CHAPTER 1

INTRODUCTION TO ANALYTICAL TECHNIQUES - COMPLEXOMETRY

AND SPECTROPHOTOMETRY

PART A

INTRODUCTION TO MASKING AND DEMASKING TECHNIQUES IN

COMPLEXOMETRIC TITRATIONS

PART B

INTRODUCTION TO SPECTROPHOTOMETRY TECHNIQUE

PART A

INTRODUCTION TO MASKING AND DEMASKING TECHNIQUES IN

COMPLEXOMETRIC TITRATION

1A.1 COMPLEXOMETRY
1A.2 SCOPE OF COMPLEXOMETRIC TITRATIONS
1A.3 ORGANIC REAGENTS IN CHEMICAL ANALYSIS
1A.4 ORGANIC THIO-LIGANDS AS ANALYTICAL REAGENTS
IA.5 MASKING TECHNIQUES
1A.6 TYPES OF MASKING TECHNIQUES
1A.7 DEMASKING TECHNIQUES
1A.8 SELECTION OF MASKING REAGENTS
1A.9 INDUSTRIAL APPLICATIONS OF MASKING TECHNIQUES
1A10. REFERENCES

2
1A.1 COMPLEXOMETRY

The last four decades have witnessed a tremendous change in the field of
analytical chemistry. The analysts have developed an extensive array of instrumental
techniques. These techniques are extremely sensitive and can yield results rapidly to a
high degree of accuracy, but they are expensive. The classical methods of gravimetry
and titrimetry have therefore faced severe competitions from the instrumental
methods of analysis. Gravimetric methods are slow and involve many steps in the
determination. They are now employed occasionally to establish the reliability of new
instrumental methods but titrimetric methods represent a lively and still growing area
in the field of analytical chemistry. This is due to its versatility, coupled with
simplicity and speed. The introduction of EDTA, a new titrant and metallochromic
indicators have given a unique face to the complexometric technique.

In the past few decades, the titrimetry has undergone a rapid change to keep
pace with the modem developments in the industry and research. Among the
titrimetric methods, complexometry enjoys a great deal of interest as an important
tool of analytical chemistry.

Chelometric titration is currently the most widely applied methods based on

formation of complexes, which is generally termed complexometry. Complexation
reaction with a metal ion involves the replacement of one or more of the coordinated
solvent molecules by other nucleophilic groups or ligands. In aqueous phase such a
reaction can be represented by the general equation

[M(HzO)nT+L Jiz- -■ [ML(H2Q)n,1p + H2Q

Where L, is the monodentate ligand, which is a neutral molecule and M

represents the metal ion with charge +m. The charge on the metal in the complex is
changed if L is a negatively charged ion. Successive replacement of water molecule
by ligand molecule finally lead to the formation of the complex ML„, where vn’ is the
coordination number of the metal ion, indicating the number of monodentate ligands
that can be bound to it.

Depending upon the donor centers, i.e., the number of points of binding to the
central metal ion, the ligands may be classified into monodentate, bidentate,

3
multidentate etc. Halide ions, water, ammonia molecule etc., comes under the
monodentate ligands, while acetylacetone, ethylenediamine etc., are classical
examples of bidentate ligands. Multidentate ligands contain many donor atoms per
molecule. EDTA is a hexadentate ligand, which has two donor nitrogen atoms and
four donor oxygen atoms. The denticity of multidentate ligands is also dependent on
the steric factors. The complexation of the metal ion with bidentate or polydentate
ligands leads to the ring formation, including the metal ion. This process is called
chelation. The ring formation offers stability to the metal complex. Generally five and
six member rings are very stable. EDTA forms five member rings with the metal
atoms. Hence a stable complex of EDTA with many of the metal ions are formed.
Steric factors also affect the denticity of ligands.

Complex formation reactions can be employed in titrimetry, especially for

determining the proportions of individual cations in the mixtures. Titrations involving
complex ions or unassociated neutral molecules fall under complexometric titrations.
The basic requirement in such titrations is that the complexes involved should be
highly soluble and stable.

The greatest development in the field of complexometric titrations have been

achieved in the past thirty years owing to the chelate forming ability of
polyaminopolycarboxylic acids. The best-known reagent used in complexometric
titrations is EDTA and a vast majority of complexometric titrations are carried out by
using it. But the reagent is very unselective and reacts with a wide number of cations
forming 1:1 soluble chelates [1]. The selectivity of EDTA can be increased by the
control of pH.

For simplification purpose, EDTA is assigned the formula H4Y and its
disodium salt is therefore, Na2H2Y. It affords the complex forming ion H2Y2' in
aqueous solution, which reacts with many metal ions in 1:1 molar ratio. The reaction
with the metal cations may be represented as

Mn+ + H2Y2 -« :*» MY(n-4)+ + 2H+

It is clear from the above equation that the dissociation of the complex
depends greatly on the pH of the solution. Lowering the pH will decrease the stability

4
of the metal EDTA complex. If the metal EDTA complex is highly stable the titration
of the metal ion with EDTA may be carried out at lower pH. Hence the selectivity of
EDTA can be increased by the control of pH. For example, nickel(II) can be titrated
in the presence of alkaline earth metal ion at pH 3.5 without any interference from the
latter, whereas interference will occur at pH 10 in ammonia buffer [2]. Similarly a
mixture of Bi(III) and Pb(II) can be successfully titrated by first titrating the Bi(III) at
pH 2 with xylenol orange as indicator and then titrating Pb(II) by raising pH to 5
using hexamine [3]. The selectivity of the complexometric titrations with EDTA can
also be achieved by using suitable metallochromic indicators [4]. The use of various
metallochromic indicators has greatly enhanced its value in titrimetry. Each of these
indicator is effective over a limited pH range.

When a solution containing two cations which complex with EDTA is titrated
without the addition of a complex forming indicator, and if a titration error of 0.1% is
permissible, then the ratio of the stability constants of the EDTA complexes of the
two metals M and N must be such that Km/Kn > 106, if N is not to interfere with the
titration of M. The constants Km and Kn considered in the above expression should be
apparent stability constants of the complexes. If complex forming indicators are used
for a similar titration the ratio Km/Kn should be > 108 [3],

There are very few cases where the ratio of stability constants of the EDTA
complex of the metal ion in a particular mixture is above 106, complication often
arises when one wishes to determine a desired metal complexometrically. However
the addition of masking agents can often achieve the desired effect. Hence the
selectivity of complexometric titrations can be greatly increased by the application of
masking agents. The masking agent is added either to prevent the interference by
unwanted cations or to displace a cation from its EDTA complex, followed by the
back titration of the liberated EDTA. A wide range of complex forming reagent is
available which form, with a particular cations, complexes that are very stable
preferably soluble, colorless, and inactive towards EDTA and the indicators that are
used. The use of masking agents in the complexometric titrations have allowed an
extension of the possibilities of determination of the metal ions especially in the
routine analysis of alloys, ores, concentrates and the other systems containing the
mixture of metals. Difficulties arise in masking the cations that have small complex

5
forming tendencies or that give rise to very intensely colored complexes, and in the
determination of chemically related cations, such as Co-Ni or Zn-Cd, because of the
lack of specific masking agents.

A limiting factor in the choice of possible indicators to be used in the

complexometric titrations is that the stability constant of the indicator with any kind
of metal ion present must not be so high that the indicator becomes blocked. Thus, in
titrations of Mn and Zn using eriochrome black-T indicator, traces of Co, Ni, Cu or Fe
block the indicator. This behavior is strictly analogous to masking, and the indicator
can be demasked if suitable reagent is added which forms an even stronger complex
with the interfering cation. In this example deblocking of eriochrome black T can be
achieved in the back titration of Co, Fe, Cu, Ni and A1 by adding water miscible
organic solvents such as ethanol to diminish the stability constants of the indicator-
metal complexes [5].

1A.2 SCOPE OF COMPLEXOMETRIC TITRATIONS

There are several advantages of complexometric titrations in comparison with

other kinds of titrations. Complexometric titrations with EDTA have been applied to
the determination of virtually every metal cation with the exception of alkali metal
ions. Because of the ambiguity of EDTA chelates, the reagent might appear at first
glance to be totally lacking in selectivity. In fact the interferences can be controlled
considerably by the judicious choice of pH. For example, trivalent cations can usually
be titrated without interference from divalent species in solutions that have a pH of
about 1.0. At this pH, the less stable divalent chelates do not form to any significant
extent, but the trivalent ions are quantitatively complexed. Mg , Cr , Ca , Ba can
be titrated at pH of 11.0, while Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+, Al3+, Pb2+, Cu2+,
Ti4+ and V5+ can be titrated in the pH range 4.0 to 7.0. Since water-soluble complexes

are formed with EDTA, the equivalence point can be readily detected in such
titrations. Another merit of complexometric titrations is the determination of several
metal ions in semi-microgram levels.

The complexometric titrations can also be used for the successive

determination of various metal ions in a mixture. By changing the condition of
titrations, such as adjusting the pH or using proper masking and demasking agents or
by heating or cooling, the amount of various ions can be determined accurately. For

6
example, in a mixture of manganese, magnesium and zinc, the sum of all the ions can
be determined by direct EDTA titration first and using fluoride and cyanide ions as
demasking agents, the amount of magnesium and zinc can be determined respectively [3].
Even by changing or readjusting the pH to different values the determination of
various metal ions in a mixture can be done. With methyl thymol blue, lead may be
titrated at a pH of 6.0 without interference by calcium; subsequently calcium is
titrated at pH 12 [3]. For the successive determination of iron, aluminium and titanium,
Li et al. used a reagent 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol [6]. Dipali and Roy
reported a simple complexometric titration with EDTA as titrant, for the determination
of calcium and magnesium in the same solution and at the same pH [7], after masking
magnesium using acetylacetone at pH 10 the amount of calcium is determined,
followed by demasking of magnesium at 90 °C and titrating with EDTA at the same
pH.

Finally, interference from a particular cation can sometimes be eliminated by

adding a suitable masking agent. This is simply an auxiliary ligand that preferentially
forms highly stable complex with the potential interference and thereby prevents
involvement of that ion in equilibria associated with the EDTA, the titrant or the
indicator.

1A3 ORGANIC REAGENTS IN CHEMICAL ANALYSIS

Organic reagents have been used for a long time in analytical work involving
inorganic species. They are highly sensitive and selective in their reaction towards
metal ions. They find extensive application in titrimetry. The most important field of
application for organic reagents are found in titrations of metal ions with complexing
agents both as titrant and as indicators in chelometry. The tests for the determination
of substances with organic reagents can be often performed with higher sensitivity
and selectivity. A fair degree of sensitivity and selectivity can be achieved by the
careful control of pH, reagent concentration and by the use of suitable masking
agents.

Many organic reagents form stable chelate with metal atoms or ions. Such
chelates, particularly of ligands containing two or more fused rings are found to be
extraordinarily stable as compared to non-chelate complexes [8]. Organic compounds
possessing a dual character, viz, of having both acidic and basic functional groups are

7
capable of forming neutral chelate with the metal ion. The acidic group usually
contains a replaceable hydrogen ion and the basic group contains a lone pair of
electrons, which takes part in coordination.

Another condition for effective chelation is that the functional group must be
so positioned as to favour the formation of a strain free ring. Five or six member
chelate rings are naturally the most stable chelates.

1A.4 ORGANIC THIO-LIGANDS AS ANALYTICAL REAGENTS

Thio-ligands, which constitute an important class of reagents, are of recent

interest in analytical chemistry. This is because sulfur anion with a radius of 1.84 A is
so large that it can get readily polarized. Similarly large sized cations in lower
oxidation states are also readily polarizable. Interaction between these less readily
polarizable cations and anions leads to the formation of ionic bond while that between
more readily polarizable anions and cations results in the formation of covalent bonds.
The higher the covalent character of the bond between the metal and the ligand, the
lower is the solubility of metal complex in water.

Further, the tendency of sulfur to take part in hydrogen bonding is less than
that of oxygen. Hence the sulfur containing complex is less soluble in water than the
corresponding complex containing oxygen in the place of sulfur. The solubility of a
ligand in water decreases as the hydrophobic nature is increased.

If the conditions are favourable, the vacant d-orbitals on sulfur, being probably
of low energy are suitable for back-bonding with metals having filled or partially
filled d-orbitals. With the help of HSAB concept one can expect that sulfur forms
stronger complexes with cations of larger size and in lower oxidation states. This
remarkable ability of sulfur atom as a potential donor has contributed a great deal to
the increasing interest in the use of sulfur containing ligand as analytical reagents.

The soft sulfur present in the thio-ligands can form stable complexes with 'soft
metal ions like Pd2+, Ag+, Tl+ etc., as well as borderline metal ions like Cu2+, Pb2+,
Hg etc. These metal ions being strong 'b’ class acceptors have greater affinity for
sulfur than for oxygen or nitrogen. The formation constants of a series of 'S’ and 'N’
containing chelates with various transition metal ions have been determined [9] and
these values indicate that sulfur is a better donor than nitrogen for 'b’ class metal ions.

8
1A.5 MASKING TECHNIQUES

Very few analytical reagents are highly selective in their action. Hence many
reagents which have otherwise useful properties may have restricted analytical
applications because they give similar reaction with many ions. The attainment of
selectivity has long been one of the principal objectives of research in analytical
chemistry. Considerable success has resulted from the development of new reagents,
which are inherently more selective. Employment of masking reagents affords one of
the most promising approaches to the solution of the problem of selectivity.

Masking is a technique by which certain ions which might normally interfere

can be prevented from interference by forming stable coordination compound with a
suitable complexing agent [10]. Masking is ordinarily more rapid than a preliminary
separation and frequently permits a greater accuracy as well. This is one of the
versatile and useful adjuncts in analytical chemistry [11].

Masking was introduced in analytical chemistry by Fritz Feigl [12] to describe

a process by which an ion or a molecule could be so transformed, usually by complex
formation, that it no longer shows its characteristic chemical reactions. The wide
range of applications of the technique of masking is because it provides a convenient
means to remove or temporarily suppress the effects of unwanted constituents in a
system without recourse to physical separation. Considerable interest has developed
in this field during recent years because of the availability of a large number of
substances largely organic, which are capable of forming complexes with metal ions.

In analytical chemistry, the technique of masking is used for improving

selectivity in a wide variety of methods such as titrimetry, spectrophotometry,
precipitation reactions, ion-exchange chromatography, polarography, solvent
extraction etc. The industrial applications of this technique are numerous and cover a
broad spectrum of fields like manufacture of soaps and detergents, food stuffs and
beverages, drugs and pharmaceuticals, dyes and paints, rubber and polymers etc.
Reagents capable of masking metal ions in vivo are used as antidotes against metal
poisoning and for the removal of excess iron and copper. However, the main
application of the masking procedure is to keep within acceptable limits the level of
free metal ions which can otherwise form turbidities or precipitates or undergo
decomposition, oxidation, color development or other undesirable changes.

9
 Recent developments in analytical chemistry such as activation and atomic
absorption spectrophotometry are diminishing the scope of conventional chemical
analysis. But the extent of this erosion will depend largely on how much can be
achieved in simplifying and speeding up the existing titrimetric, spectrophotometric
and gravimetric procedures without sacrificing accuracy and reliability.

It is in this field that much can be hoped for, from the simple and elegant
masking technique designed to enhance the selectivity of chemical reactions and in
many cases to achieve rapid analysis of mixtures that would otherwise take quite long
time by the conventional techniques of analysis. Employment of masking techniques
afford one of the most promising approaches to the solution of the problem of
selectivity, because of simplicity with which it may be carried out in practice.

Masking by complexation has long been employed in inorganic analysis using

reagents such as cyanide, tartrate, oxalate, citrate and fluoride ions. Cyanide ion is
often employed as a masking agent to permit the titration of magnesium and calcium
ions in the presence of ions like cadmium, cobalt, copper, nickel, zinc and palladium.
Fluoride ion can be used as masking reagent to arrest aluminium from reacting with
EDTA [13]. Similarly it masks cations such as Fe3+, Sb3+, Sn4+, Zr4+ and rare earths.

Thiols are often proposed in place of cyanide for masking because they are less
hazardous. Reagents such as 1,2-dimercaptopropanol (BAL) [14], thioglycollic acid [15] and
unithiol (2,3-dimercapto propane sulfonic acid) [16] are being used successfully in
practical applications. The determination of Ca2+ and Mg2+ in presence of Hg2+, Pb2+
'jt

and Zn was done by masking the metal ions using unithiol [17]. Using 0-
aminoethylmercaptan [18] the titrations of alkaline earth metals are possible in the
presence of bivalent heavy metals except Pb2+ and Mn2+. a-Hydroxy acids [19]
are used in the direct titration of Bi3+ with EDTA. Thiosulfate is used as a selective

masking reagent for the determination of copper [20] and palladium [21].

1A.6 TYPES OF MASKING TECHNIQUES

Masking reactions that received special importance include precipitation,

oxidation and reduction, soluble complex formation, kinetic masking, mixed masking
and indirect masking.

10
1A.6.1 Masking by Precipitation

When the species to be masked reacts with the masking agent to yield a
precipitate, it is known as masking by precipitation. Sulfate was used to precipitate
lead in the determination of Bi3+ [22]. Using xylenol orange and cetyl pyridinium
bromide as mixed indicator, lead was determined at pH 5-6 from lead-EDTA complex
by selectively masking lead with sulfate ions [23, 24]. The precipitation is often
accompanied by coprecipitation of the species to be determined resulting in obscure
end points with low results or less accuracy.

1A.6.2 Masking by Soluble Complex Formation

Masking by soluble complex formation has been employed in inorganic

analysis by using reagents such as cyanide, oxalate, citrate and tartrate. The selective
precipitation of yellow cadmium sulfide can be achieved by masking Cd2+ by the
addition of cyanide ion. Tartrate has been used as an auxiliary complexing agent to
mask magnesium during the titration of calcium at pH 12 [25]. Fritz and co-workers
achieved selectivity in EDTA titrations by using citrate [26].

Masking agents with sulfur as donor atom are selective towards many metals.
Copper can be masked using thiourea [27], while cadmium and zinc can be masked
with cysteine [28]. 3-Mercapto propionic acid [29] is used as a masking agent for
lead.

In recent years, EDTA and related compounds, polyamines and certain sulfur
containing reagents [30] have greatly increased masking probabilities for cations.
Majority of the masking agents for inorganic ions involve soluble complex formation.
Almost every ion can be converted into a water-soluble complex.

1A.6.3 Masking by Oxidation and Reduction

Different oxidation states of a metal ion show entirely different reaction

tendencies. Consequently, oxidation or reduction of a species may be suitable for
achieving effective masking. However, care should be taken to see that excess of the
oxidant or reductant should be removed to avoid interferences with the principal
reaction. For example, oxidation of Cr3+ to Cr6+ permits its clean separation from iron
and aluminium as used in classical procedure. Reduction of Fe3+ to Fe2+ by the action
of stannous chloride, sulfite, hydroxylamine or ascorbic acid is often an effective

11
means of eliminating the interference of Fe3+ [31]. However, this type of masking by

oxidation and reduction has limited application since very few of the common
elements can exist in different oxidation states in aqueous solution. In addition to that
some of the possible oxidation states are so unstable that metals cannot be preserved
in those oxidation states. For example, Co3+ is highly unstable in aqueous phase

unless it is suitably complexed.

1A.6.4 Mixed Masking

At times, if need arises, more than one of the above said types of masking
processes may be applied simultaneously or concurrently to advantage. For example,
Hg2+ can be masked towards a number of reagents by boiling its solution with ascorbic

acid thereby reducing it to the metal which precipitates. Thiosulfate ion [20] and thiourea
[32] in a weakly acidic medium reduces Cu2+ to Cu+ and forms a stable complex with

the latter. Iron(III) is often masked by the addition of cyanide ion followed by
reduction with ascorbic acid to form hexacyanoferrate(II) ion. Both these examples
involve simultaneous reduction and complexation.

1A.6.5 Kinetic Masking

If the principal reagent reacts at significantly different rates with the various
species of interest, selectivity can sometimes be achieved by taking advantage of the
differences in the kinetics of the reaction. This approach is termed as kinetic masking [31].

The complexation of Cr3+ ion with many complexing agents proceeds slowly

at room temperature and boiling of the solution is often required to ensure completion
of the reaction. Hence a number of metal ions can be titrated with EDTA without
interference from Cr3+ in the cold condition. Fe3+ and Cr3+ in combination can be

determined [3] by the addition of an excess of standard EDTA to the cold mixture at
pH 5-6 followed by back titration of the solution with lead nitrate solution using
xylenol orange as indicator. If the solution is kept cold, chromium does not interfere
at all. The same solution is then acidified to a pH of 1-2 and treated with excess of
EDTA and boiled for 15 minutes when the red violet Cr3+-EDTA complex is

produced. After cooling the solution to room temperature and readjusting the pH to 6,
the excess of EDTA is titrated with lead nitrate and the used up EDTA corresponds to
Cr3+.

12
 By cooling the sample solution, the effective kinetic masking can be achieved
sometimes. For example, the displacement of Ni2+ from its EDTA complex by Bi3+ in

ice cold solution at pH 2.0 is very slow, while cations like Cd , Co , Pb , Cu and
Zn2+ are displaced rapidly. Hence the amount of co-ions can be determined at ice cold
solution while the amount of Ni2+ can be determined at elevated temperature by
adding excess of EDTA to the mixture and titrating the solution with Bi3+.

1A.6.6 Indirect Masking

Masking agents especially those acting via soluble complex formation may be
added after the species has reacted with the principal reagent. This approach, which
may be called indirect masking, sometimes allows the determination of several
species with single aliquot of the sample solution.

For example, Al3+ can be determined in the presence of other cations which

interact with EDTA by complexing all the cations including aluminium with known
excess of EDTA under optimum conditions and back titrating the excess EDTA
followed by the addition of fluoride ion to mask Al3+ and release the EDTA from Al-
EDTA complex and determining the EDTA so released by using standard Zn2+ or
Pb2+ solution.

Thallium(III) can be determined in the presence of other cations which interact

with EDTA by complexing all the cations including thallium with known excess of
EDTA under optimum conditions and back titrating the excess of EDTA followed by
the addition of semicarbazide hydrochloride [33] or ascorbic acid [34] or
hydroxylamine hydrochloride [35] or sodium sulfite [36] or thiosulfate [37] to mask
Tl3+ and release the EDTA from Tl-EDTA complex and determining EDTA so
released by using standard Zn2+ or Pb2+ solution. Similarly Cu2+ can be determined in

the presence of other cations which interact with EDTA by complexing all the cations
including copper with known excess of EDTA under optimum conditions and back
titrating the excess EDTA followed by the addition of 2-mercaptoethanol [38] or DL-
cysteine [39] to mask Cu2+ and release the EDTA from Cu-EDTA complex and
determining the EDTA so released by using standard Zn2+ or Pb2+ solution.

13
1A.7 DEMASKING TECHNIQUES

Demasking is the term used to describe the process by which a masked species
regains its ability to take part in its normal reactions, in systems in which, the cations
have been masked by complex formation with a suitable ligand. This can be often
achieved by adding a sufficient amount of another reagent, which has a greater
affinity for the ligand concerned, so that initially masked cation is once again
liberated.

The common demasking methods can be classified as

1. Displacement reaction
2. Conversion of masking agent into non-reacting species
3. Adjustment of pH
4. Destruction of the ligand
5. Physical removal of the ligand
6. Change in oxidation state of the metal ion

1A.7.1 Displacement Reaction

The metal ion forming stronger, more stable complex with the masking agent
is added to the system. The masking agent sets free the metal ion under consideration
and preferentially bonds to the metal ion added. For example in ammoniacal
[Ni(CN)4]2' solution, Ni2+ is demasked by Ag+ ions, since the [Ag(CN)2]' complex
formed is more stable than [Ni(CN>4]2' complex [41,42]

[Ni(CN)4f + 2Ag+----------------- -- 2[Ag(CN)2]' + Ni2+

The displaced nickel ions can be determined by the titration with EDTA using
murexide indicator.

Bismuth can be titrated with EDTA in the presence of Hg2+, if Hg2+ is masked
by adding slight excess of potassium thiocyanate to form soluble [Hg(SCN)4]2' [40].

After the titration Hg is demasked by adding slight excess of silver nitrate and the
Hg2+ is titrated with EDTA.

14
1 A.7.2 Conversion of Masking Agent into Non-reacting Species

The reagent, which preferentially reacts with the masking agent, is added to
the system. The reagent sets free the masking agent and combines with it to form non­
reacting species. For example in a mixture containing Zn2+ and Cd2+ selectivity can be
achieved by masking Zn2+ in an alkaline medium with potassium cyanide and then

demasking the metal ions with formaldehyde. [42,43]

[Zn(CN)6f + 4HCHO + 4H20 --------------- ► Zn2+ + 4 HOCH2CN + 4 OH

Chloral hydrate [44] or acetone can also be used in the place of formaldehyde.

1A.7.3 Adjustment of pH

Because of the marked effect of pH on the apparent stability constant of most

metal complexes demasking can often be achieved by simple adjustment of pH. For
example at pH 7 barium is masked by EDTA against precipitation as its sulfate, but at
pH 5 it is no longer masked. The blue color of the cuprammonium ion is discharged if
the solution is made acidic.

The nature of the reagent used to lower the pH of the solution may also be
important. Dilute acetic acid is unsuitable for demasking the complexes of W, Mo, Ce
and the rare earths with EDTA because colloidal precipitates are obtained and
appreciable reduction occurs.

Conditional stability constant for EDTA complex formation decreases

markedly as the pH is lowered so that EDTA is much weaker ligand at pH 4 than at
pH 10. Thus in a pyridine buffer ammonium borofluoride masks calcium and
magnesium, where as manganese can be directly titrated with EDTA against methyl
thymol blue.

1 A.7.4 Destruction of the Ligand

In some cases heat, chemical reaction or both can destroy the masking agents.
Masking by thiocyanate ion can be demasked by the oxidation of the ligands with the
alkaline hydrogen peroxide and heating. The excess of hydrogen peroxide can be

15
catalytically decomposed by adding a small amount of Fe3+. EDTA can be destroyed
in acid solutions by permanganate or other strong oxidizing agents

1A.7.5 Physical Removal of the Ligand

Ligands, which form volatile compounds by heating with the mineral acids or
form precipitates with some reagents can be removed from the reaction mixture.
Prolonged boiling with strong mineral acids can be used to remove stable but volatile
masking agents such as hydrogen fluoride, hydrogen chloride, hydrogen bromide,
hydrogen iodide. On the other hand silver iodide, bromide, and chloride are
selectively and sequentially precipitated, in that order from a concentrated ammonia
solution. When the ammonia is allowed to escape progressively the three halides can
be separate by filtration over suitable pH range.

1A.7.6 Change in the Oxidation State of the Metal Ion

When a metal ion can exist in more than one oxidation state there will be
usually differences in stability constants of corresponding complexes with a given
ligand. A change in the oxidation state of a metal ion may so decrease the stability of
a complex that the metal ion may be no longer masked, e.g. Thiosulfate ion forms
very stable complex with copper(I) in slightly acidic solution. If the solution is made
alkaline copper(I)-thiosulfate is readily oxidized to copper(II)-thiosulfate where the
metal is only weakly complexed.

1A.8 SELECTION OF MASKING REAGENTS

Masking agents by suppressing the effects of interfering species will make the
reaction much more selective or even in some cases specific under chosen conditions.
Masking technique depends largely on complex formation, and the study of factors
governing the stability of metal complexes plays a major role in the selection of
masking agents for new application.

In any particular system many different factors are involved in determining the
effectiveness with which an ion or neutral molecule needs to be masked if it is not to
cause interference, e.g. Relatively weak complex formation may be sufficient to
prevent precipitation or to reduce an already low concentration of a foreign ion below
the level at which it causes detectable interference, but if the same ion is present at

16
very high concentrations much more effective masking may be necessary.
Considerations such as these make it difficult to lay down firm rules for masking
agents. A reagent that is suitable under one set of conditions may be quite inadequate
at different pH and at different metal ion concentration. In general however, stability
constants of the complexes must be sufficiently high, the reaction must be rapid and
the precipitation must be avoided wherever possible.

The best ligands to use as masking agents would be those which form strong
colorless complexes with the ions to be masked, but relatively weak complexes with
other cations that are present. Thus copper(II) is reduced to copper(I) by thiourea with
the formation of complexes that are more stable than those of calcium, where as the
calcium complexes with EDTA are stronger than those of copper(I). Hence calcium is
titrated preferentially with EDTA in the presence of copper.

The selectivity of masking agents can often be improved by careful pH

control. It is also necessary to consider the particular situation in which a masking
agent is also to be used. Highly colored complexes are unsuitable if end point in the
titrimetric determination is to be determined by the color change of the indicator.
Thioglycollic acid forms an intensely colored complex with Fe3+, hence is an
unsatisfactory masking agent for use when much iron is present, unless
triethanolamine or some other alternative complexing agent is added to mask iron
against this reaction.

Considerations other than purely of chemical ones have also sometimes to be

taken into account. For example use of cyanide ions as masking agent should be
restricted. Cyanide ion is highly poisonous and acidification of it leads to the
evolution of hydrocyanic acid. Although the common masking agent mercaptoethanol
is nontoxic, it has a most objectionable smell.

Only a small number of groups commonly take part in metal complex

formation in aqueous system, and these subdivided into those where the coordination
of the metal is through the lone pair of electrons on nitrogen, oxygen or sulfur. The
groups that involve oxygen are the carboxylate ion, -COO" and the enolate ions, -O',
with weaker bindings to ether oxygens and carbonyl oxygens. For nitrogen amino
groups are of most important, followed by diazo and ring nitrogens. Sulfur ligands

17
bind most strongly when thiolate ions are involved but there is much weaker bonding
through thioether and -S-S- groups.

There is however, three factors that are directly responsible for the great range
that can be achieved in the stabilities of the metal complexes formed by ligands
containing these groups

1. The preferred stereochemical arrangement of the bonds about metal ions.

2. The ability of many ligands to coordinate to metal ion through more than
one center, to give five or six member chelate rings.
3. The compatibility of metal and ligand donor atoms as expressed in the
concept of hard and soft acids and bases.

One of the most useful concepts for a qualitative discussion of the intrinsic
factors that govern the strengths of the bonds formed between metal ions and
eomplexing agent is that of “hard” and “soft” acids and bases developed by
Pearson[45]. “Hardness” is a characteristic of bonds that have a high ionic character
where as “softness” is related more to covalent bond formation.

Lewis [46] defined a ‘base’ as a species which can donate a pair of electrons
to form a coordinate bond and an ‘acid’ as a species which can accept a pair of
electrons. In terms of these definitions all ligands are Lewis bases and all metal ions
are Lewis acids.

A ligand is described as a ‘soft base’ if its donor atom is of high polarizability,

with empty, low lying molecular orbitals. Such a donor atom is usually of low
electronegativity and easily oxidized. That is, the orbitals of valence electrons are
easily distorted; often to such an extent the electrons themselves can also be removed.
Conversely, in a ‘hard base’ the donor atom is of low polarizability, hard to oxidize
has a high electronegativity, and its empty molecular orbitals are of high energy,
hence are much less rapidly accessible. Similarly, a metal ion is classified as a ‘soft
acid’ if it is of low charge, large in size, and has several easily excited outer electrons.
Conversely, a metal ion is a ‘hard acid’ if it is of high positive charge, small size and
lacks easily excited outer electrons.

This approach has led to the generalization that hard acids form stronger bonds
with hard bases, where as soft acids prefer to coordinate to soft bases. Such

18
complexes are usually much more stable than related ones formed by a hard acid with
a soft base or soft acid with a hard base.

In general the stability of complexes of a hard metal ion with a hard base
increases with the charge on the metal ion, so that Al3+>Mg2+>Na+. With the soft
metal ions and soft bases the opposite is usually true i.e. Ag+> Cd2+>Au3+>Sn4+

Hard metal ions coordinate best to the lightest atom in a family of elements in
the periodic table. Soft acids coordinate best to one of the heavier atoms of the same
family, possibly because these atoms have vacant d-orbitals which may be used for
the ^-bonding of some of the d electrons of the metal ions that are soft acids.

Many of the masking agents mask all related cations rather than individual
species in isolation. By considering all the above points, the important requirements
that determine the suitability of a reagent as a masking agents can be summarized as:-

a. The kinds of atoms through which masking agent bond the metal ions.
b. The stability constant of the complex formed by the masking agent with
metal ion should have a high value.
c. The complex formed after masking should not have a polar group or a net
electronic charge and color.
d. The masking agent should be much more soluble in water than the metal
complex so that the former is not co-precipitated and excess of masking
agent should not affect on the method of analysis.

1A.9 INDUSTRIAL APPLICATIONS OF MASKING TECHNIQUES

The most effective cations, which catalyze chemical reactions, are usually
those of transition metals such as iron, copper and manganese. These may be present
in raw materials used in industry and also be introduced by corrosion, physical or
mechanical wear out and breaking down of non-ferrous alloys and surface coatings.

Traces of heavy metal ions catalyze aerial oxidation of fats, oils, proteins and
many other organic compounds, probably by a free radical peroxide chain reaction
mechanism. Such auto-oxidation reactions led to undesirable effects like rancidity,
turbidity, disagreeable taste and odor, in natural products or manufactured items like
food stuffs and beverages. The auto-oxidation of sunflower or other unsaturated oils is

19
explained by a two-step mechanism involving the formation of R-O-O-H followed by
decomposition. The second step is metal catalyzed, so that complex forming agents
such as EDTA or tartrate ion can inhibit the reaction. The oxidative destruction
includes the loss of important constituent like ascorbic acid.

EDTA has been tried extensively in the preservation of fresh fish. Iron,
vanadium and copper are the most active among the cations which enhance rancidity
in blended fish muscles. By using EDTA, dephosforylation of inosine-5-phosphate
present in the fish muscle is inhibited and although there is little effect on bacterial
growth and hence the storage life of the haddock is increased.

Many medicinal and organic chemicals undergo oxidative attack in aqueous

solutions in the presence of heavy metal ions unless masking agents are used as
preservatives. EDTA protects aqueous solutions of adenosine triphosfate and other
polyphosfate derivatives of adenosine by binding traces of heavy metal ions that
catalyse their dephosforylation. It also reduces the deterioration of the weakly basic
salt of carboxy methyl cellulose formed by the action of Cu2+ by masking the latter.

Because of the chelating tendency of most of the dyes, they will form
complexes, if metal ion impurities are present. This will result in some undesirable
properties like change in color, variation in shade, tint, hue etc., between successive
batches of dyeing. So masking of various trace metal ion impurities become necessary
to ensure consistency and uniformity in the shades, tints etc.

The interference of ferric ion and other metal ions during the manufacture of
paper causes discoloration of unbleached pulp and paper. It also reduces the strength
of the paper because of localized oxidation during bleaching. These unwanted effects
can be prevented by the application of EDTA as masking agent. A detergent with
metal-sequestering properties that has been suggested for removing bloodstains
comprises a mixture of an organic acid such as dodecylbenezene- sulfonic acid with
mono-, bis-, tris- and tetra (hydroxypropyl) ethylenediamines [46].

20
1A.10. REFERENCES

1. G. Schwarzenbach, “Complexometric Titrations”, London, Methuen and Co. Ltd.,

(1957).
2. R. A. Day and A. L. Underwood, “Quantitative Analysis”, 5th Edn., Prentice-Hall,
Inc., USA
3. A. I. Vogel, “A Text Book of Quantitative Inorganic Analysis”, 4th Edn.,
Longmans, London (1978)
4. A. Ringbom, “Complexometric Titrations”, Inter Science New York (1963)
5. T.A. Kiss, Z. Anal. Chem.,2m (1965) 334.
6. Z. Y. Li, F. L. Zhou and L. Zhou, Huaxue Fence, 31 (1995) 95.
7. D. Kundu and S. K. Roy, Analyst, 118 (1993) 905.
8. J. C. Bailar, ‘Chemistry of Coordination Compounds', Reinhold
Publishing Corporation, New York (1956).
9. Gonic Femelins and Doughlas, Technical Report to O. N. R. October 15 (1953).
10. V. Raj and Brumblay, “A First Cause in Quantitative Analysis”, Addenson
Wesley Publishing Co., London (1970) 66.
11. D. D. Perrin, “Masking and Demasking of Chemical Reactions”, Wiely, New
York (1970).
12. Fritz. Feigl, Anal, Chem., 21 (1949) 1298.
13. R. Pribil and V. Vesely, Talanta, 9 (1962) 23.
14. R. Pribil and Z. Roubal, Coll. Czech. Chem. Comm., 19 (1954) 1162.
15. R. Pribil and V. Vesely, Talanta, 8 (1961) 880.
16. Y. V. Morachevskii and L. A. Volf, Zhur. Analit. Khim., 15 (1960) 656.
17. L. A. Volf, Zavod. Lab., 25 (1959) 1438.
18. K. Yamaguchi and K. Uena, Talanta, 10 (1963) 1041.
19. K. M. Parida, R. S. Thakur, J. R. Rao and S. B. Rao, Indian J. Chem., 26A (1987)
87.
20. K. L. Cheng, Anal. Chem., 30 (1958) 243.
21. S. Raoot and K. M. Raoot, Talanta, 33 (1986) 544.
22. J. I. Vsatenko and M. A. Vitkina, Zavod. Lab., 26 (1960) 542.
23. X. Wang, Analyst, 115 (1990) 1611.
24. X. Wang, Yankuang Ceshi, 9 (1990) 238.
25. A. Bieber and Z. Veera, Anal. Chem., 6 (1961) 17.

21
26. J. S. Fritz, M. J. Richard and S. K. Karaker, Anal. Chem., 30 (1958) 1347.
27. J. Kinnumen and B. Wennwestrand, Chem. Anal., 46 (1957) 34.
28. W. Bemdt and J. Sara, Talanta, 8 (1961) 653.
29. S. Hara, Busekikagaku, 10 (1961) 633.
30. H. R. A. Gadiyar, R. V. Gadag and M. R. Gajendragad, Talanta, 29 (1982) 941.
31. A.N. Reily and A. J. Bam ad, “Handbook ofAnalytical Chemistry”, L. Meites
(Edn.), Me Graw Hill, New York (1963).
32. O. B. Budesky and L. Sinova, Talanta, 9 (1962) 769.
33. B. Mathew, B. Narayana, B. M. Rao, C. H. R. Nambiar and B. Ramachandra,
Mikrochim. Acta, 122 (1996) 295.
34. B. M. Rao and B. Narayana, Tr. J. Chem., 17 (1993) 138.
35. B. M. Rao and B. Narayana, Ann. Chim (Rome), 85 (1995) 105.
36. C.H. R. Nambiar, B. Narayana, B. M. Rao and B. Mathew, Analyst, 120 (1995)
1843.
37. A. Joseph, B. Mathew and B. Narayana, J. Indian Chem. Soc., 73 (1996) 633.
38. B. M. Rao, B. Narayana and K. Subramanya Bhat, Mikrochim. Acta, 112 (1994)
109.
39. B. Narayana and M. R. Gajendragad, Analyst, 117 (1992) 203.
40. L. Barcza and E. Koros, Chem. Anal., 48 (1959) 94
41. S.J. Gedansky and L Gordon, Anal. Chem., 29 (1957) 566.
42. H. Flaschka and F.Huditz, Z. AnaLChem, 137 (1952) 104,172.
43. H. Flaschka, Z. AnaLChem, 138 (1952) 332.
44. R. Pribil, Chem. Listy., 47 (1953) 1173.
45. R.G. Pearson, J. Am. Chem. Soc., 85 (1963) 3533.
46. G.N. Lewis, “Valency and the Structure ofAtoms and Molecules”, Chemical
Catalog Co., New York, (1923)141.

22
PART B

INTRODUCTION TO SPECTROPHOTOMETRY TECHNIQUE

1B.1 SPECTROPHOTOMETRY
1B.2 CALIBRATION CURVE
1B.3 RINGBOM’S PLOT
1B.4 CHOICE OF WAVELENGTH
1B.5 SENSITIVITY OF SPECTROPHOTOMETRIC METHOD
1B.6 PRECISION AND ACCURACY
IB.7 COLOR DEVELOPEMENT
IB.8 CHOICE OF SOLVENT
1B.9 LIMITATIONS
IB. 10 APPLICATIONS
1B.11 PRESENT INVESTIGATION
IB. 12 REFERENCES

23
1B.1 SPECTROPHOTOMETRY

Newly emerging fields like pollution control, toxicology, food adulteration

and forensic sciences, etc., demand simple, rapid and accurate methods for routine
analysis. In the past few years, analysts have developed an extensive array of
instrumental techniques. These techniques are extremely sensitive and can yield
results rapidly to a high degree of accuracy. Among the 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 modem analytical chemists [1]. It is the most
important method 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.

The act of identifying materials based on their 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. The first measurements were made by using the human eye as
the detector and undispersed sunlight or artificial light as the light source. 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 readout electronic systems.

Absorption spectrophotometry 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

24
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 and / or electron acceptor groups. 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 [2].

1B.2 CALIBRATION CURVE

The common method of using the spectrophotometer requires the construction

of a calibration curve for the constituents being determined. For this 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.

IB .3 RINGBOM’S PLOT

Ayres [3] pointed out that a straight line obtained in Beer’s law curve does not
show directly the concentration range within which accurate determination of the
colored species is possible. The optimum range for highest precision is determined by
plotting percentage transmittance against log of the metal ion concentration as was
suggested by Ringbom [4]. The optimum concentration range corresponding to a
nearly constant and high rate of change in transmittance with concentration is
indicated by a virtually linear portion of the Ringbom’s plot.

1B.4 CHOICE OF THE WAVELENGTH

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

absorptivity (£) 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 [5]. Therefore, it is necessary to select the wavelength corresponding to

25
the maximum of e. When the transmittance of the solution increases continuously
over the wavelength range covered by the light filter, Beer’s law will not be obeyed.

1B.5 SENSITIVITY OF SPECTROPHOTOMETRIC METHODS

Sensitivity is often described in terms of the molar absorptivity (e, lmor’cm'1) of

the metal ligand complex. The awareness of the sensitivity is very important in
spectrophotometric determination of trace metals. The numerical expression [6-8] is the
molar absorptivity (e) at the wavelength of maximum absorbance of the colored
species.

A
Molar absorptivity (e) = — -------- (1)
c1

Sensitivity depends on the monochromaticity of the radiation. With

monochromatic light of very narrow bandwidth corresponding to the wavelength of
Xmax, the maximum value of molar absorptivity is obtained.

Sawin [9] suggested a relation between sensitivity and molar absorptivity. He

suggested the following criteria for describing the sensitivity.

Low sensitivity, e < 2xl04,1 mol1 cm'1

Moderate sensitivity e = 2 - 6xl04,1 mol1 cm'1

High sensitivity e > 6xl04,1 mol'1 cm'1

It is generally stated [10] that the molar absorptivity will not exceed
approximately 105.

Other way of specifying sensitivity are as specific absorptivity [11] or the

Sandell’s sensitivity [12]; 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 sensitivity is
the concentration of the analyte (in pg/ml) which will give an absorbance of 0.001 in
a cell of path length 1 cm and is expressed as pg cm"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

26
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.

1B.6 PRECISION AND ACCURACY

Precision describes the reproducibility of results where accuracy denotes the

nearness of a measurement to its accepted value. The accuracy and precision of
spectrophotometric method depends on three major factors. Instrumental limitations,
chemical variables and 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.

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 calculation of coefficient of variation is
given below

27
 CV = s>,0°
(2)
X

Where s = Standard deviation and x = Arithmetic mean of a series of measurements.

1B.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 [13, 14]. 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 reagent blank and the slope of the calibration curve of
the analyte, the detection limit (DL) may be expressed as:

DL= ------------(3)
S

Where a = 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 estimate
of o may be measured based on the standard deviation of the reagent blank.

1B.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 [15, 16]. Based on the standard deviation of the reagent blank samples and
the slope of the calibration curve of the analyte, the quantitation limit (QL) may be
expressed as:

10 cr
QL = (4)
S

where o = 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 estimate
of o may be measured based on the standard deviation of the reagent blank.

28
1B.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 and
(b) the variance ratio test (F-test) [17,18].

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

degrees of freedom. In statistical terms this is the number of independent values
necessary to determine the statistical quantity. Thus a sample of n values has n

degrees of freedom, whilst the sum 2 (x-x) is considered to have n-1 degrees 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.

(a) Student’s t-test

This is a test 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. It is also used to test the difference between the means

of the two sets of data Xl and X2.

---------------- (5)

Where s = Standard deviation, x = Arithmetic mean of a series of measurements, p is

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

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

29
(b) The Variance Ratio Test (F-test)

This is used to compare the precisions of two sets of data of two different
analytical methods or the results from two different laboratories. It is calculated from
the following equation [17,18]:

(6)

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 [17]
corresponding to the numbers of degrees of freedom for the two sets of data.

1B.6.4 Comparison of the Means of Two Samples

When a new analytical method is being developed it is usual practice to

compare the values of the mean and precision of the new (proposed) method with
those of an established (reference) procedure [18].

There are two common methods for comparing results: (a) t-test and (b) the
variance test (F-test).

(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 method [17, 18]. The value of ‘t’ when
X X
comparing two sample means 1 and 2 is given by the expression:

t= (7)

Where Sp is the pool standard deviation, is calculated from the two samples standard
deviations SI and S2 as follows :

(8)

ni and n2 are the number of trials of first and second method.

30
(b) Variance Ratio Test (F - Test)

The F-test must also be applied to establish that there is no significant

difference between the precision of the two methods. The value of F calculated from
equation (6). 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 [17]
corresponding to the numbers of degrees of freedom for the two sets of data. Thus, the
calculated value of F is less than the tabulated value, therefore the method have
comparable precisions (standard deviations).

1B.7 COLOR DEVELOPMENT

There are only a few elements, which give sufficiently 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.

1B.7.1 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.

1B.7.2 The Criteria for Satisfactory Spectrophotometric Analysis

Though spectrophotometric methods are 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.

31
IB.7.2.1 Specificity of the Color Reactions

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.

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.

1B.7.2.2 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.

1B.7.2.3 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

32
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.

1B.7.2.4 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.

1B.8 CHOICE OF SOLVENT

The solvent, which is to be used in colorimetric or spectrophotometric

determinations must meet certain requirements. It must be a good solvent for the
substance under determination. Before using a particular solvent, it must be ensured
that it does not interact with the solute. The solvent must not show significant
absorption at the wavelength to be employed in the determination.

For inorganic compounds, water normally meets these requirements, but for
majority of organic compounds, it is necessary to use an organic solvent. All solvents
show absorption at some point in the ultraviolet region and care must be taken to
choose a solvent for a particular determination which does not absorb in the requisite
wavelength region. Any impurities present in the solvents may affect the absorption at
certain wavelength and it is therefore, essential to employ materials of the highest
purity.

1B.9 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

33
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

Tobs = lfis+2- -------------(9)

1 +P

Where p is the fraction of all the light coming from the wavelength isolation
device, which is stray light, and Tobs and 7^ are the observed and true transmittances,
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 is measured.

A common error is encountered when making the measurements is called

finite slit width effect. 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 a 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. Even when the incident light is highly collimated and falls on the cell

34
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.

1B.10 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 these 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.

35
 Many compilations of methodology for a variety of analytes and sample types
that are regularly updated are available [19-21]. Other general sources for
spectrophotometric analysis are commonly consulted and found helpful [22-24].
Methods specific for metals [25], and nonmetals [26] should be consulted when
dealing with these analytes. Standard methods specific to certain industries and areas
of study are very useful sources when specific sample types are being considered,
such as water and waste water [27] and pharmaceuticals [28].

1B.11 PRESENT INVESTIGATION

The work described in chapter-2 presents two new releasing agents such as
thioglycollic acid and 2,2'-bipyridyl used for the complexometric determination of
nickel and thallium respectively. Chapters 3-10 deal with the spectrophotometric
determination of chromium, arsenic, thallium, vanadium, nitrate, iodate and periodate,
selenium and endosulfan. Chapter-3 describes spectrophotometric determination of
chromium using thionin, azure B, variamine blue and saccharin as chromogenic
reagents. Chapter-4 deals with the spectrophotometric determination of arsenic in
various environmental samples using variamine blue and azure B as reagents.
Chapter-5 presents spectrophotometric determination of thallium using thionin, azure
B and leuco xylene cyanol FF as analytical reagents. Chapter-6 includes
spectrophotometric determination of vanadium in synthetic mixtures and alloy
samples using variamine blue and thionin as reagents. Chapter-7 describes
spectrophotometric determination of nitrite in microgram levels based on the
diazocoupling reaction between p-nitroaniline with malanonitrile, ethoxyethylene-
malonicester and ethyleyanoaeetate. Chapter-8 deals with spectrophotometric
determination of iodate in table salts samples and periodate in river water samples
using thionin, azure B and variamine blue as analytical reagents. Chapter-9 describes
spectrophotometric determination of selenium in vegetables, water and soil samples
based on the oxidative-diazocoupling reaction between phenylhydrazine-p-sulfonic
acid with ethylacetoacetate and acetylacetone. Chapter-10 deals with the
spectrophotometric determination of endosulfan in water and soil samples using azure
B and variamine blue as reagents.

36
The structures of the reagents used are given below.

SI. No. Name of the reagent Structure

1. Thioglycollic acid (TGA) HS- CH2-COOH

2. 2,2'-Bipyridyl

°2n—^ ^—nh2
3. p-Nitroaniline

0X
0z

0z
4. Malanonitrile

CM
5. Ethoxyethylene H5C2OOC
h5c2o—^
malonicester
COOC2H5

6. nc-ch2-cooc2h5
Ethylcyanoacetate

7.
Thionin

ch3
L
8.
Azure B CH3

J& tx
JYH

9.
Variamine Blue

37
 SOgNa

y^s°3H

10. Xylene Cyanol FF

hn
JL J y
A
NH

H3(T
J *
ch3 ch3
3 ^CH3

11. Phenylhydrazine-p- H03S—^ NH—NH2

sulfonic acid

O
12. Acetylacetone
h2ct
A XH,

13. Ethylacetoacetate
<A,„

38
1B.12 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. A. H. Ayres, Ind. Eng. Chem. Anal., 21 (1949) 652.
4. A. Ringbom, Z. Anal. Chem., 115 (1939) 332.
5. I. M. Kolthoff and E. B. Sandell, “Text Book of Quantitative Inorganic Analysis ”,
3rd Edn., The Macmillan Company, New York, (1959).
6. A.B. Blank, Z. Anal. Chem., 17 (1962) 1040.
7. I.S. Mustafin, Zavod. Lab., 28 (1962) 664.
8. I.E. Banney, Talanta, 14 (1967) 1363.
9. S. B. Savvin, CRC Crit Rev. Anal Chem., 8 (1979) 55.
10. H. Muller, Anal Chem., 13 (1982) 313.
11. A.H. Ayres and B. D. Narang, Anal. Chem. Acta., 24 (1961) 241.
12. E. B. Sandell, Colourimetric Determination of Traces of Metals, 3rd Edn., Inter
Science, New York, (1959) 83.
13. J. M. Green, Anal. Chem. News & Features, May 1 (1996) 305A.
14. B. Renger, H. Jehle, M. Fischer and W. Funk, J. Planar Chrom., July/Aug 8
(1995)269.
15. J. Vessman, J. Pharm & Biomed Analysis, 14 (1996) 867.
16. D. Marr, P. Horvath, B. J. Clark and A.F. Fell, Anal. Proceed. 23 (1986) 254.
17. D.A. Skoog, D.M. West and F.J. Holler, Fundamentals ofAnalytical Chemistry
5th Edn., (1988) P. 38 and 29.
18. A.I. Vogel, Text Book of Quantitative Chemical Analysis, 5th Edn. (1989) 139-
141.
19. ASTM, Annual Book OfASTM Standard (66 volumes in 16 sections), American
Society for Testing and Materials, Philadelphia, 1987.
20. S. Williams, ed., Official Methods of Analysis of the Association of Official
Analytical Chemists, 16th Edn. Arlington, 1995.
21. J. A. Howell and L. G. Hargis, Anal. Chem., 68 (1996) 169R.
22. D. Eckroth, ed., Encyclopedia of Chemical Technology, 3rd Edn., Wiley, New
York, (1984).

39
23. L. C. Thomas and G. J. Chamberlin, Colorimetric Analytical Methods, 9th Edn.,
Tintometer press, Salisbury, England, (1980).
24 Z. Marczenko, Spectrophotometric Determination of Elements, Halsted, New
York, (1975).
25. F. D. Snell, Photometric and Fluorimtric Methods ofAnalysis, part 1 & 2, Wiley,
New York, (1978).
26. D. F. Boltz and J. A. Howell, Colorimetric Determination of Nonmetals, 2nd
Edn., Wiley, New York, (1978).
27. American Public Health Association, Standard Methods for the Examination of
Water and Wastewater, 17th Edn., American Public Health Association,
Wasington, D.C, (1992).
28. United States Pharmacopoeial Convention, United States Pharmacopoeia, 23rd
Rev., New York, (1995).

40