 0.03

11.98 ±0.04

100.25

99.75

99.83

3.96 ± 0.05

7.95 ± 0.02

11.93 ± 0.05

99.00

99.38

99.42

1.234

0.832

1.231

1.56

2.25

1.56

Mozax

(5 mg/tab)

4.0

8.0

12.0

3.95 ± 0.08

7.99 ± 0.03

11.99 ± 0.06

98.75

99.88

99.92

3.97 ± 0.06

8.01 ± 0.05

11.97 ± 0.05

99.25

100.13

99.75

0.376

0.542

0.404

1.77

2.77

1.44

Mosid

(5 mg/tab)

4.0

8.0

12.0

3.97 ± 0.04

7.99 ± 0.06

12.00 ± 0.04

99.25

99.87

100.08

3.95 ± 0.07

7.96 ± 0.05

11.98 ± 0.02

98.75

99.50

99.83

0.392

0.607

0.707

3.06

1.44

4.00

Moza

(5 mg/tab)

4.0

8.0

12.0

4.02 ± 0.06

7.95 ± 0.04

11.94 ±0.08

100.05

99.37

99.50

4.00 ± 0.08

7.98 ± 0.05

11.92 ± 0.10

100.00

99.75

99.33

0.316

0.741

0.246

1.77

1.56

1.56

Mozasef

(5 mg/tab)

4.0

8.0

12.0

4.03 ± 0.03

7.98 ± 0.06

11.93 ± 0.05

100.75

99.75

99.42

3.99 ± 0.04

7.97 ± 0.05

11.91 ± 0.04

99.75

99.62

99.25

1.264

0.202

0.490

1.77

1.44

1.56

a. Mean (SMP-Samples) ± standard deviation( n=5)

b. Tabulated t-value for 8 degree of freedom at P(0.95) is 2.306.

c. Tabulated F-value for (4,4) degree of freedom at P(0.95) is 6.39.

265

TABLE 9A3

DETERMINATION OF MOSAPRIDE IN DIFFERENT PHARMACEUTICAL

SAMPLES USING ETHYL ACETOACETATE


Samples

SMP

added

-1

)

SMP found ±

SDa -1

)

Recovery

%

SMP found ±

SDa -1

)

Recovery

%

t- testb

F-testc

Somapride

(5 mg/tab)

5.0

10.0

15.0

4.96 ± 0.01

9.99 ± 0.02

14.95 ± 0.06

99.20

99.90

99.67

4.94 ± 0.02

9.98 ± 0.04

14.96 ± 0.05

98.80

99.80

99.73

1.414

0.353

0.202

4.00

4.00

1.44

Mozax

(5 mg/tab)

5.0

10.0

15.0

4.96 ± 0.07

9.93 ± 0.08

14.98 ± 0.05

99.20

99.30

99.86

4.94 ± 0.05

9.95 ± 0.06

14.97 ± 0.08

98.80

99.50

99.80

0.367

0.396

0.167

1.96

1.77

2.56

Mosid

(5 mg/tab)

5.0

10.0

15.0

4.99 ± 0.04

9.97 ± 0.07

14.93 ± 0.11

99.80

99.70

99.53

4.97± 0.05

9.98 ± 0.10

14.95 ± 0.13

99.40

99.80

99.66

0.493

0.129

0.185

1.56

2.04

1.39

Moza

(5 mg/tab)

5.0

10.0

15.0

5.04 ± 0.06

9.96 ± 0.10

14.94 ± 0.09

100.80

99.60

99.60

5.00 ± 0.08

9.97 ± 0.08

14.91 ± 0.06

100.00

99.70

99.53

0.632

0.123

0.438

1.77

1.56

2.25

Mozasef

(5 mg/tab)

5.0

10.0

15.0

4.97 ± 0.06

9.86 ± 0.04

14.84 ± 0.08

99.40

98.60

98.93

4.96 ± 0.05

9.88 ± 0.05

14.79 ± 0.06

99.20

98.80

98.60

0.202

0.493

0.790

1.44

1.56

1.77

a. Mean(SMP-Samples) ± standard deviation( n=5)

b. Tabulated t-value for 8 degree of freedom at P(0.95) is 2.306.

c. Tabulated F-value for (4,4) degree of freedom at P(0.95) is 6.39.

266

TABLE 9A4




-1

)

ε (Lmol-1

cm-1

)

-2

)

λmax

(nm)

Ref.

No.

N-(1-Naphthyl)

ethylenediaminedihydro

chloride

Spectrophotometry 20-160

--------

540 27

Phloroglucinol Spectrophotometry 2.0-10 -------- 434 29

Chrotropic acid(in

alkaline medium)

Diphenylamine(in acidic

medium)

Spectrophotometry

Spectrophotometry

2.0-16

0.5-8.0

ε = 1.90×104

ss = 2.21×10-3

ε = 3.92×104

ss = 1.07×10-3

560

540

37

37

Proposed Method

Acetylacetone

Ethyl acetoacetate

Spectrophotometry

Spectrophotometry

0.4-13.5

0.5-17.0

ε = 1.026×104

ss = 4.484×10-3

ε = 1.097×104

ss = 4.193×10-3

402

422

267

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270

LIST OF RESEARCH PAPERS PUBLISHED AND PRESENTED IN

CONFERENCES

1. C. Pasha and B. Narayana, A highly sensitive spectrophotometric method for the

determination of iodate using leuco xylene cyanol FF, Acta Chem Slov., 53 (2006)

77-80.

2. C. Pasha and B. Narayana, Spectrophotometric method for the determination of

iodate using methylene blue as a chromogenic reagent, Bull. Chem. Soc. Ethiopia,

20 (2006) 143-147.

3. C. Pasha and B. Narayana, A facile spectrophotometric determination of

hypochlorite using rhodamine B as a chromogenic reagent, J. Braz. Chem. Soc., 18

(2007) 167-170.

4. C. Pasha and B. Narayana, Determination of arsenic in environmental and

biological samples using toluidine blue or safranine O by simple

spectrophotometric method, Bull. Environ. Cont. Toxicol., 81 (2008) 47-51.

5. C. Pasha and B. Narayana, A simple method for the spectrophotometric

determination of cephalosporins in pharmaceuticals using variamine blue, Ecl.

Quim., 33 (2008) 41-46.

6. C. Pasha and B. Narayana, Determination of selected cephalosporins in

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determination of vanadium in steel, pharmaceutical, environmental and biological

samples using safranine O, ICFCR-2008 (Accepted).

271

OTHER PUBLICATIONS

1. C. Pasha and B. Narayana, Spectrophotometric determination of endosullfan using

thionin and methylene blue as reagents, Bull. Environ. Cont. Toxicol. 80 (2008)

85-89.

2. C. Pasha and B. Narayana, Selective complexometric determination of mercury

using sodium dithionate or methylacetoacetate as masking agent. Chem. Environ.

Res.,14 (2005) 195-205.

3. C. Pasha and B. Narayana, A new method for the spectrophotometric determination

of iodate using rhodamine B as a chromogenic reagent. 24th

ICC Conference, AO-

03 (2005).

4. C. Pasha and B. Narayana, Selective complexometric determination of gadolinium

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using 2,2’-bipyridyl acid as a masking agent, EMTIC-2005, National Seminar on

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