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Analytical Chemistry
Analytical Chemistry
DR. H. KAUR
READER
Postgraduate Department of Chemistry
N.A.S. (P.G.) College, Meerut, India.
~PRAGATI PRAKASHAN
PRAGATI PRAKASHAN First Edition : 2008
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Contents
1. INTRODUCTION................................... 1-39
Role of analytical chemistry 1
Types of analysis 2
Analytical methods 3
Classification of classical and instrumental methods of analysis 3
Selecting an analytical method 9
Steps involved in quantitative analysis 10
Neatness and cleanliness in laboratory 10
Selecting and handling of reagents 11
Organic reagents used in inorganic analysis 12
Safety in the analytical laboratory 14
Laboratory note book 15
Laboratory operations and practices 16
Sample preparation, dissolving the sample and sample decomposition 16
Stoichiometry 23
Volumetric glasswares 24
Gravimetric techniques 24
Analytical balance 33
Techniques of weighing using analytical balance 36
Weighing errors 36
Cleaning and calibration of glasswares 37
ANALYTICAL CHEMISTRY
Analytical chemistry is a scientific discipline that develops and applies
methods, instruments and strategies to gain infonnation about the
composition and nature of matter. It is an interdisciplinary branch of science
which plays an important role in nearly all streams of chemistry such as
inorganic, organic, physical, industrial and biochemistry. It also finds
extensive applications in environmental science, agricultural science,
oceanography, clinical chemistry, solid state research and electronics. The
scope of analytical chemistry is very broad and embraces a wide range of
manual, chemical and instrumental techniques.
Role of Analytical Chemistry.
Analytical chemistry seeks ever imposed means of determining the
chemical composition, purity and quality of substances by qualitative and
quantitative analysis.
• It is the science of chemical characterisation of matter. It provides the
techniques and tools needed for insight into our material world.
• Manufacturing industries rely upon analytical chemistry for
testing raw materials and for assuring the quality of finished products
whose composition is critical. The quality of manufactured products
often depends on proper chemical proportions and measurement of
the constituent is a necessary part of quality control. The
semiconductor industry is an example of an industry whose very
existence depends on accurate determination of substances present in
extremely minute quantities.
• Several industrial and household products, alloys, polymers, fuels,
paints, perfumes, and pharmaceuticals are analysed by the
procedures developed by analytical chemists before being sold to the
consumer.
• Many industrial processes give rise to pollutants which can create a
health problem. Quantitative analysis of air, water and soil samples
must be carried out to determine the levels of pollution and to
establish safe limits for pollutants.
• Environmental quality is often evaluated by testing for suspected
contaminants using the techniques of analytical chemistry.
• In medicine, analytical chemistry is widely used for clinical laboratory
tests to assist in diagnosis of illness and in monitoring the conditions
of patients. The composition and purity of drugs detennine their
efficacy.
(1)
2 ANALYTICAL CHEMISTRY
TYPES OF ANALYSIS
Analysis involves the resolution of a chemical compound into its
proximate or ultimate parts, the determination of its elements or of foreign
substances it may contain. The first requirement in a completely unknown
sample is to ascertain what substances are present in it and which type of
impurities are contained in the sample. The solution of such problems lies
within the province of qUalitative analysis. Having ascertained the nature
of the constituents of a given sample, the next step is to determine how much
of each (or specified) component is present. Such determinations lie within the
realm of quantitative analysis and a variety of techniques are available to
supply the required information. Depending upon the nature of information
sought, chemical analysis may be classified into four types.
1. Proximate analysis, in which the amount of each element in a
sample is determined with no concern as to the actual compounds
present.
2. Partial analysis deals with the determination of selected
constituents in the sample.
3. Trace constituent analysis, a specialised instance of partial
analysis, deals with the determination of specified components
present in very minute quantity.
4. Complete analysis, when the proportion of each component of the
sample is to be determined.
On the basis of sample size, analytical methods are often classified as
follows:
INTRODUCTION 3
Continued ..... .
6 ANALYTICAL CHEMISTRY
them to emit energy. The intensity of this energy is then measured. Here are
some of the common excitation techniques.
• Flame photometry uses a solution of the sample injected into a flame.
• Emission spectroscopy subjects the sample to an inductively
coupled plasma and then examining the emitted light (which may
extend into the ultraviolet region).
• Fluorimetry takes a substance in a fluorescent reagent and excites
it using visible or ultraviolet radiation.
(iii) Magnetic Resonance Spectroscopy. Nuclear magnetic
resonance (NMR) spectroscopy is concerned with the study of interaction of
energy with spin-active nuclei which have permanent magnetic moments and
quanti sed nuclear spin states.
• Electron spin resonance (ESR) spectroscopy. Electrons in the
free radicals, atoms, ions or molecules (having unpaired electrons)
change their spin under the influence of applied magnetic field and
spectra arising is called ESR or EPR.
(iv) Photoelectron Spectroscopy (PES). In PES, a beam of photons
of known energy is allowed to fall on the sample and kinetic energy of the
ejected electrons is measured. PES can be studied either using X-ray photons
(XPES) or UV photons (UVPES).
(v) Scattering Methods. Nephelometric and turbidimetric methods
measure the amount of light stopped or s<:attered by a suspension.
C. Chromatographic and Electrophoretic Methods. These are
essentially separative processes for mixtures of substances but equipped with
modern detector systems, they are also adapted to identifY components of
mixtures.
3. Non-Destructive Methods (X-ray Methods).
(i) Electron Probe Micro Analysis. Primary X-rays are produced
when high speed electrons collide with a solid target. It is possible to identify
certain emission peaks which are characteristic of elements contained in the
target. The wavelengths of the peaks can be related to the atomic number of
the elements producing them, so they provide a means of identifYing elements
present in the target sample. Further, under controlled Gonditions, the
intensity of peaks may be used to determine the amounts of the various
elements present. This forms the basis of electron probe micro analysis,
in which a small target area of the sample is selected for identification.
(ii) X-ray Fluorescence. When a beam of short wavelength primary
X-rays strikes a solid target, it emits X-rays at wavelengths characteristic of
the atoms involved. This is referred to as secondary or fluorescence
radiation. It is possible to get an idea of sample composition by examining
the peak heights of the .fluorescence radiation. X-ray fluorescence analysis
is a rapid process which finds applicatlons in metallurgical research, in
processing of metallic ores and in cement industry. Crystalline material will
diffract a beam of X-rays. X-ray powder diffractometry can be used to
identifY components of mixtures.
8 ANALYTICAL CHEMISTRY
4. Radioactive Methods.
Radioactive methods involve measuring the intensity of radiation from
a naturally radioactive material and also measuring radioactivity induced by
exposing the sample to a neutron source (activation analysis), or isotope
dilution and radio immunoassay. Typical applications include
determination of trace elements and quality control in semiconductor
manufacturing.
5. Special Methods.
(i) Optical Methods. Refractometry can be used to measure the
refractive index of liquids and to check the purity of the compound. In
conjunction with a calibration curve, it can be employed to analyse a mixture
of two liquids. Optical rotatory dispersion and circular dichroism
coupled to polarimetry correlate measurement of optical rotation and
absorption.
(ii) Kinetic Methods. Kinetic methods are based on the fact that the
speed of a given chemical reaction may be increased by the addition of a small
amount of catalyst, and within limits, the rate of catalysed reaction will be
governed by the amount of catalyst present. If a calibration curve is prepared
showing variation of reaction rate with the amount of catalyst used, then
measurement of reaction rate will make it possible to determine how much
catalyst has been added in certain instance. This provides a sensitive method
for determining sub-microgram amounts of appropriate substances.
Non-destructive methods, radioactive methods, optical and kinetic
methods also require the use of an instrument, hence may be considered as
instrumental methods.
Advantages of Instrumental Methods.
• Instrumental methods are general1y more sensitive and selective than
the classical methods.
• These techniques are normally applicable at concentrations far too
small to be amenable to determination by chemical methods.
• Determination is much faster than purely chemical procedures.
• Measurements obtained are reliable. Accuracy is obtainlild.
• Even complex samples can be handled easily.
• Finds wide application in industry.
• In most cases a microcomputer can be interfaced to the instrument so
that absorption curves, polarograms, titration curves etc. can be
plotted automatically. In fact, by the incorporation of appropriate
servo-mechanisms, the whole analytical process may be completely
automated.
Limitations of Instrumental Methods.
Despite the advantages possessed by instrumental methods in many
directions, their wide-spread adoption has not rendered the purely chemical
or classical methods obsolete, the situation is influenced by the following
factors:
INTRODUCTION 9
and laundered frequently. The cloth should not be used on the inside
of the vessels.
3. The working surface of the .bench must not be cluttered with
apparatus. All the apparatus associated with some particular
operation should be assembled together on the bench. This is
extremely essential to avoid confusion when duplicate determina-
tions are performed. The apparatus which are not in use should be
returned to the locker.
4. The container must be labelled so that the solution, precipitate or
filtrate can be readily identified. The vessel must be covered to
prevent contamination of the contents by dust.
5. For temporary labelling, a chinagraph pencil or a felt-tip pen (glass
marker pen) is preferred to wax pencils or adhesive labels. Adhesive
labels are employed for long term use.
6. Reagent bottles should never be allowed to accumulate on the bench,
these must be replaced on the reagent shelves immediately after use.
7. Reagent solutions should be poured in the testing vessel.
8. Never insert spoon, spatula or knives into the reagent bottle. Use
clean porcelain spoon for taking solid chemicals.
9. All determinations should be performed in duplicate.
10. A stiff covered note book should be used for recording experimental
observations.
SELECTING AND HANDLING OF REAGENTS
The purest reagents should be used for quantitative analysis, wherever
possible use reagents of analytical quality. Analar chemicals from BDH Ltd.
(Merck) conform to ACS specifications. Certain manufacturers market
chemicals of high purity and each package of these analysed chemicals has a
label giving the manufacturer's limits of certain impurities. Following points
should be considered while handling the reagents.
• Liquid reagents should be poured from the bottle; a pipette should
never be inserted into the reagent bottle. Avoid contamination of the
stopper of reagent bottle.
• When the liquid reagent is poured from a bottle, the stopper should
never be placed on the working bench or shelf.
• The stopper should be placed upon a clean watch glass.
• The stopper should be returned to the bottle immediately after the
reagent has been removed.
• The reagent bottles must be kept scrupulously clean, particularly
round the neck or mouth of the bottle.
• If a reagent of adequate purity for a particular determination is not
available then the purest available product must be purified. Liquids
can be purified by fractional distillation and solids by
recrystallisation.
• The reagents should be tested by standard methods for the impurities
that might cause errors in the determination.
• Use specially purified reagents such as the BDH Aristar chemicals to
detect lower limits in various types of instrumental analysis.
12 ANALYTICAL CHEMISTRY
~OH
l2JSJ
a-Nitro so !3-naphthol solution reacts with slightly acidic solution of
Co2+ ion to form its red brown chelate of octahedral geometry. This test is not
shown by Ni 2+ ion.
3. S-Hydroxy quinoline (Oxine).
~
yN~
OH
Oxine is employed for the separation of AI from Be in acidic medium, Mg
from Ca in alkaline medium and for gravimetric estimations of Cu, Ni, Co,
Zn, Pb, Cd and Mg etc.
4. Cupferron.
/NO
~N'ONH4
Cupferron, the salt of nitrosophenyl hydroxylamine reacts with Fe(ITI)
to form bidentate inner complex of iron in which a labile hydrogen (or NH;t
ion) is replaced and oxygen of the nitroso group co-ordinates. The reagent is
highly selective for Nb, Ta, W, Hg(I), Fe(III) and Zn in acid solution.
5. Dithizone or Diphenyl thiocarbazone.
N=N-C-NH-NH
@ll@
INTRODUCTION 13
&CH=NOH
Salicylaldoxime has been employed for the determination of Cu. Tartaric
acid is used as the masking agent.
9. Magneson (p-Nitrobenzene azo resorcinol).
02N--@-N~N-(jj)-OH
HO .
Magneson forms a blue lake with Mg(OH)2 m alkaline medium.
Ammonium salts reduce the sensitivity of the test.
10. Benzidine.
H2N-@-@-NH2
Benzidine is used as the dihydrochloride for the precipitation of
sulphate in acidic solution.
11. Anthranilic acid.
~NH
&COOH
Sodium salt of anthranilic acid is useful for quantitative determination
of Cu, Cd, Ni, Zn and Co at controlled pH.
12. 1, 10-Phenanthroline.
r0n
t:N">-<"NJ
14 ANALYTICAL CHEMISTRY
The reagent yields extremely stable red colour with Fe2+ ion
[Fe(C 12H s N 2)s]2+. The test is highly sensitive over a wide pH range (2 to 9)
and conform to Beer's law. The ferrous complex of 1, 10-phenanthroline
(ferroin) is a valuable redox indicator.
13. Acetyl acetone.
o °
II
CHs-C-CH2-C-C " HS
Acetyl acetone reacts with Cu2+ ions to form neutral inner chelate which
can be separated from Fes+ ions by solvent extraction. Trifluoroacetyl acetone
has been used in the separation of Zr and H£
14. Ethylene diamine tetra-acetic acid.
HOOCH2C " /CH2COOH
N-CH2-CH2-N
HOOCH2C / '-.....CH2COOH
Disodium salt of EDTA has been used for the estimation of Mi+ and
Ca2+ ions by complexometric titrations at pH 10 using Eriochrome Black T as
an indicator.
15. Alizarin red.
cQ5:::iN
o
a
To the Als+ salt solution add NH 40H, alizarin red and acetic acid in
excess. Red colouration is obtained which is destroyed by F- ions. Hence the
reagent is very sensitive for the detection of V- ions.
SAFETY IN THE ANALYTICAL LABORATORY
There is always a potential hazard in a chemistry laboratory and
regrettably, accidents do occur on occasions. However, they can be minimised
by adhering to the laboratory safety code, by taking all necessary safety
precautions and by carrying out a hazard assessment before any
experimentation. Following points should be borne in mind.
• Many chemicals encountered in analysis are poisonous and must be
handled carefully.
• It is necessary to carry out a safety assessment of the poisonous
chemicals, concentrated acids, PARs, PCBs, KCN and halogenated
solvents.
• Many operations involving chemical reactions are potentially
dangerous, so recommended procedures must be carefully followed
and obeyed.
• Always clean up spilled chemicals. Do not leave broken or chipped
glassware lying around working bench.
INTRODUCTION 15
LABORATORY NOTEBOOK
• A stiff covered notebook should be used for recording experimental
observations as they are made.
• Never use loose leafs. Number pages consecutively.
• Never tear out pages. If not used, put a line through the page.
• Date each page and record the name of the project.
• Devote a double page to each determination. Use one page for the
experimental observations and the other for brief description of the
procedure followed with special features associated with the
determination.
• The record must conclude with the relevant calculations, so the
equations for the principal chemical reactions should be shown
together with a clear exposition of the procedure for calculating the
result.
• Finally, appropriate comments should be made about the degree of
accuracy and the level of precision.
• Now the modern laboratory instruments are frequently interfaced or
they possess built-in computers to produce printed records of the
analytical results in the form of spectra or chromatogram. These
equipments process the data and compare it automatically with
standards and previous results stored in computer memory.
16 ANALYTICAL CHEMISTRY
SAMPLE DECOMPOSITION
Methods generally adopted for decomposing and dissolving an analytical
sample include:
1. Heating with strong acids or bases. 2. Fusion reagents (fluxes).
3. Microwave heating. 4. Combustion methods.
1. Decomposing Samples with Acids in Open Vessels.
Mineral acids are generally employed to decompose inorganic samples in
open vessel. The suspension is heated by flame, hot plate or heating mantle.
Following reagents may be used to dissolve difficult materials.
(i) Concentrated acids. Concentrated HCI dissolves many metals
(above H2 in the electrochemical series) and metallic oxides. Hot conc-
entrated HN03 dissolves most metals except Sb, Sn and W which are
converted to slightly soluble acids. Hot concentrated H 2S04 dissolves several
substances, many organic materials are charred and then oxidised by this
treatment.
(ii) Aqua regia (75 vol % HCI and 25 vol % HN0 3 • Due to its strong
oxidising character, it is a very potent solvent. The effectiveness of the acid is
increased by adding other oxidants such as Br2 and H20 2 .
(iii) Hydrofluoric acid. HF is used mainly to decompose silicates,
minerals and steel. Excess of HF is removed by evaporation with H 2S0 4
leaving a residue of metallic sulphates. Use HF with great care since it
causes painful skin burns.
(iv) Perchloric acid. HCI0 4 attacks stainless steels and a number of
iron alloys that do not dissolve in other acids. Hot concentrated HCI0 4 gives
explosive reactions with organic substances or easily oxidised inorganic
compound. Evaporation involving perchloric acid should be performed in a
fume cupboard free from combustible organic materials. For safety, the
compound should be treated first with concentrated nitric acid, the mixture
heated and then add small quantities of HCI0 4 until the oxidation is
complete. When 60 vol % HN0 3, 20 vol % HCI04 and 20 vol % H 2S04 is used,
the perchloric acid is also evaporated along with HN03 leaving a sulphuric
acid solution of the components for analysis. The organic part of the
material under investigation is destroyed and the process is called
wet ashing. Use perchloric acid with great care since it is dangerously
explosive.
2. Decomposing Inorganic Substances by Fusion Reagents (Fluxes).
Fusion reagents (alkali metal salts), commonly known as fluxes are used
to decompose sub!;!tances (e.g., mineral oxides, silicates and some iron alloys)
20 ANALYTICAL CHEMISTRY
that are not soluble in normal solvehts or acids. Typical fluxes include
anhydrous Na2C03' either alone or mixed with KNOa or Na202, NaOH, KOH,
K 2S 20 7 (potassium pyrosulphate).
Procedure of Fusion of Fluxes.
To carry out the fusion, a layer of flux is placed at the bottom of the
crucible, then an intimate mixture of the flux and finely powdered substance
added. The crucible should be about half-filled and kept covered during the
whole process. The crucible is heated gradually at first and the temperature
slowly raised to the required temperature. The final temperature should not
be higher than is actually necessary.
When the fusion has been completed (30 to 60 minutes), the crucible is
gently rotated and tilted by tongs so that molten material distributes itself
around the walls of the container and solidifies as a thin layer. When cold,
the crucible is placed in a pyrex beaker, porcelain dish or platinum basin and
covered with water. Acid is added, if necessary. The vessel is covered with
clock glass and temperature is maintained to 368-373 K until solution is
achieved. Typical fluxes used to decompose inorganic materials are illustrated
in Table 2.
Types of Fluxes.
The flux employed will depend on the nature of the insoluble substance.
(i) Acidic fluxes such as pyroborates, pyrosulphates and acid fluorides
are used to decompose basic materials.
(ii) Basic fluxes (carbonates, hydroxides, peroxides, metaborates)
attack acidic materials.
(iii) Oxidising fluxes (Na202 or Na2C03 mixed with KN0 3) are used
in oxidising medium.
Characteristics of some fluxes.
(i) Sodium carbonate. Sample containing silicates or refractory
material can be decomposed by Na2C03 at 1273 K. The cationic constituent of
the sample is cunverted to acid soluble carbonate and non-metallic constituent
to soluble sodium salts.
(ii) Lithium metaborate. Anhydrous LiB0 2 is especially suitable for
materials containing silica, when the resulting mass is dissolved in dilute
acids.
Advantages claimed for LiB02 •
• Fusion with lithium metaborate are usually quicker (15 min) and can
be performed at a lower temperatures than with other fluxes.
• The loss of Pt from the crucible is less with LiB02 fusion than with a
Na2C03 fusion.
• No gases are evolved during the fusion or dissolution of the melt. So
there is no loss of material due to spitting.
• Many elements can be determined directly in the acid solution of the
melt without the need for tedious separations.
3. Microwave Decomposition of the Sample.
Microwave digestion of the sample can be carried out by several devices.
(i) Atmospheric pressure vessels.
Atmospheric pressure vessel utilises open vessel system, has a focussed
microwave cavity and a tubing for the insertion and removal of reagents.
Since the reagents operates at atmospheric pressure so there is evolution of
gases during the digestion process.
(ii) Moderate pressure microwave vessels.
Microwave digestion vessels are constructed from teflon (m.p. 573 K) or
quartz or borosilicate glass vessels. A closed digestion vessel of teflon body
consists of a cap and a safety valve. It can operate at 120 ± 10 Psi pressure
to decompose silicates.
(iii) High pressure microwave vessels (Microwave bombs).
Many of the substances which require fusion treatment to render them
soluble will in fact dissolve in mineral acids if the digestion with acid is
carried out under high pressure and high temperature. Such drastic
treatment requires a container capable of withstanding requisite pressure
and also resistant to chemical attack. Such conditions are met in acid
digestion vessels (microwave bombs). Digestion vessels comprise a stainless
steel pressure vessel (50 cm3) with a screw-on lid and fitted with a teflon
liner. They can withstand pressures of 8-9 MPa (80-90 atm) and may be
heated to 453 K. Under these conditions, the refractory material can be easily
decomposed in about 40 minutes.
22 ANALYTICAL CHEMISTRY
Advantages.
• Decomposition of the sample is time and cost saving.
• There is no need for expensive platinum ware.
• No losses can occur during the treatment.
• The resulting solution is free from the heavy loading of alkali metals.
(iv) Microwave ovens.
Microwave ovens are now being used extensively for decomposing
several compounds. In the oven, 12 moderate pressure vessels can be heated
at a time. The vessels can rotate through 360· so the average energy received
by each vessel is nearly the same. Decomposition is much rapid and drying
times for precipitates is greatly reduced. Further, the loss of volatile
components of the sample is virtually eliminated. Microwave decomposition
is often easy to automate thereby reducing operation times required to
prepare samples for analysis.
(v) Microwave furnaces.
Microwave furnaces consist of a small chamber to accomodate only a
crucible. The chamber is made up of SiC which is surrounded by quartz
insulation. The advantage of microwave furnace over muffle furnace is the
speed, at which the high temperature (1273 K) is reached within two minutes.
4. Decomposing Organic Compounds by Combustion Methods.
(i) Combustion in an open crucible (Dry ashing).
The organic sample is heated in an open crucible until all carbonaceous
matter has been oxidised, leaving a residue of inorganic components as oxide.
The residue is dissolved in dilute acid and the solution can be analysed by
suitable procedure. The technique is inapplicable when the inorganic
component is volatile.
(ii) Combustion-tube method.
In automated combustion tube analysers, the sample is ignited in a
stream of helium. Oxygen is passed over an oxidation catalyst consisting of a
mixture of silver vanadate and silver tungstate. Halogens and sulphur are
removed with a packing of silver salts. A packing of hot copper is placed at
the end of combustion train to remove 02 and to convert NOx to N 2. In this
method C, H, N can be determined quickly. The exit gases consisting of a
mixture of H 2 0, CO2 , N and helium is collected in a glass bulb.
(iii) Combustion with O2 in a sealed container (Schoniger method).
Analysis of organic compounds for elements (S, P or halogens) is
performed by combustion of organic material in 02; the inorganic constituents
are thus converted to forms which can be determined by titrimetric or
spectrophotometric methods.
Procedure. Schoniger flask, containing few em3 of absorbing solution
(e.g. aqueous NaOH), is filled with oxygen and sealed using the stopper with
the platinum basket attached (Fig. 1a). About 5-10 mg of sample is weighed
iNTRODUCTION 23
STOICHIOMETRY
Analytical procedures may either be stoichiometric or non-stoichio-
metric.
1. Stoichiometric Methods.
Here the constituent whose amount is being measured (R A ) undergoes a
reaction with another substance (R B ).
RA +RB ---t PC+PD
The amount of desired constituent, R A can be calculated by applying the
law of definite and combining proportions, provided the amount of any of the
products (Pc or PD ) or of the reagent is known. Wet chemical methods
(gravimetric and volumetric), coulometric methods, gas analytical methods
and certain separation techniques are stoichiometric.
2. Non-stoichiometric Methods.
They can not be written as exact well-defined reactions. These methods
are based on the accurate measurement of a physical property which changes
in a proportion to the concentration of the desired constituent. Here it is only
necessary to calibrate the procedure. Most instrumental methods are
non-stoichiometric. Some examples are :
• Optical methods based on
(a) absorption of energy (UV and IR spectrometry) and
(b) emission of energy (flame photometry, fluorescence,
chemiluminescence ).
• Electrical methods, e.g., potentiometry, polarography, conductivity
etc.
24 ANALYTICAL CHEMISTRY
VOLUMETRIC GLASSWARES
Volumetric glasswares (flasks, burettes, pipettes, syringes) are used for
the measurement of volumes.
Volumetric Flasks. They are employed in making up standard
solutions to a given volume and for obtaining (with the aid of pipette), aliquot
portions of a sample solution to be analysed. The mark extends around the
neck. In order to avoid errors due to parallax, the lower edge of the
meniscus of the liquid should be tangential to the graduation mark and both
the front and back of the mark should be seen as a single line.
Pipettes.
(i) Transfer pipettes have one mark and deliver a constant volume
of liquid under certain specified conditions.
(ii) Graduated or measuring pipettes are employed to deliver various
small volumes as required.
(iii) Syring pipettes or micro pipettes have fixed or variable volume
and are used for dispensing large numbers of identical volumes
very quickly.
Use of transfer pipette. A suitable pipette filler is attached to the
upper end of the pipette (filling should never be carried out by mouth suction).
Rinse the pipette with a solution and discard it. It should then be filled with
the liquid to 2 cm above the mark. Any adhering liquid is removed by wiping
with a filter paper, the liquid is allowed to run out slowly until the bottom of
the meniscus just reaches the mark. The pipette must be held vertically and
with the mark at eye level. The liquid is then allowed to run into the receiving
vessel with the tip touching the wall of the vessel.
When the discharge has ceased, the jet is held in contact with the vessel
for a draining time of 15 s. The liquid in the jet of pipette must not be
removed by blowing.
Burettes.
Burettes are used to deliver fractional volumes of a solution of known
concentration which reacts stoichiometrically with another reactant. Burettes
should be clamped vertically in the holder. To deliver liquid from a burette in
a conical flask during titration, one hand is used to swirl the vessel and the
other is used to control the stopcock. To read the position of the meniscus,
the eye must be at the same level as the meniscus in order to avoid errors
due to parallax.
Piston burettes can provide automatic plotting of titration curves.
They also allow a variable rate of delivery as the end point is approached. So
there is no risk of over shooting.
GRAVIMETRIC TECHNIQUES
Gravimetric analysis employs preparation of the sample (described
earlier), precipitation, digestion, filtration, washing, drying, incineration and
weighing of the product.
INTRODUCTION 25
PRECIPITATION "'
Many methods exist for the precipitation of compounds. When the ionic
product exceeds the solubility product, the solution is supersaturated and
precipitation occurs.
Factors which Determine Successful Analysis by Precipitation.
1. The precipitate should be so insoluble that no appreciable loss occurs
when it is collected by filtration.
2. The precipitate must be coarse, dense, granular and can be readily
separated from the solution by filtration.
3. The precipitate must be convertible into a pure substance of definite
chemical composition.
Procedure for Efficient Precipitation.
1. Precipitation should be carried out in dilute solution, due regard
being paid to the solubility of the precipitate, time required for
filtration and the subsequent operations to be carried out with the
filtrate. This will minimise errors due to co-precipitation.
2. Precipitation is effected in hot solutions, provided the solubility and
stability of the precipitate permits. At higher temperature
(i) solubility is increased with a consequent reduction in the degree of
supersaturation. (ii) Coagulation is assisted' and sol formation
decreased. (iii) The velocity of crystallisation is increased thus
forming better crystals.
3. The precipitant is added slowly with constant stirring (without
splashing). A slight excess of the reagent should be added to take
advantage of common ion effect. Large excess may lead to complex
formation.
4. Mter the precipitate has settled, a few drops of the precipitant should
be added to determine whether further precipitation occurs.
5. Crystalline precipitates should be digested over night, except in those
cases where post-precipitation may occur. As a rule, digestion on the
steam bath is desirable. This process decreases the effect of
co-precipitation and yields readily filterable precipitates. Digestion
has little effect upon gelatinous or amorphous precipitates.
6. The precipitate should be washed with the appropriate dilute solution
of electrolyte. Pure water causes peptisation.
7. If the precipitate is contaminated by co-precipitated impurities, it can
be purified by reprecipitation.
8. Precipitation from a homogeneous solution is generally employed to
prevent supersaturation.
9. Precipitates so formed may be crystalline (e.g., BaS04), curdy (AgCl),
gelatinous [(Fe(OHhl or flocculent.
Co-precipitation.
The contamination of precipitate by substances that are normally
soluble under the conditions of precipitation is called co-precipitation. Thus
the soluble impurities become incorporated into the crystal lattice of the
26 ANALYTICAL CHEMISTRY
DIGESTION (AGEING)
The precipitate is allowed to stand at room temperature or at a flame,
hot plate or on a water bath. Digestion has little effect upon amorphous or
gelatinous precipitates. The net result of digestion is to reduce the extent of
co-precipitation and to increase the size of particle, rendering filtration easier.
FILTRATION
Filtration involves the isolation of the precipitate from the mother liquor.
The object is to get the precipitate and the filtering medium quantitatively
free from the solution. The systems used for filtration include
INTRODUCTION 27
splashing. The liquid to be filtered is then poured down a glass rod into the
filter paper. The lower end of the stirring rod should be very close to the filter
paper but not quite touching on the side. The paper is never filled completely
with the solution. The last traces of the precipitate in the beaker should be
transferred to the funnel by holding the glass rod across the beaker, tilting it
and directing a jet of water from a wash bottle.
Filtration by suction is rarely necessary with gelatinous and fine
precipitates. The suction will draw the particles into the pores of the paper
and the speed of filtration is reduced.
(ii) Crucibles.
The precipitate may react with the cellulose of the filter paper so it is
convenient to collect the precipitate in a crucible for direct weighing.
Following types of crucibles are employed.
Gooch crucibles. A mat of asbestos (filtering medium) is placed on a
perforated porcelain plate (witt plate) in Gooch crucible.
Sintered glass crucibles. These crucibles are made of resistance glass
and have a porous disc of sintered ground glass fused into the body of the
crucible. The filter d~sc is made in varying porosities, zero is the coarset and
five the finest. The range of pore diameter for various grades is as follows.
Porosity 0 1 2 3 4 5
Pore diameter (J.UU) 200-250 100-120 40-50 20-30 5-10 1-2
Depending upon the pore size these crucibles are graded as G-l, G-2, G-3
etc. Porosity 4 (or G-4, G is a kind of glass) is suitable for very fine
precipitates (such as BaS04), porosity 3 for precipitates of medium particle
size (such as AgCl).
Advantages.
• Sintered glass crucibles are resistant to most reagents except HF and
hot concentrated acids.
• These crucibles can be heated to 773 K with gradual heating. They
can be readily cleaned and dried at 373 K
Porcelain crucibles. Porcelain crucibles are often used for igniting
precipitates and heating small quantities of solids because of their cheapness
and ability to withstand high temperatures. Some reactions, such as fusion
with Na2C03 and evaporation with HF cannot be carried out in porcelain
crucibles due to resultant chemical attack. A slight attack also occurs with
pyrosulphate fusions.
Fused-silica crucible is resistant to heat shocks because of its very
small coefficient of expansion. It is not attacked by acids at a high
temperature except HF and H 3P04.
However, fused-silica crucible is attacked by fused alkalis and
carbonates. It is more brittle than ordinary glass and requires longer time for
heating and cooling than platinum crucible.
Platinum crucible. Crucibles made of 95% Pt and 5% Au (non-wetting
alloy) are used in preparing samples for X-ray fluorescence investigation.
INTRODUCTION 29
WASHING
The object of washing is to free the precipitate from the dissolved
substances or absorbed ions with the minimum amount of washing liquid and
to reduce the loss of precipitate by solution. The washing solution should have
a minimum solvent effect on the precipitate and should not help in peptizing
it even after electrolytic ions have been washed off.
It is better to wash with a number of small portions of the washing
liquid, which are well drained between each washing, than with one or two
large portions or by adding fresh portions of the washing liquid while solution'
still remains on the filter paper. An ideal washing liquid should possess
the following qualities.
• It should have no solvent action on the precipitate but dissolve foreign
substances easily.
• It should have no dispersive action on the precipitate.
• It should form no insoluble product with the precipitate.
• It should be easily volatile at the drying temperature of the precipitate.
• It should not contain any substance which may interfere with
subsequent determination in the filtrate.
In general, pure water should not be used unless it is certain that it will
not dissolve appreciable amounts of the precipitate. If the precipitate is
soluble in water, a common ion is usually added, since any electrolyte is less
soluble in a dilute solution containing one of its ions than it is in pure water.
Categories of Wash Solutions.
(i) Solutions which prevent the precipitate from becoming
colloidal and passing through the filter..Ammonium nitrate solution is used
for washing iron (III) hydroxide and 1% HN03 for washing AgCl.
(ii) Solutions which reduce the solubility of the precipitate. The
wash solution containing a common ion with the precipitate may reduce the
solubility of the precipitate.
(iii) Solutions which prevent the hydrolysis of salts of weak acids
and bases.The addition of an acid to the wash solution prevents the
INTRODUCTION 31
INCINERATION
The well drained filter paper and precipitate are carefully detached from
the funnel, folded and ignited in the weighed crucible to a constant weight.
In case the precipitate (e.g., Filter
BaS04, PbS04, Bi20 3, CuO) is
reduced by the burning paper
or decomposed on strong
heating, the filter paper is
separately incinerated. The
precipitate from the filter
paper is carefully transferred
to the watch glass placed over
a glazed paper and covered
with an inverted funnel Fig. 3. Incineration of the filter paper.
(Fig. 3).
The filter paper is folded several times to make it like a cone and is held
just above the crucible by a clean tongs. It is burnt with a non luminous
bunsen flame. The ash is collected in a weighed crucible on a glazed paper.
32 ANALYTICAL CHEMISTRY
Particles falling on the crucible are collected with the help of a feather and
transferred to crucible. The crucible is heated strongly so as to burn all carbon
to a white ash. It is cooled in the desiccator. The main part of the precipitate
is transferred to the crucible. Finally, the precipitate is brought to constant
weight of heating to the necessary temperature.
ANALYTICAL BALANCE
Weighing is an essential part of chemical analysis, both for measuring
the sample and for preparing standard solutions. Standard laboratory
weighings are typically made to three or four significant figures so the
weighing device must be accurate and sensitive. There are various
sophisticated ways of achieving this, but the most accurate and versatile
device for measuring mass is analytical balance.
Analyst actually deals with masses rather than weights. The weight (w)
of an object is the force of attraction due to gravity that is exerted upon the
object, i.e.,
w=mg
where m is the mass and g is the acceleration due to gravity. Since the
gravitational attraction varies with altitude, the weight of the object is
variable. Mass is the quantity of matter of which the object is composed and
is invariant.
Recently the design of analytical balance has been fundamentally
altered and the traditional free-swinging, equal arm, two pan chemical
balance together with its box of weights is uncommon.
Weight control
knobs
Damping
counter weight
Pan arrest
Pan
release
A three-position beam arrest knob is used to protect the knife edges and
beam. The centre position arrests the pan and beam, a second position
releases the pan for use and third position completely releases the pan to
allow the balance to come to rest. From a typical single-pan balance (Fig. 7).
weighings can be made in less than a minute. Mechanical balances are being
replaced by electronic balances but many are still in use.
2. Electronic Balances.
Modern electronic balances provide convenience in weighing and are free
from mechanical failure and sensitivity to vibration. It eliminates the
operation of dailing weights, smooth release of balance beam and pan support,
turning and reading micrometers etc.
Operating Principle of an Electronic Balance.
Electronic balances operate by applying an electromagnetic restoring
force to the support for the balance pan. The pan rests on the arm of a movable
hanger whose position is monitored by an electrical position scanner (Fig. 8).
Position
scanner
Movable ha:ng~e:r3~U~[i~;~~~L___
Coil Temperature sensor
Fig. 8. Operating principle of electronic balance.
When the sample is placed on the pan, the resultant displacement of the
support is cancelled out. The magnitude of restoring force is governed by the
value of current flowing in the coils of the electromagnetic compensation
system. This compensation current is proportional to the weight placed on the
pan. A microprocessor converts the value of
current to the corresponding weight value (in
grams) which appears as a digital display.
Electronic balances incorporate a tary facility
where the weight of the container can be
automatically subtracted.
Working. A single control bar is used to
switch the balance on and off, to set the display
to zero and to tare a container automatically on
the pan. The majority of balances incorporate a
self-testing system and a built-in weight
calibration system. These balances can be
coupled to a printer which gives a printed record
ofthe weight. A digital-display electronic balance
is shown in Fig. 9. Fig. 9. Electronic balance.
36 ANALYTICAL CHEMISTRY
WEIGHING ERRORS
Inspite of careful weighing, some errors may persist due to following
causes.
1. Defective balance.
INTRODUCTION 37
o
2
ERRORS AND EVALUATION
INTRODUCTION
The function of analyst is to obtain a result as near to the true value as
possible by the correct application of the analytical procedure employed.
Quantitative analysis is not simply a case of taking a sample, carrying out a
single determination and then claiming that the value obtained is irrefutable.
It requires a sound knowledge of the chemistry involved, of the possibilities
of interferences from other ions, elements and compounds as well as of the
statistical distribution of values. In chemical analysis, when numerical data
and numerical results are measured with the maximum accuracy that the
instrument, method and analyst are capable of, it has been observed that the
results of successive determination differ among themselves to some extent.
The values obtained may be correct within the possible limits of
measurements. The average value of a series of measurements is accepted as
the most probable value. The reliability of the result depends upon the
magnitude of the difference between the average value and the true value.
(40)
ERRORS AND EVALUATION 41
- -
432101234
-va Error +ve Error
Fig. 1. Magnitude of errors.
3. Errors in Measurements.
(A) Errors in weighing may be due to insensitivity of the balance, wrong
suspension of the ring-riders, placing the weights at edge of the pan and use
of non-calibrated weights etc. Difference in temperature between the object
weighed and the balance also causes error in weighing. It has been estimated
that a difference of 1K causes an error of about 0·15 mg.
(B) Errors in measuring solutions may be due to incorrect use of
glasswares.
4. Gross Errors.
Common gross errors due to carelessness of the analyst are :
(i) Use of numerically incorrect conversion factors.
(ii) Wrong selection of the method.
(iii) Unsuitable storage of samples.
5. Other Errors.
(i) Errors in radiometric analysis.
(ii) Errors in chromatography.
(iii) Photometric errors.
• One way of reducing the effect of constant error is to increase the size
of sample. Consider the following example.
A 0.50 mg of precipitate is lost when it is washed with 200 em3 of wash
liquid. If the precipitate weighs 500 mg, the relative error due to
solubility loss is (0.50/500) x 100% = 0.1%. Loss of same quantity
from 50 mg of precipitate results in a relative error of 0.1%.
• The presence of interference contaminants in the sample affect the
results. High results are observed for the percentage of Cu (in
presence of Fe) because the 12 produced will be a measure of Cu and
Fe in the sample.
• Gross errors affect results that differ markedly from all other data in
a set of replicate measurements.
ACCURACY
Accuracy has been defined as the concordance between a measured value
and the accepted true or most probable value. It follows, therefore, that
systematic errors cause a constant error (either too high or too low) and thus
affect the accuracy of a result.
Measure of Accuracy.
Accuracy can be expressed in terms of absolute and relative error.
Absolute Error. The absolute error (d) of a measured value is a
numerical difference between the true value ()1) and the measured or observed
value (x), i.e., d = )1- x. Absolute error can be positive if)1 > x and negative
if)1 < x. Sometimes absolute error is expressed by the absolute value, without
respect to the sign.
Relative Error. The relative error(e) is the absolute error divided by
the true value. Thus,
PRECISION
Precision may be defined as the concordance of a series of measurements
of the same quantity. It is expressed in terms of numerical difference between
a given experimental value and the mean value of all the experimental
results. Accuracy expresses the correctness of a measurement and
precision the reproducibility of a measurement. Precision always accompanies
accuracy but a high degree of precision does not imply accuracy. Precision
may be expressed as the standard deviation, the coefficient of variation, the
range of the data or as a confidence interval about the mean value.
Example. A substance was known to contain 49·10 ± 0·02% of a constituent
X. The results obtained by two analysts using the same substance and the
same analytical method were as follows :
Analyst 1. % X: 49·01; 49·25; 49·08; 49·14.
The arithmetic mean is 49·12% and the results range from 49·01% to
49·25%.
46 ANALYTICAL CHEMISTRY
MINIMISATION OF ERRORS
Systematic errors can often be materially reduced by one of the following
methods:
1. Calibration of Apparatus and Application of Corrections.
All instruments such as burettes, pipettes, measuring flasks and weights
etc., should be calibrated, and the appropriate corrections applied to the
original measurements. In cases, where an error cannot be eliminated, it is
possible to apply a correction for the effect that it produces. Thus an impurity
in a weighed precipitate may be determined and its weight deduced. Also
recalibrate instruments frequently.
2. Running a Blank Determination.
This consists in carrying out a separate determinatipn, the sample being
omitted, under exactly the same experimental conditions as are employed in
the actual analysis of the sample. The object is to find out the effect of the
impurities introduced through the reagents and vessels and to determine the
excess of standard solution necessary to establish the end point under the
conditions met within the titration of the unknown sample. A large blank
correction is undesirable because the exact value then becomes uncertain and
the precision of the analysis is reduced.
3. Running a Control Determination.
The method consists in carrying out a determination under (as nearly as
possible) identical experimental conditions upon a quantity of a standard
substance which contains the same weight of the constituent as is contained
in the unknown sample. The weight of the constituent x in the unknown can
then be calculated from the relation :
Result found for standard = Weight of constituent in standard
Result found for unknown x
Here it must be pointed out that National Bureau of Standards in
USA supply standard samples of irons, steels, non-ferrous alloys and primary
standards e.g., sodium oxalate, potassium hydrogen phthalate, benzoic acid
and arsenic (III) oxide.
ERRORS AND EVALUATION 47
SIGNIFICANT FIGURES
The term digit denotes anyone of the ten numerals, including zero. A
significant figure is a digit which denotes the amount of the quantity in the
place in which it stands. The digit zero is a significant figure except when it
is the first figure in a number. For example, in the quantities 1·3680 and
1·0082 g the zero is significant but in the quantity 0·0035 kg the zeros are not
significant figures. They serve only to locate the decimal point and can be
omitted by suitable choice of units, i.e., 3·5 g. The first two numbers contain
five significant figures but 0·0035 contains only two significant figures.
Observed quantities should be recorded with one uncertain figure retained.
Thus the weights are determined to the nearest tenth of a milligram.
Consider the weight 2·2756 g. This means that the weight is less than
2·2757 g and more than 2·2755 g. A weight of 2·270 g would signify that the
weight is nearer to 2·270 g than it is to either 2·271 g or 2·269 g.
The digits of a number which are needed to express the precision of the
measurement from which the number was derived are known as significant
figures. The significant figurt'ls in addition and subtraction are based on
absolute uncertainty which is equal to the largest absolute uncertainty of any
individual number. The concept of significant figures in multiplication and
division operations is based on relative uncertainty.
Exercise 2. How many significant figures does each of the following numbers
have?
(a) 2500 (b) 0·004 (c) 5000·0 (d) 0·02765 (e) 4·20 x 1010.
Solution. (a) 4 (b) 1 (c) 5 (d) 4 (e) 3.
or
:r.fi Xi 360
Arithmetic mean = x = - - = - =9
'if;, 40
Exercise 13. If the mean of distribution is 1·46, find the missing frequencies
in the following frequency distribution.
x=0 1 2 3 4 5 Total
f = 46? ? 25 10 5 200
Solution. Suppose the missing frequencies are 11 and f2.
ERRORS AND EVALUATION 53
Calculation of mean.
Xi 0 1 2 3 4 5
Ii 46 fl f2 25 10 5 n =86 +fl +f2
'-
fixi 0 fl 2f2 75 40 25 r.. Ii xi = 140 + fl + 2f2
Since n=200, So 200=86+/\ +/z
or 114 =f1 +f2 ... (2)
"Lfi xi
Also Mean = 1·46 = - -
n
2f2
= 1·46= 140 +f1 + -
200
or 292 = 140 + f1 + 2 f2
152 =f1 +2f2 ... (3)
Solving equations (2) and (3) we get '1 = 76 and '2 = 38.
Mean Deviation.
The mean deviation of a single measurement is the mean of the
deviations of all the individual measurements. It can be calculated by :
(i) Determining the arithmetical mean of the results.
(ii) Calculating the deviation of each measurement from the mean.
(iii) Dividing the sum of the deviations (regardless of sign) by the
number of measurements. The mean or average deviation d is calculated by
- "LIMn-ml
d = -'--~-":'"
n
where, IMn -ml = absolute value of the deviation of the Mnth number from
the mean.
Exercise 14. Analysis of a given quantity gave the following nine values.
Assuming the errors to be random ones calculate mean. 46·62, 46·47, 46·64,
46·76, 46·53, 46·60, 46·71, 46·60, 46·71.
"LMn
Solution. The mean m =-n-
= 46·62 + 46·47 + 46·64 + ... 46·71 = 46.627
9
The difference between anyone of the values and the mean (46·627) is
the deviation Xi of that value from the mean. These deviations regardless of
the sign are: 0·007, 0·157, 0·013, 0·133, 0.:997, 0·027, 0·083, 0·027, 0·083
respectively. The mean or average deviation d is
- "L IMn - m I "LXi 0·007 + 0·157 + 0·013 + ...
d= =-= =0·070
n n 9
where "LXi is the sum of the individual deviation from the mean.
54 ANALYTICAL CHEMISTRY
MEDIAN
Median is the middle result when replicate data are arranged according
to increasing or decreasing values. Or median is a value about which all the
other values are' equally distributed.
Median of an ungrouped data. For an ungrouped data xl> x2' ... x n '
the median is computed as follows :
1. Arrange the data in ascending or descending order of magnitude.
2. Determine the total number of observations (n).
3. If the data, n is odd, then
Median = Value of [ n ; 1
4. If the data, n is even, then
r observation.
.
MedIan =
Value of
(2'n]th observation + Value
2
of (n
2' + 1]th observation
... (4)
Exercise 15. Calculate the median of the following data :
37, 31, 42, 43, 46, 25, 39, 45, 32
Solution. Arrange the data in ascending order.
25, 31, 32, 37, 39, 42, 43, 45, 46
No. of observations = 9 (odd).
..
9
Medi= Value of ( ; 1
= Value of 5
r observation
th observation
=39
Note. If the 'data is an odd numbered set, the median is the middle
value.
Exercise 16. Calculate the median of the data given below,'
15, 24, 21, 13, 12, 16, 25, 18, 10, 22
Solution. Arrange the data in ascending order
10, 12, 13, 15, 16, 18, 21, 22, 24, 25
Here n = 10 (even). Applying equation (4), we get
Value of 5th observation + Value of 6th observation
Me di an=
2
= 16 + 1812 = 17
ERRORS AND EVALUATION 55
. = Value of [N
MedIan + 1J = 1002+ 1 = 50· 5th Item.
-2- .
Median =7
Characteristics of Median.
• Median can be calculated graphically, while mean cannot be.
• Median is not affected by absolute value.
• Median is the only average which is used while dealing with the
qualitative data which cannot be measured quantitatively.
STANDARD DEVIATION
Standard deviation (also called root mean square deviation) cr
measures how closely the data are clustered about the mean. The smaller the
standard deviation, the more closely the data are present about the mean.
S.D. of a single measurement can be obtained by extracting the square root
of the quotient obtained by dividing the sum of the square of the individual
deviations of the number of measurements made.
[ r
/2
22·8 0·5329 = 3.9~43 = 0.8077.
154·5 3·9143
RELIABILITY OF RESULTS
Statistical figures obtained from a set of measurements are of limited
value by themselves. Analysis of the results can be considered in two main
categories.
(1) The reliability ofthe results; and
(2) Comparison of the results with other set of data or true value.
The values should be rejected only when a suitable statistical test has
been applied or when there is an obvious chemical or instru.mental reason
that could justify exclusion of a result. Consider the following example.
Exercise 22. Lead was determined in a sample of dust. Following values
were obtained 4·3, 4·1, 4·0, 3·2 fJg g-l. Should the last value, 3·21lg g-l be
rejected?
Solution. Here Q test may be applied to solve this problem.
1Questionable value - Nearest value 1
Q= Largest value - Smallest value
13 .2 - 4 .0 1 0·8
Q= 4.3-3.2 =TI=0.727
Thus Q calculated is O· 727 but the critical value of Q for a sample size
of four is 0·831. Hence the result 3·21lg g-l should be retained. If three more
measurements were made i.e., 4·3, 4·1,4·0,3·2,4·2, 3·9,4·0 Ilg g-l then,
13 .2 - 3·91 0.7
Q = 4.3 - 3·2 = 1.1 = 0·636.
ERRORS AND EVALUATION 59
REJECTION OF RESULTS
When a series of replicate analysis are performed, one of the results
appears to differ markedly from the rest. It is now to be decided whether to
reject or retain such a result. There is no uniform criteria to do so. Experience
is the best basis for judging the validity of a particular observation as a
statistical test would be. However, following tests are available for
determining whether or not a rejection is justified.
1. Average Deviation.
After the average deviation for a series of measurements have been
obtained, the data may be tested as follows. Reject the doubtful value and
determine the mean and average deviation of the retained value. If x ;;:: 4d
i.e., if the deviation ofthe suspected value from the mean is atleast four times
the average deviation, then the rejection is justified. Consider exercise 14.
Suppose that a tenth value is 46·34. Deviation of this suspected value from
the mean is 46·627 - 46·34 =0·287. Average deviation is 0·070. Since o· 287 is
more than 4 times the average deviation, the rejection is justified.
2. Standard Deviation.
A normal distribution curve is plotted (Fig. 2) for 100 measurements of
a sample. Here frequency of occurrence of a measurement is plotted against
the value of the measurement. The curve shows that upon taking another
measurement there would be 68·26% chance and it would fall between ± 10"
of the mean. The chance would be 95·46% if ± 20" were taken, while ± 30" would
give a 99· 7% chance that the result would fall within this range. Thus a value
of 30" is used as a criteria for rejecting a measurement.
IMPORTANT RELATIONS
• Average deviation of a single measurement is the mean of the
deviations of all the individual measurements, i.e., d = L Bin where
L B is the sum of all deviations from the mean, n is the total number
of values.
• Average deviation of mean CD) is equal to the average deviation of a
single measurement divided by the square root of the number of
measurements made. D = d/--!n where D = average deviation of
mean, n = no. of readings, d = average deviation of a single
measurement.
• Standard deviation is a precise and reliable measure of deviation.
~fU,2
Standard deviation of a single measurement(8) is 8 = -" ~--1 where
n-
L B2 is the sum of squares of all deviations from mean and n is the
total number of measurements.
• Standard deviation of mean(S) is obtained from the standard
deviation (8) by dividing it by the square root of the number of
measurements. S = 81--!n. Usually (28) is taken as a reasonable limit
within which the true value is likely to lie.
o
3
FOOD ANALYSIS
INTRODUCTION
Chemical analysis of food is done to determine the acceptability,
nutritive value, quality, composition and authencity of the food products.
Major steps in the analysis include
(i) to select and prepare samples,
(ii) to perform the assay
(iii) to calculate and interpret the data.
The choice of the analytical method is usually based on the nature of
sample, the specific reason for the analysis and characteristic of the method
itself, such as specificity, speed, accuracy, precision, cost of equipment and
training of personnel.
Sampling and Sample Preparation.
Sampling is the most variable step in the overall analysis of food. It
involves following steps :
1. Selection of Sampling Procedures.
The first requirement in sampling is to clearly define the population to
be sampled. The sampling procedure selected depends on the purpose of
inspection, nature of the product, nature of the test method and the nature of
the population being investigated. Increasing the sample size will increase the
reliability of the final results.
2. Sampling for Attributes or Variables.
Attributes are those characteristics that are present or not present.
Variables are those characteristics that are measured on a continuous scale.
The actual value obtained is compared to the expected value and the deviation
determined.
Sampling plans. These may be single, double or multiple. Selection of
appropriate sampling plan depends on the overall quality of the lot and the
cost of sampling. Multiple sampling plans reduce sampling costs by rejecting
low-quality lots or accepting high-quality lots. Every sampling plan has
inherent risks associated with it. Sampling plans depend on whether the
population is homogeneous or heterogeneous.
Preparation of Food Samples.
Sample preparation depends on the nature of the food and the type of
analysis. Very small samples should not be used, as this leads to moisture loss
during preparation and subsequent handling. Sample preparation involves :
(62)
FOOD ANALYSIS 63
ASH ANALYSIS'
Ash refers to the inorganic residue remaining after complete oxidation
of organic matter in a food stuff. Determination of ash content is a part of the
proximate analysis for nutritional evaluation. Ashing is the first step in the
preparation of a food sample for specific elemental analysis.
Ash Content in Foods. The ash content of most fresh food rarely
exceed 5% while dried beef may contain 11·6% ash (wet weight basis).
FOOD ANALYSIS 67
Eggs, roast beef, fish fillet, hamburger contain 1·0, 3·0, 1·3 and 1·1
percent ash content respectively. Fats, oils and shortenings have 0·0 to 4·09%
ash, starch contains 0·3% and wheat germ 4·3% ash. Meat, poultry and sea
foods contain 0·7 to 1·3% ash.
Calculations.
nt A h (d
-to S ry b aSIS
.) = Wt. after ashing - tare wt. of crucible
.
Wt. of sample x dry matter coefficIent
where dry matter coefficient = % solids/100.
For example, if corn meal is 87% dry matter, the dry matter coefficient
would be 0·87. If ash is calculated in wet-weight basis, delete the dry matter
coefficient. If moisture was determined in the same crucible prior to ashing,
the denominator becomes (dry sample wt. - tared crucible wt.).
However, if carbon is still present after the initial incineration, several
drops of HN03 or H 20 should be added and the sample re-ashed. If the carbon
persists, such as with high-sugar samples then
• Suspend the ash in water.
• Filter through ashless filter paper because this residue tends to form a
glaze.
• Dry the filtrate.
• Re-ash the paper and dried filtrate in muffle furnace.
Note that:
• Glycerin, alcohol and hydrogen will accelerate ashing.
• Samples such as jellies will spatter and can be mixed with cotton
wool.
• Ashing of cereals can be accelerated by adding alcoholic solution of
magnesium acetate. An appropriate blank determination is necessary.
• High fat samples should be extracted either by using the crude fat
determination procedure or by burning off prior to closing the muflle
furnace .
• Use crucible cover to prevent spattering of salt-rich foods.
2. Wet Ashing.
Wet ashing or wet digestion is a procedure for dissolving minerals and
oxidising substances with high fat content (meats etc.) using oxidising
agents.
Procedure.
• A 1 g dried, powdered sample is placed in 150 mL Griffin beaker.
• Add 10 mL HN03 and allow it to soak overnight.
• Add 3 mL of 60% HCI04 (Place a beaker under pipette tip during
transport) and heat on a hot plate upto 350°C until frothing stops and
RN0 3 is almost evaporated.
• Continue boiling until perchloric reaction occurs. Place watch glass on
beaker. Sample should be colourless. Do not evaporate the liquid to
dryness.
• Cool the beaker. Wash watch glass with minimum. deionised water.
Add 10 mL 50% HCI.
• Transfer to 50 mL volumetric flask and dilute with deionised water.
• Wash the hood pre cautiously after last sample.
Preparation for iron analysis in a meat. Boil 2 g sample in 30 mL
HN0 3 at 350·C on hot plate until 10 mL remain. Add 10 mL of 60% HCI04
FOOD ANALYSIS 69
and continue boiling until copious fumes occurs. Place watch glass in beaker.
Dilute to 100 mL in a volumetric flask following oxidation.
Advantages of Wet Ashing.
• Minerals usually remain in solution.
• There is little or no loss from volatilization because of lower
temperature.
• The oxidation time is short and requires a hot plate, hood, tongs and
safety equipments.
Disadvantages.
• Wet ashing requires constant operation attention.
• Corrosive reagents are necessary.
• Only a few samples can be handled at one time.
• Perchloric acid reacts with iron (in the assay for iron) to form ferrous
perchlorate. It forms an insoluble complex with o-phenanthrolene in
the procedure. It should not interfere with atomic absorption
spectrophotometry.
3. Modified Dry-wet Ash Oxidation.
• Evaporate moist samples (25 to 50 mL) at 100°C overnight or in a
microwave oven.
• Heat on a hot plate until smoking ceases.
• Ash at 525°C for 3 hours.
• Cool and wet with deionised distilled water and 3 mL HN03 .
• Dry and incinerate at 525°C for 1 to 2 hours.
• Weigh sample after cooling in desiccator.
4. Low Temperature Plasma Ashing.
Principle. Low temperature plasma ashing refers to a specific type of
dry ashing method whereby foods are oxidised in a partial vacuum by nascent
oxygen formed by radiofrequency electromagnetic field generator. Highly
volatile elements are preserved by this method.
Instrumentation. The equipment consists of a glass system with a
variable number of chambers for samples that may be evacuated by a vacuum
pump.
Procedure.
The ground material is inserted into individual glass boats which are
inserted into separate glass chamber. The chambers are sealed and a vacuum
is applied. A small flow of 02 or air is introduced into the system maintaining
minimum vacuum. The frequency generator is then activated at a frequency
less than 14 mHz and adjusted by the amount of wattage applied (50 to 200
watts) to control incineration. The progress of ashing may be observed
through the chambers.
Advantages.
(i) There is less chance of losing trace elements by volatilization.
(ii) The low temperature (I50°C) used with plasma ashers keeps the
microscopic and crystalline structures unaltered.
70 ANALYTICAL CHEMISTRY
ANALYSIS OF PROTEIN
Protein is a common ingredient of all food materials. Analysis of protein
is required to know
FOOD ANALYSIS 71
Digestion.
Place accurately weighed sample in Kjeldahl flask. Add H 2S04 and
catalyst (Hg, Cu or Se or Se02 : CuS04 in 3: 1 ratio) for complete breakdown
of organic matter. During digestion, protein N is liberated to form NH! ions.
H 2S0 4 oxidises organic matter and combines with ammonium formed. Carbon
and hydrogen are converted to CO 2 and H20.
H 2S04 ,d
Protein(N) ) (NH4)2S04
catalyst
~~. '''; ... protein contain 16% N, so the conversion factor is 6·25 (1~~ = 6.25).
~~ 'f~
Alternate Procedures.
In place of distillation and titration with acid, NHa or N can be
quantitated by :
(i) Nesslerisation.
NH40H + 2HgI2 + 2KI + 3KOH ~ NH4Hg2I + 7KI + 4H20
Red-orange, 440nm
FOOD ANALYSIS 73
ANALYSIS OF FAT
Fats are esters of fatty acids with triacylglycerols. To analyse food for fat
contents accurately, it is necessary to know the general compositions of the
lipids in the foods and the physical and chemical properties of lipids and
foods. An accurate analysis of lipids in foods is important for nutritional
labelling, to determine whether the food meets the standard of identity and
is uniform.
Percent lipids (wet weight basis) of some foods are : Lard, oils
(-100), butter and margarine (80), salad (40-70), almonds (54), walnuts (64),
soybeans (18), milk (4·3), grains (3-5), germ (10), bacon (65), eggs (12), apples
(0·4), avocados (26·4) and asparagus (0·2).
Methods.
Methods used for fat analysis are :
1. Solvent extraction methods (continuous solvent extraction,
semicontinuous solvent extraction and discontinuous solvent
extraction methods).
2. Non-solvent wet extraction methods (Babcock method, Gerber
method, detergent method).
3. Refractive index method.
4. Instrumental methods.
5. Calorimetric method.
MOJONNIER METHOD FOR THE ANALYSIS OF MILK FAT
1. Discontinuous Solvent Extraction Method.
Principle. Fat is extracted with a mixture of ethyl ether and petroleum
ether. Extracted fat is dried to a constant weight and expressed as percent fat
by weight.
Preparation of Sample. Weigh or measure the test portion of a
homogeneous milk sample. If lumps of cream do not disperse, warm the
sample to 38°C and cool the warmed sample to 20°.
First Extraction.
• Weigh 10 g milk into a Mojonnier fat extraction flask.
• Add 1·5 mL NH4 0H and shake vigorously. NH40H neutralises acidic
sample and dissolves protein.
• Add 10 mL of 95% ethanol to prevent gel formation and shake for 1
minute ..
• Add 25 mL ethyl ether to dissolve the lipid and shake well.
• Add 25 mL petroleum ether and shake. It removes moisture from the
ethyl ether extract and dissolves more nonpolar lipid.
• Centrifuge for 30 s at 600 rpm.
• Decant ether solution from the flask into the previously weighed
Mojonnier fat dish.
Second Extraction.
• Add 5 mL of 95% ethanol and shake vigorously for 15 s.
• Add 15 mL ethyl ether and shake for 60 s.
FOOD ANALYSIS 75
+
Digest starch with amyloglucosidase for 16 hours.
+
Centrifuge and filter to separate soluble and insoluble fibre.
i
+ +
Filtrate. Pellet and filter retentate.
+
Collect soluble polysaccharides ppt.
+
Wash with ethanol, acetone and
with ethanol or by dialysing dry overnight under vacuum (40°C).
filtrate and freeze-drying the
dialysate. ~
Hydrolyse cellulose with
+
Hydrolyse soluble fibre with
12N H 2S04 (lhr).
This method for measuring fibre provides the most accurate estimate
of fibre over a wide range of foods.
ANALYSIS OF CARBOHYDRATES
Carbohydrates play an important role in human nutritions as energy
reserves. These are classified as monosaccharides (simple sugars),
oligo saccharides and polysaccharides.
Conversion factor from glucose (mol. wt. 180) to starch (mol. wt.
162).
10 parts of glucose are equal to 9 parts of starch.
. 162 9
·. ConverSIOn from glucose to starch = 180 = 10 = 0·9
• Wash the residue with hot water and collect the washing in the same
beaker.
• Add to the solution in the beaker 0·5 mL of bromocresol green
indicator and then NH 40H till the colour changes to blue. Adjust the
pH at 4·5 by adding acetic acid until the colour changes to distinct
green.
• Filter the solution. Wash with hot water. Collect the washing in the
same beaker and heat to boil.
• Add saturated ammonium oxalate solution dropwise till the
precipitate appears and then add excess solution. Heat to boil.
• Digest for 3 hours. Decant the solution through ashless filter paper.
• Pour 20 mL of hot water on the precipitate and again decant the clear
solution.
• Dissolve any precipitate remaining on the filter paper by washing
with hot dilute HCI into the original beaker. Wash the filter paper
with hot water.
• Reprecipitate by adding NH40H and a little ammonium oxalate
solution.
• Digest for 3 hours. Filter through the same filter paper. Wash with
hot water until it is chloride free.
• Perforate the apex of the filter cone. Wash precipitate into the beaker.
Wash the filter paper with hot dilute H 2S04 and titrate with standard
KMn04 solution at 70·C.
Calculation.
2·8 NY
Calcium (as CaO) percent by mass = M where
N = Normality of standard KM:n04 solution.
V = Volume (mL) of the standard KM:n04 solution used for titration.
M = mass (g) of the material taken for the test analysis.
Calcium can also be calculated as follows :
Ca mg/100 g
Titre x 0·2 x Total volume of ash solution x 100
=~~--~~~~~~~~~~~==~~==~~~~~-
Volume taken for estimation x wt. of sample taken for ashing
ANALYSIS OF PHOSPHORUS
Principle.
Phosphorus reacts with molybdic acid to form a phosphomolybdate
complex. It is then reduced with aminonaphthol sulphonic acid to the complex
molybdenum blue which is measured colorimetrically.
Reagents. 1. Molybdate solution. Dissolve 25g of ammonium
molybdate in 400 mL of water. Add 500 mL of 10 N H 2S04 and make up the
volume to 1 L with water.
2. Aminonaphthol sulphonic acid solution. Dissolve in water 0·5 g
1-amino-2-naphthol-4-sulphonic acid, 30 g NaHSO g and 6 g Na2S0g. Make up
the volume to 250 mL.
FOOD ANALYSIS 83
Principle.
Sodium in solution is atomised into an oxyhydrogen flame. Excited
sodium atoms emit radiations at specific wavelengths. The amount of
radiation emitted is proportional to the concentration of sodium in solution.
Reagents.
1. NaCI stock solution. Dissolve 2·5418 g of NaCI in 1 L of distilled
water in volumetric flask (1 mL = 1 mg Na).
. 2. Standard solution. Measure 10 mL of stock standard solution
(containing 10 mg of Na) and 5 mL of HCI into alL volumetric flask and
make up the volume with water. This solution contains 10 ppm of Na.
In order to compensate for minute interference produced by other ions
in the det~rmination of sodium, precautions identical to the case in potassium
should be taken.
Standard Curve. Draw standard curve between concentration and
percent luminosity of sodium as in potassium.
Procedure.
Dilute an aliquot of plant extract so that it contains less than 10 ppm of
Na. Add sufficient HCI so that the concentration of acid is the same as that
in the standard solution. Atomise the diluted extract in a calibrated flame
photometer with the wavelength dial set at 589 nm and the transmittance at
100% for the top standard solution of sodium.
Calculation.
N~/100g=
_ ppm found from the standard curve x Volume made up x Dilution x 100
- Weight of sample x 1000
FOOD ADULTERATION
Food adulteration implies the addition of foreign matter or removal of
certain valuable ingradient from it. A food article shall be deemed to be
adulterated if
FOOD ANALYSIS 85
Experimental Technique.
Extraction of the food sample. Extract the sample (1 L) in a
separatory funnel and acidify to pH 2 with H 2S04 . Add 60 mL of acetone and
shake. Then extract with 60 mL dichloromethane and hexane (1 : 1) by
shaking for 2 minutes. Water layer is transferred in the original sample
container and organic phase is collected in Kudema Danish flask. Extraction
is repeated twice with 50 mL each of dichloromethane and hexane and the
solvent is treated as above. Solvent extract volume is then reduced to 0·5 mL
under reduced pressure. Few microlitre of this concentrate is injected to
HPLC for the analysis of organophosphates.
Standard Conditions for Operating HPLC.
• The column of the HPLC is packed with 5 )lm C-8 bonded-phase
particles.
• The width and height dimensions of column are 4·5 x 250 mm.
• Flow rate of eluting solvent methanol (67 mL CH30H + 33 mL H 20)
is maintained at 2 mL per minute.
• A UV detector is attached to the instrument.
Process. The organophosphates are separated by using the gradient
elution system of mobile phase and
the elute monitored by UV detector Peak identification
at 254 nm. Chromatograms showing 1. Methyl parathion
2. Ciodrin
the peaks of various
3. Parathion
organophosphates in food sample so 4. Dyfonate
obtained are shown in Fig. 2. 5. Diazinon
Calculations are based on 6.EPN
peak height measurement of the 3 7. Ronnel
sample and the standard for 8. Trithion
determining the concentration of 1 8
4
species. 2 5 6
Analysis of Chlorinated
Insecticides in Milk by HPLC. 7
Automated HPLC pesticide analyser
can be used for the analysis of
chlorinated insecticides in milk by
injecting raw milk onto a short silica
precolumn where the fat was
retained and the pesticide eluted
with hexane. The less polar fat
eluting organochlorines such as DDT, Time,min
DDE and a-BHC were stored at the Fig. 2. Chromatograms of various
organophosphates in food sample.
top of a longer analytical column.
Pesticides such as f3-BHC, y-BHC and dieldrin which eluted later were
resolved from each other by the precolumn and passed directly into an
electron capture detector by means of a computer controlled
pneumatically-operated switching value for determining their concentration
in the milk.
92 ANALYTICAL CHEMISTRY
HPTLC.
High-performance thin layer chromatography is a new technique in
which TLC plates are coated with smaller, more uniform particles of
controlled porosity. This permits better detection of organochlorines in food
stuffs.
o
4
TYPES OF WATER POLLUTION
WATER POLLUTION
Water is a vital natural resource which is essential for multiplicity of
purposes. About 80% of the earth surface (i.e., 80% of the total 50,000 million
hectares area) is covered by water. Out of the estimated 1011 million km 3 of
the total water present on earth, only 33400 m 3 of water is available for
drinking, agriculture, domestic, power generation, industrial consumption,
transportation and waste disposal. In the chemical process industrial water
is used as a reaction medium, a solvent, a scrubbing medium and a heat
transfer agent. As a source of life for man, plants and other forms of life, it
cannot be replaced by any other solvent. But today water resources (rivers,
lakes, ponds, streams) are founding it more and more difficult to escape from
pollution.
The signs of water pollution are obvious to all such as bad taste of
drinking water, offensive odours from water bodies, unchecked growth of
aquatic weeds in water, decrease in number of fish in fresh water, oil and
grease floating on water surfaces. These factors disturb the normal uses of
water for public water supply, recreation and aesthetics, aquatic organisms
and wild life, agriculture and industry.
The problem is-what would happen when the water pollutants
gathered in the sea water reach the threshold levels of toxicity at which
phytoplanktons cease to function? Also what would happen when dissolved
oxygen would be depleted by biological oxygen demand? The plant and animal
life would disappear, because of death. Bacterial decomposition shifts from
aerobic to anaerobic conditions. The products of metabolism will change.
Under aerobic conditions, the products so formed are not so toxic.
C ~ CO2 , S ~ H 2S0 4, N ~ NH3 + HN03, P ~ H3P04
Under anaerobic conditions, the products tend to be odouriferous,
turbid and likely to be more toxic.
The main worry is-How is this situation to be faced? What arguments
should be proposed to the concerned pullution problems? Industrial societies
do indulge a lot of water pollutants making it foul, unpleasant, turbid and
unfit for drinking purposes.
(95)
96 ANALYTICAL CHEMISTRY
o o
II /H .. II
R-C-O- R-N:"H/N~ R-O-P-O-
I
Carboxylate Phenoxide Aliphatic and aromatic o
group group amino group Phosphate group
Humic compounds. Humic substances occur naturally during the
decomposition of vegetation. They occur as deposits in soil, marsh sediments
and decayed vegetables. These non-degradable materials are classified into
humin, humic acid and fulvic acid on the basis of solubility.
The properties of water are influenced by the humic substances due
to their acid-base, adsorptive and complexing properties. The soluble fulvic
acid has an effect on the properties of water, while the insoluble humin and
humic acids affect water quality through exchanges of cations, organic
materials etc., with water.
Humic substances are high molecular weight polyelectrolytic macro-
molecules. They consist of a carbon skeleton with a high degree of aromatic
character and bulky functional groups containing oxygen. Humic substances
have elementary composition: C 45 to 55%, H 3 to 6%, 0 30 to 45%, N 1 to
5%, S 0 to 1%. They may also consist of protein-like materials and carbo-
hydrate fraction. The decomposition products of humic acid are :
OH H-C=O COOH
@yoH
H3CO~OCH3 OH
HoJ§lOH
Catechol Syringaldehyde 3, 5-Dihydroxy benzoic acid
98 ANALYTICAL CHEMISTRY
lQr
C'-...o
o O./M
I
solids and bacterial contamination. It is the source of water for wells and
springs i.e., the recommended source of rural domestic use. It is replenished
by precipitation through rain, snow, sleet and haiL
The major portion of water (about 1650 cubic kms) which goes to earth
crust is retained by its upper layer as soil moisture. Only 500 cubic kms
percolate down to the ground water deposits. About 120 cubic kms of water
applied to agricultural fields moves down to ground water table and about 50
cubic kms of surface flow also end up as ground water. Thus a total of 670
cubic kms of fresh water enters the ground annually.
Factors Affecting Ground Water Pollution.
Today human activities are constantly adding numerous wastes to
ground water reservoirs at an alarming rate. Ground water contamination is
generally irreversible i.e., once it is contaminated, it is difficult to restore the
original water quality of the aquifer. The extent of water pollution depends
on the following factors :
1. Rain fall pattern.
2. Depth of water table.
3. Distance from the source of contamination.
4. Soil properties such as texture, structure and filtration rate.
Sources of Contamination in Ground Water.
Underground sources of drinking water, especially in outskirts of larger
cities are highly polluted. Ground water is threatened with pollution from the
following sources.
1. Domestic wastes.
2. Industrial effluents.
3. Agricultural discharges.
4. Run off from urban areas.
5. Soluble effluents.
6. Waste water treatment lagoons.
7. Mine spills.
8. Seepage pits.
9. Earthen septic tanks.
10. Urban and rural garbages.
11. Refuse dumps.
12. Barnyard manures.
13. Transport accidents.
14. Leaching of pollutants.
Protecting Ground Water from Pollution.
Following steps are suggested for the protection of ground water :
1. The contaminant sources should be carefully surveyed.
2. Location of industrial and municipal disposal sites should be decided
keeping in view the ground water levels and flow pattern in the area.
3. In case of toxic industrial effluents, steps should be taken for
predisposal treatment by the industry itself.
TYPES OF WATER POLLUTION 101
from land. Good quality water is inadequate even for the normal living and
is getting polluted due to numerous discharges. Major Indian rivers such as
Ganga, Yamuna, Tapti, Narmada, Sone, Chambal, Daha, Damodar,
Krishna, Cauvery, Brahmaputra, Mahi are severely polluted. Table 1
gives an idea of the quantum of discharge in major rivers of the world.
MARINE POLLUTION
Marine pollution is defined as the discharge of waste substances into the
sea resulting in harm to living resources, hazards to human health, hindrance
to fishery and impairment of quality for use of sea water. Marine pollution is
associated with the changes in physical, chemical and biological conditions of
the sea water. This water is also unfit for human consumption and industrial
purposes because of high salt content. Chemically it is a solution of 0·5 m NaCI
and 0·005 m MgS0 4 containing traces of all conceivable matter in the universe.
Like the land, the air, the rivers, the lakes, our seas and oceans also suffer from
pollution. The oceans are deemed to be our last and endless dust bin.
Sources of Oil Pollutants in Marine Water.
At present annual consumption of refined petroleum products is more
than 25 billion barrels (800 billion gallons). Such large scale consumption is
associated with some losses in water. The total annual influx of petroleum
hydrocarbons into the ocean is about 10 million metric tonnes by tankers. Oil
tankers are also responsible for oil spill in seas.
• Cargo tanker washings at sea. About 3 million tonnes of oil are
added annually to the sea by using sea water as ballast for empty
tankers. It is mixed into water when the ballast gets dumped and
carries residual oil from the tanker. It can coat 20 feet wide and half
an inch deep oil layer for 8700 miles in a beach.
• Import oil losses. Collisions in port contribute one million tonne of
oil in sea water annually.
• Bilge pumping at sea. The dumping of bilge contents by ships adds
nearly 5,00,000 tonnes of oil per year in sea water, while total influx
of oil into ocean has been 5 to 10 million tonnes annually.
• Recent oil based technology and vessel accidents near sea shore add
to extensive marine oil pollution. It is estimated that two million
tonnes of used lubricating oil are added every year in coastal waters.
Maritime accidents due to collision, fire, explosion or grounding
also result in oil release in water.
• International discharge of oily wastes from tank washings and
accidental spillages pollute the sea water severely. From Indra Dock
basin alone, more than 90,000 litres of waste oil was collected in 1984.
A recent report shows that about 20 billion tonnes of wastes per year
from industries, homes, farms and municipalities end up in sea.
106 ANALYTICAL CHEMISTRY
• Oil leakage from 20,000 miles of pipe lines which cross water ways
may undergo corrosion, cracks or punctures and would lead to oil
pollution in sea water.
• The blowout of wells, disposal of drilling muds, accidental damages
to offshore, drilling rigs add to oil pollution in water.
• Oily wastes from oil fields or refineries near the coast produce
problems in coastal water. Shipping operations at the coastal belt add
light diesel oil and crude petroleum to sea water.
• Today refined petroleum products meet more than 60% of the world's
energy requirement. Annually about 25 billion barrels (800 billion
gallons) of petroleum products are consumed on a global basis. Such
large consumption results in some losses which severely pollute the
marine environment.
• The hazardous crude petroleum is a complex mixture of paraffins
(25%), cyclo paraffins (20%), aromatics (5%) and several toxic organic
compounds. The total influx of petroleum hydrocarbons into oceanic
water is 10 million metric tonnes per year generated due to
transportation activities which result in acute oil pollution in sea.
• Oil spills mixed with urban sewage, silt, plastics, pesticides and
insiduous toxic compounds are pervasive and complex the pollution
problems in sea. Toxic substances abound oil spilling into the oceans
at 10 times the rate of natural seeps, while lead being deposited on
soil and in waterways 100 times. Cadmium being released 40 times,
radionuclides many times and acidity of precipitation over millions of
square kilometres increasing 10-fold, are ultimately added to oceans.
Transportation of Oil-Spill in Marine Environment.
Oil is transported mainly by wind currents, waves and tides in marine
ecosystem. The dispersal of oil and its persistence in sea water are affected
by the kind of oil, chemical composition, specific gravity, temperature and the
state in which it is discharged into the sea. The distributed oil is then
subjected to natural processes like evaporation, dissolution, emulsification,
oxidation, sedimentation and uptake by marine organisms.
Due to these interactions, the volatile components of oil, such as, low
boiling aromatics (benzene, phenanthrene), paraffins (n-hexane, 2, 3-dimethyl
hexane), cycloparaffins (cyclohexane, 2, 3-bicyclo octane) readily evaporate.
Highly soluble aromatics can be removed by dissolution. Less resistant
paraffins get readily degraded by bacteria. Heavy oil residues disintegrate as
tar lumps or tar bills washed into the beaches.
Major Accidents.
World's ocean system has been subjected to 9,151 cases of oil spills
between 1967 and 2003. A total of 5,432,000 tonnes of oil has been split into
the ocean.
• Emulsified oil may reach the bottom of sea damaging aquatic animals
and plants. Oil may serve as a concentration medium for fat soluble
poisons like pesticides. These poisons may seriously accumulate in
aquatic biota posing deleterious effects.
3. Effects of Oil Pollution on Man.
Oil pollution in marine water also affects man critically in the following
ways:
• Paraffins, like methane and ethane are asphyxiants, i.e., they cause
acute suffocation. Some paraffins are central nervous system
depressants. Liquid paraffins can remove oil from exposed skin
causing dermatitis and pneumonia in lung tissues.
• Breathing higher concentrations of unsaturated cycloparaffins can
result in irritation and anaesthesia. Aromatic thiophenes,
benzothiophenes and mercaptans damage liver and kidneys.
• Crude oil contains sulphur compounds, small amount of nitrogen,
little olefins, metals like iron, nickel and vanadium. These are
extremely lethal. Carbonyl sulphide is dangerously poisonous. It is
also toxic to rats at 2900 ppm. Actually it dissociates to hydrogen
sulphide which acts on central nervous system resulting in death
mainly from respiratory paralysis.
4. Effects of Oil Pollution on Birds.
Ironically the oil that drives millions of vehicles around the world,
sometimes drives countless birds and animals to a most cruel death.
• Birds are specially vulnerable to damage from oil coating. The spilled
oil break down their natural insulating oils and waxes which shield
the birds from water. Ultimately they lose insulation, start shivering
and may freeze to death in winter. About 25,000 birds died in Torry
Canyon incident.
• Oil spilling in sea water causes abnormally low body temperature in
birds resulting in hypothermia. Nearly 150 rare species of·bald
eagles also became victims, when they ingested oil during Exxon
Valdez accident, scavenging an oily sea bird c~rasses.
• About 1000 sea otters died when their fur became saturated with oil
by losing insulation. Several birds developed respiratory ailments
because volatile components of oil weakened membranes in their
lungs. Others suffered from liver and kidney damage caused by
ingesting oil while cleaning their coats.
In addition to these severe effects, oil and tar coated beaches are
anaesthetic.
5. Burning the oil slick. Burning oil slicks. on the open seas is
comparatively less successful because the more volatile light fractions
evaporate quickly from the oil slick. The water also removes heat much faster
and the process leads to extensive air pollution.
6. Using chemical additives. Chemical additives can be used to
solidify oil from water surface. Mechanical methods involving the use of
additives and skimmers have been satisfactorily used to remove oil slicks.
7. Floating booms. Now floating booms are in common use in
harbours and areas where transfer of petroleum products occur.
S. Improved navigation aids. Hazard warning instrumentation and
offshore drilling operations can effectively protect the water from oil pollution.
Development of submerged pyramid shaped canopies to cover the drill hole
area, use of mechanical or pneumatic (air curtain) walls around the drill site
and physical encapsulation of drill and its hole can be suggested to escape
from the pollution hazards of sea water.
9. Using micro-organism in oil clean up. Microbes can be deployed
as voracious scavengers removing all kinds of oil pollutants. Various varieties
of Pseudomonas can consume esteric compounds and hydrocarbons from the
oil. The gene secreting enzymes are found on plasmids, small and
semi-autonomous rings of DNA. Some microbes can ingest dispersed oil
droplets and subsequently deposit them as faecal pellets.
o
5
SOURCES OF WATER POLLUTION
INTRODUCTION
Water is essential for the survival of any form of life. Today water
resources have been the most exploited natural system since man strode the
earth. Pollution of water bodies is increasing tremendously due to rapid
population growth, industrial proliferations, urbanisation, increasing living
standards and wide spheres of human activities. Time is, perhaps not too far
when pure and clean water, particularly in densely populated and
industrialised water scarce areas may be inadequate for maintaining the
normal living standards. Water pollution is mainly caused by natural
processes (volcanic eruptions, decomposed vegetable and animal matter) and
anthropogenic sources such as domestic, agricultural, mining and industrial
etc.
(111)
112 ANALYTICAL CHEMISTRY
residues along with organic debris from the remains of harvested crops are
trapped by run-off water causing pollution problems in receiving waters. The
agricultural run-off is considerably rich in NPK nutrients, organic matter and
pesticides. While NO) and PO~- cause eutrophication, pesticides have been
reported to get bioaccumulated and biomagnified through food chains
resulting in secondary poisoning to man, animals and predatory birds.
3. Radioactive Pollutants.
Living organisms are continuously exposed to a variety of radiation
sources illustrated below :
Natural sources of radiation are:
• Solar rays, gamma rays, cosmic rays.
• Electromagnetic radiations
• Environmental and particulate radiations.
• Radionuclides in the earth crust, and
• Internal radiations.
Anthropogenic sources. Recently man made sources have begun to
add large doses of radiation to the existing natural radioactive pollution to
which our bodies have got accustomed with several ill effects. Major sources
of radioactive pollutants are :
• Nuclear power plants and nuclear reactors.
• Radioactive materials used in nuclear weapons.
• Mining and processing of ores to produce radio-isotopes.
• Radioactive fall out from nuclear bombs.
• Emission from the industrial use of nuclear energy.
• Leakage from underground nuclear detonations.
• Use of radio-isotopes in medicine, industry, agriculture and research
operations.
• Major radio-pollution in water is caused by uranium, radium and
plutonium.
Once the radionuclides find access into the aquatic environment, they
enter the ecocycling processes and entirely disrupt the metabolic pathways.
As compared to organic poisons, injurious effects of radionuclides are
exceedingly high.
4. Thermal Pollutants.
Thermal pollution involves warming up of an aquatic ecosystem to the
point where desirable organisms are adversely affected. Following sources
contribute to thermal pollution.
• The operation of nuclear power plants.
• Nuclear fuel processing units.
• Coal-fired thermal power plants.
• Industrial effluents.
• Domestic sewage etc.
Release of heat from vario~s sources is :
Thermal power plants 75%, effluents 6%, sewage 9% and biological
activities 4 %. Hydroelectric power stations have negative effects of thermal
loading. Discharge of unutilised heat adversely affect the aquatic biota.
SOURCES OF WATER POLLUTION 113
5. Industrial Effluents.
Industrial activities generate a variety of waste products which are
generally discharged into water streams. The nature of industrial wastes
depends upon the industrial processes in which these are generated. The
pollutants usually associated with industrial effiuents are organic matter,
inorganic dissolved salts, suspended solids, fertilizing materials, thermal
constituents in the form of heat, micro-organisms and pathogens (Table 1).
Contd. ...
116 ANALYTICAL CHEMISTRY
Suspended Other
BOD
COD solids Consti- Pollution
Industry pH (mgIL)
(mgIL) (SS) tuents Aspects
(20°C)
(mgIL) (mgIL)
o
6
WATER POLLUTANTS AND
THEIR EFFECTS
INTRODUCTION
Water, the most abundant and natural resource, is extremely essential
for the survival of all organisms. But today clean water has become a precious
commodity and its quality is threatened by numerous water pollutants.
... (2)
Reaction (2) proceeds slowly at pH below 3·5 but can be catalysed by iron
bacterium Thiobacillus ferroxidant, while at pH 4·5 bacterium Metallogenium
accelerates the reaction. Ferrobacillus ferro-oxidants also favour the reaction.
(117)
118 ANALYTICAL CHEMISTRY
It has been estimated that 0·5 x 10-3 M H 2S04 decreases the pH of stream
water upto 3·0. An estimate showed that 4 x 109 kg. of H 2S04 enters into
United States streams every year and its 60% originates in abandoned mines
causing massive fish kill. The water beds contaminated with acid mine
eftluents get coated with yellow deposit of amorphous semigelatinous ferric
hydroxide. Large fluxes of strong acids have been able to overwhelm the
buffering capacity of water causing drastic drop in pH. However, carbonate
rocks and bicarbonate ions can maintain the natural buffer system in water
as follows:
CaC0 3 + H 2S04 ~ Ca2+ + SO~- + H 20 + CO2
But as the pH increases, the ferric hydroxide in water covers the
particles of carbonate rock with impermeable membrane which inhibits
further neutralization of sulphuric acid.
(v) Other chemicals. The inorganic metals and organic species
interact with each other forming toxic organo-metallic compounds. The
interaction of these compounds play a major role in redox equilibria,
neutralization, colloid formation and micro-organism's mediated reactions in
aquatic ecosystem. Various industrial products, plastics and synthetic fibres
when consigned to incinerators emit highly toxic vapours and particulates in
air. They ultimately enter the water systems with rain fall. Similarly,
discarded plastic materials, poly vinyl chloride (PVC) produce toxic
hydrochloric acid. These chemicals are highly irritating even in low
concentrations and extremely lethal in higher doses to aquatic organisms.
TOXIC METALS
Toxic metals are added in aquatic system from industrial processes,
domestic sewage discharge, street dust, land run off and fossil fuel burning.
Traces of heavy metals such as Hg, Cd, Pb, As, Co, Mn, Fe and Cr have been
identified as deleterious to aquatic ecosystem and human health. Hard water
contains carbonate and sulphate ions which combine with lead forming
insoluble protective coating of lead carbonate and lead sulphate.
The major sources of lead poisoning have' been the steel and paint
industries. About 80% of lead retained in the body enters the bone affecting
the metabolic activities. Another major source of lead pollution is the burning
of gasoline containing the anti knock additive lead tetra ethyl, Pb(C2H 5)4' In
fish, Hg is present as dimethyl mercury, (CH3)2Hg, a toxic substance which
is known to concentrate in the food chain. This compound is synthesised from
elementary mercury by certain anaerobic bacteria living at the bottom of
lakes and rivers. Dimethyl mercury appears to concentrate in brain tissues
and remain their for long periods of time. Symptoms of Hg-poisoning arise
when concentration of (C2H5)2Hg in the brain reaches 5 ppm. 12 ppm is
highly fatal. Manganese also enters the water bodies through domestic
wastes, industrial eftluents and dry cell batteries. Its chronic exposure leads
to neurological disorders. Selenium content of most drinking waters is found
as 10 ppb. Its excessive amount creates' carcinogenic effect on man and
animals.
WATER POLLUTANTS AND THEIR EFFECTS 119
surface run off than that of sewage discharge deteriorate the water quality
broadly.
Water bodies get easily flooded. Sediments make the rivers,
streams, channles and reservoirs to overflow. They also change the flow rates
and depths of water systems as well as destroy the life of reservoirs. However,
expensive treatments are needed to counteract these effects.
5. SYNTHETIC DETERGENTS
Detergents include ingredients like surfactants, builders and additives
such as anticorrosive sodium silicate, stabilizers and soil suspending
carboxymethyl cellulose etc. The surfactant is a surface active agent which
dissolves partly in water and partly in organic solvent because of its dual
hydrocarbon and polar character. Alkyl benzene sulphonates (ABS) are
considered as surfactants. They decrease the surface tension of water so that
they penetrate the surface and interstices of the object being cleaned. Oils and
greases also absorb the hydrophobic end of surfactants.
The builder is usually a sodium phosphate (polyphosphate) of the type
Na5P301O or Na4P207 acting as a sequestering agent. The builders form
chelated complexes with calcium and magnesium ions and react with water
forming alkaline solutions. Both the surfactants and builders of detergents
create serious pollution problems in water. Additives may consist of
enzymes, perfumes and bleaching agents. The active components of
detergents are double headed amphipathic molecules usually consisting of a
bulky water insoluble hydrocarbon chain to which is attached a highly polar
group. The hydrolytic enzymes used in detergents act by solubilizing the
biological stains by degrading the large molecules into smaller water soluble
compounds which do not adhere to fabric. Structure of alkyl benzene
sulphonate is as follows :
CHa CHa CHa CHa
I I
16
I
CH a-C-CH2 -C-CH2 -C-CH 2 -C-H
HI HI H
1#
I
SOaNa
Alkyl benzene sulphonate (ABS) showed remarkable resistance to
biodegradation (the so called hard detergents) and has been subsequently
replaced by a new surfactant called linear alkyl sulphonate (LAS) which is of
comparable cost and cleaning potential but is rapidly biodegradable. At
present, one of the most pressing problems is not the effects of surfactant
itself but the release of polyphosphate builders into natural waters.
These detergent builders contribute significantly to the phosphate
present in sewage effiuents. The main builder Na5P301O undergoes fast
biodegradation and hydrolyse to orthophosphate.
P30~O + 2H20 ~ 2HPO~- + H 2P04
124 ANALYTICAL CHEMISTRY
dL =-k L
dt 1
2N02" + O2 ~ 2NO a
The oxidation process speeds up towards the end of the first stage and
slows down again as ammonia is oxidised. Ammonia exerts a very high oxygen
demand requiring over 4·5 times its own weight of oxygen for complete
oxidation. Thus, if nitrification is allowed to occur in the receiving stream, a
further decrease in the oxygen resource will be observed.
Example. Calculate the BOD of a water sample which contains one gm of
urea for every 100 litres of water. The reaction between urea and oxygen is
NH2 CONH2 +402 ~ CO2 +2N03+2W+H2 0
WATER POLLUTANTS AND THEIR EFFECTS 127
2·13 x 103 mg 02 .
Thus BOD = 100litres = 21·3 mg Oz/htre
7. DISEASE-CAUSING AGENTS
Water has been a potential carrier of toxic inorganic and organic
materials, non-biodegradable matters and pathogenic microbes which can
endanger health and life. The potable water contaminated with municipal
sewage and industrial wastes is the root cause of dangerous diseases in living
beings.
Effects of Pathogens.
• Parasites are considerably harmful for man. Egg of nematodes, hook
worms and tape worms occur mostly in crude sewage. When such
sewages are discharged into water bodies without treatment,
contamination of water occurs with resultant danger to man and
aquatic life.
• The enteric diseases are transmitted mainly by drinking
contaminated water or swallowing food. The pathogens most
frequently transmitted through water cause infections of intestinal
tract like typhoid, paratyphoid, amoebic dysentry, cholera, polio and
infectious hepatitis.
• The disease causing organisms present in the faeces of infected people
get ultimately mixed with the water supply spreading chronic
diseases.
• Intestinal helminthes, i.e., Ascaris lumbricoides and
Trichuristrichiura are also water borne. Entamoeba histolytica is the
casual agent which causes internal amoebiasis and several
extra-intestinal diseases.
• The guinea worm, responsible for dracontiasis, is transmitted through
open village wells and ponds infested with the copepod intermediate
host.
• Several human diseases whose epidemics recurrently detrimate
human population, get contaminated by water.
Infections Transmitted from Man to Man.
• Typhoid fever, bacillary dysentery, cholera, poliomyelitis and hepatitis
occur from contaminated water and food through inhalation.
128 ANALYTICAL CHEMISTRY
8. RADIOACTIVE POLLUTANTS
Radioactivity is a physical type of environmental pollution caused by the
increase in natural back ground radiation emerging from the activities of
man.
Sources of Radioactive Pollutants in Water.
• Use of radioactive materials in nuclear weapons.
• From nuclear power plants and nuclear reactors.
• Mining and processing ores to produce radio-isotopes.
• Radioactive fall out from nuclear bombs.
• Use of radio-isotopes in medicine, industry, agriculture and research
operations.
• Uranium ore which contains 2 to 5 pounds of U 30 S per tonne.
Large quantities of uranium tailing's are produced during extraction
which find their way into water. Once these radionuclides find access into the
aquatic environment, they entirely disrupt the metabolic pathways.
WATER POLLUTANTS AND THEIR EFFECTS 129
9. PLANT NUTRIENTS
Plant nutrients constitute an important limiting factor for plant growth.
Nitrogen and phosphorus are the main nutrient species which enter in to
fresh and marine systems changing oligotrophic water to intensely
productive eutrophic conditions. According to Wetzel each phosphorus
molecule promotes the incorporation of 7 molecules of nitrogen and 40
molecules of carbon in aquatic algae. These nutrients ultimately tend to
accumulate in ground water.
Why do small addition of Nand P promotes algal growth?
Continued addition of phosphorus into water may lead to nitrogen depletion
in the photic zone. Under these conditions, blue green algae fIxes sufficient
nitrogen to main eutrophic conditions. Phosphates and essential inorganic
nutrients, i.e., iron and manganese form highly insoluble compounds and are
effectively lost to the lake sediments. Carbonates and associated cations lead
to artifIcial oligotrophy. Many pathogens begin to grow on products in water
bodies under anaerobic conditions and spread fatal water borne diseases.
EUTROPHICATION
Eutrophication is a natural process, derived from the Greek word
eutrophos meaning well nourished or enriched. This enrichment leads to
other slow processes referred to as natural aging of lakes. C.H. Weber
described eutrophication as nutrient rich conditions used to determine the
flora of German peat bogs as eutrophe, mesotrophe and oligotrophe. It is a
phenomenon through which a nutrient rich bog in a shallow depression
changes to leached bog deficient in nutrients. Eutrophication escalates rapidly,
however when normally high amounts of nutrients from fertilizers, domestic
and industrial wastes, urban drainage, detergents, animal wastes and
sediments enter water streams. Eutrophication was fIrst studied in lake
located in north western Ontario.
Lake Erie in USA. Lake Erie in USA is an excellent example of
eutrophication due to human's activities. Nearly 80 tonnes of phosphates were
added in this lake daily in 1965. Each 400 g of P0 4 encourages 350 tonnes of
algal slime. Due to extensive algal growth, the lake appeared as big mounds
producing unpleasant odour, clogging pipes and interfering with fIshing and
navigation.
Types of Eutrophication.
(i) Natural Eutrophication. The process of lake aging characterised
by nutrient enrichment is called natural eutrophication. During this process
oligotrophic lake is converted into an eutrophic lake. It permits the production
of phytoplankton, algal blooms and aquatic vegetation including water
hyacinth, aquatic weeds, water fern and water lettuce which in turn provide
ample food for herbivorous zooplankton and fIsh.
(ii) Cultural Eutrophication. This process is generally speeded up by
human activities, which are responsible for the addition of 80% nitrogen and
WATER POLLUTANTS AND THEIR EFFECTS 131
75% phosphorus to lakes and streams. Lake Mendota and Lake Washington
have undergone rapid eutrophication due to man's activities. In India,
recreational value of Kashmir lakes is reduced while Nainital lake is
undergoing a rapid eutrophication as a result of sewage, domestic waste and
detergent addition.
Effects of Eutrophication.
Eutrophication causes several physical, chemical and biological changes
which considerably deteriorate the water quality.
• During eutrophication, algal bloom release toxic chemicals which kill
fish, birds and other aquatic animals causing the water to sink.
• Decomposition of algal bloom leads to oxygen depletion in water. Thus
with a high CO2 level and poor oxygen supply, aquatic organisms
begin to die and the clean water turns into a stinking drain.
• When 02 level falls to zero (anaerobic zone), some bacteria drive
oxygen through reduction of nitrates. On complete exhaustion of
nitrate, oxygen as a last resort be obtained by reduction of sulphate
yielding hydrogen sulphide causing foul smell and putrefied taste of
water.
• Many pathogenic microbes, viruses, protozoa and bacteria etc. grow
on sewage products under anaerobic conditions. It results into spread
of fatal water-borne diseases such as polio, dysentery, diarrhoea,
typhoid and viral hepatitis.
• Algae and diatoms attain high degree of dominance due to over
fertilization. Algae and rooted weeds interfere with the hydroelectric
power, clog the filters, retard the water flow and affect water quality
and water works.
• Macrophytes, particularly Hydrilla, Potamogeton, Ceratophyllum and
Myriophyllum assume high population densities making near shore
and shallow regions unsuited for any purpose.
• During eutrophication midge Chironomous plumosus and tubificid
worms develop extremely high populations creating anaesthetic and
economic problems in water.
" Phytoplankton communities are most sensitive to eutrophication.
Investigations on lake Wisconsin showed that their population in
eutrophic lake is smaller as compared to oligotrophic water.
• The lake undergoing eutrophication may become oxygen deficient
destroying fish habitats leading to the elimination of several desirable
aquatic species in water.
• Prolonged eutrophic conditions lead to dystrophic state. The lake
receiving huge amounts of organic matter from alloethonous source
are called dystrophic. These lakes contain bog flora and high amounts
of humic acid while planktonic productivity is very low.
• In India, Dal, Nagin, Loktak lake and Hussain sagar are seriously
chocked by aquatic weeds affecting fisheries production, utility for
aquatic flora and aesthetic value.
132 ANALYTICAL CHEMISTRY
Control of Eutrophication.
• The waste water must be treated before its discharge into water
streams.
• Recycling of nutrients can be checked through harvest.
• Eutrophication can be minimized by removing nitrogen and
phosphorus at the source, division of nutrient rich waters from the
receiving bodies and dilution of these elements. In lake Tahoe in
California and lake Washington in Seattle, total removal of sewage
effluents produced marked reversal of eutrophication.
• Algal blooms should be removed upon their death and decomposition.
• Algal food web should be disrupted to stimulate bacterial
multiplication.
• Algal growth can be controlled by limiting the dissolved nutrients.
The most suitable, feasible and effective method involves the use of
chemicals to precipitate additional phosphorus. Such precipitants
include alum, lime, iron and sodium aluminate.
• Physico-chemical methods can be adopted to remove dissolved
nutrients. For example, phosphorus can be removed by precipitation
and nitrogen by nitrification or denitrification, electrodialysis, reverse
osmosis and ion exchange methods.
10. THERMAL POLLUTANTS IN WATER
Thermal pollution involves warming up of an aquatic ecosystem to the
point where desirable organisms are adversely affected. Coal fired power
plants, electric power plants, chemical industries as well as nuclear energy
plants discharge their heated effluents into nearby lakes or rivers. A coal fired
power plant at 40% efficiency generates 16·7 joules of waste heat for every
41·8 joules of fuel burnt. All these processes increase the temperature of water
by lOoC to 15°C. A single 100 MW power plant may use one half million
gallons of cooling water per minute. The heated waters have reduced amount
of dissolved oxygen content which results into killing of marine life.
Effects of Thermal Pollution on Aquatic Ecosystem.
• Reduction in dissolved oxygen. DO content is decreased in the
warm water. Normal, biological reduction of DO level of the
atmospherically unreplenished lower layer of water may give rise to
anaerobic conditions leading to fish mortality.
• Direct fish mortality. There appears to be particular temperature
ranges that are tolerated by fish and other related species. For
example, lethal temperature for trout is 77°F, for yellow perch 88°F
and for carp it is 85°F. Thus thermal death of fish may occur due to
the action of heat on nervous system, inactivation of enzymes and
coagulation of cell protoplasm.
• Interference with reproduction. The increased temperature
triggers deposition of eggs by female. Other activities like nest
building, spawning, hatching and migration etc. get disturbed by
rising temperature.
WATER POLLUTANTS AND THEIR EFFECTS 133
to over 24000 million pounds (1975). Of these 6000 million pounds were
organochlorine insecticides which persist in the environment.
Classification of Pesticides.
Herbicides are used to kill weeds. Fungicides are toxic to fungi.
Insecticides are meant to kill insects. Rodenticides are used against rodents
(cats and mice). Nematicides inhibit nematodes.
Molluscicides are used to kill molluscs (snails and slugs).
Piscicides are used to control undesirable fish species.
Synthetic pesticides are more effective that penetrate into plant
tissues. Chlorophenoxy acid compounds, i.e., 2, 4-D, 2, 4, 5-T and MCPA are
known as hormone weed killer. 2,4, 5-T and picloram were used as defoliants
by US forces in Vietnam in 1968-69. Since weeds are not pests like insects,
fungi or bacteria so a broader term biocide can be used to include herbicides.
Structural Classes of Insecticides. Insecticides are :
(i) Chlorinated hydrocarbons
(ii) Organophosphates
(iii) Carbamates
(iv) Chlorophenoxyacids.
Among them chlorinated hydrocarbons and organophosphates serve
mainly as insecticides. Structure and permissible limits of pesticides are
illustrated in Table 4.
Dieldrin
o
Cl
Cl
Chlordane Effective against O·Olllg/L
Cl Cl
termites. Potential
Cl:¢CrCl carcinogen. Use
Cl2 I banned in USA in
1975.
Cl.
Cl
Contd . ...
136 ANALYTICAL CHEMISTRY
Trade Permissi-
Formula Uses
name hIe limit
Lindane Control of cotton 0·01 ~g/L
CI
insects and rice stem
Cl*Cl H
H
H
H
borer.
CI H Cl
CI
Hexa chlorocyclohexane
Cl:¢Q
I I Cl2
use suspended due
to potential
carcinogenicity.
CI
CI CI
o ~Cl
CH Cl I 2 2
mite, also used as
Zoocide. Precautions
CI to be taken to avoid
skin contact during
CI application.
Metho- Popular DDT 0·03 ~g/L
xychlor CCla .
substitute,
reasonably
CHao-@-t-@-OCHa
biodegradable,
H low-toxicity to
mammals.
Contd. ...
WATER POLLUTANTS AND THEIR EFFECTS 137
Trade Permissi-
Formula Uses
name hIe limit
II. Organophosphates
Malathion Control some O·ll!g/L
S 0 pests of fruits and
II II vegetable-little
(CHaOh-P-S-CH-C-OC2H5
hazard to
I ~
CH 2-C-OC2H5
mammals.
02 N -@-
0 0 - PII (OC 2H5)2
mosquito control,
also
spectrum
broad
0, o-Diethyl-o-p-nitrophenyl phosphorothionate
insecticide for fruit
and vegetable
pests.
Methyl Control of plant O·OOll!g/L
S
parathion
02 N -@-
0 0 - PII (OCHah
pests; ranks second
in
consumption
pesticide
in
USA.
III. Carbamates
Carbaryl Used on 0·005l!g/L
(Sevin) O-C-NH-CHa
crops-cotton,
oo~
00
forage, fruits and
vegetables;
and
lawn
garden
I-Naphthyl-N-methyl carbamate
insecticide; low
toxicity to
mammals.
Baygon Control of flies, O·OOll!g/L
<Q{-II
0
o O-C-NH-CHa
mosquitoes,
and cockroaches.
ants
O-CH(CHah
2-Isopropyl-N-methyl carbamate
Iv. Chlorophenoxy acids
2,4-D Herbicide-control 100llg/L
CI
of broad leave
weeds, aquatic,
Cl-@-O-CH,-COOH vegetation;
military defoliant
2, 4-Dichlorophenoxy acetic acid (May contain
highly toxic TCDD
as impurity)
Contd. ...
138 ANALYTICAL CHEMISTRY
Trade Permissi-
Formula Uses
name hIe limit
2,4,5-T Weed control, 100llg/L
Cl
military defoliant.
Cl-!fi)-o-CH 2-COOH
CI
about 25% get entered to oceans. From 1 ppt (10-6 ppm) of DDT in sea water
there is more than one million biomagnification in birds of prey (10 ppm). A
man stands at the top of every food chain. The average level of DDT is
estimated at 5 to 10 ppm in human tissues. DDT and its metabolites are not
readily degraded in the environment. The stability of organochlorines, coupled
with their high solubility in fatty tissues, causes them to concentrate in the
food chain.
DDT-The Most Widespread Molecule.
DDT was first synthesized by Othmar Zeidler of Germany in 1874 and
rediscovered by Swiss entomologist Paul Mueller in 1939 who won Noble
Prize for uncovering its powerful insecticidal properties. In World War II,
Allies used it as delousing agent to replace pyrethrum. It showed spectacular
success in controlling malaria, yellow fever and typhus. Due to continuous
use, 12 insect species developed immunity in 1948 which rose to 165 by 1967
and the number is still increasing rapidly. DDT is partially soluble in water
and carried by air and rain. Disquietingly large residues of DDT are
appearing every where.
Bioaccumulation of Pesticides.
Pesticides present in water get concentrated in tissues of plants and
animals. Aquatic animals like protozoa-Blepherisma concentrate DDT by
about 3000 folds in 12 hours. The fish Gambusia affinis accumulates DDT
to 25000 folds in a few hours.
Factors. Bioaccumulation of pesticides depends on :
• Lipid and water partition coefficient.
• Nature of aquatic organisms.
• Environmental conditions, i.e., temperature, pH, salinity.
• Concentration and duration of exposure of pesticide.
Organochlorines accumulate to a greater extent than organophosphorus
. and· carbamates. They are hydrophobic and concentrate 1000 times over the
levels in water.
Measurement of Bio-accumulation. The extent of bio-accumulation
can be measured by (i) Model ecosystem (direct method). (ii) By determining
octanol per water partition coefficient of the pesticide (indirect method).
Accumulation of DDT in Food Chain.
DDT from air and water accumulates in animal tissues, soil and humus
in concentrations thousands of times higher than it is found in water through
food chain. DDT is a persistent chemical. Once introduced into the
environment, it keeps circulating for many years.
The cycle of food chain consists of plankton in sea water which contains
0·04 ppm of DDT. The clams that consume plankton concentrate it ten times,
i.e., they contain 0·4 ppm DDT. Fish feeding on clams contain 2·4 ppm DDT
while its level goes upto 76 ppm in birds which eat fishes. In man, DDT
content rose to 15 ppm which feed on fishes in India, 2·2 ppm in England and
11 ppm in United States (Table 5).
140 ANALYTICAL CHEMISTRY
The main worry about DDT contamination in man is that since DDT
stores in the fatty tissues and there is always a risk of its being massively
released into body fluids when the fat cells are metabolised during starvation.
It is thought to dissolve in the fatty membrane surrounding nerve fibres and
interfere with ion transport in and out of the nerve membrane. DDT affects
in a remarkable fashion. If there are 10 ppm DDT in plant tissues, it would
rise to 100 ppm in cattle and 1000 ppm in man. Oysters living in sea water
with 0·001 ppm of DDT show highest DDT level upto 700 ppm in their bodies.
Biological Magnification or Biological Amplification.
Aquatic plants and animals can accumulate certain pesticides in their
body tissues in greater concentration than can in water. For instance, DDT
not only circulates in its original form in aquatic organisms but its
concentration continuously increases in successive trophic levels in a food
chain. Woodwell et al., reported that DDT level in estuary water is only
0·00005 ppm but the food chain consisting of plankton (0·04 ppm), minnow
(0·94 ppm), heron, (3·5 ppm), fish (5 ppm) increased the amount of DDT in
fish eating birds (mergansers and geills) to 75·5 ppm. Organochlorine
insecticides have the greatest magnification because they have a high affinity
for lipids and are quite persistent ecopoisons. The degree of magnification
of insecticides is usually proportional to their persistence and inversely
related to their solubility in water.
Biodegradation of Pesticides.
The biodegradation of pesticides varies depending on the chemical
nature of the pesticide itself. In soil a pesticide may be transported into
various sectors of the environment by different physical processes. It may be :
(i) absorbed by soil, (ii) leached by rain water,
(iii) picked up by plants and animals (iv) carried away by wind.
The processes that actually play important roles in reducing total
amount of pesticide residues are those mediated by micro-organisms,
actinomycetes, fungi, bacteria, plants, animals and sunlight.
WATER POLLUTANTS AND THEIR EFFECTS 141
Cl-@+@-cn CCl3
DDT DDE
CI CI
Cl@CI
CI Cl
CI
--
-Hel
CIO
CI
Cl
Cl
• Endrin is extremely toxic to fishes like blue gills and rainbow trout
with LC 50 values being 0·006 and 0·007 ppm respectively, while DDT
is much less toxic to these fishes with LC 50 values being 0·016 to
0·018 ppm. respectively.
• Even the raptorial hawks and falcons are severely affected by DDT
which causes breeding failure and death in them.
• Recent studies have proved that extremely low quantities of pesticides
which enter the aquatic environment can affect productivity of
organisms, kill eggs and larvae of clams and oysters, influence the
behavior of fishes such as schooling and feeding and deteriorate water
quality.
• Pesticides induce changes in blood chemistry and enzymatic functions
of marine invertebrates. They reduce their backbonic collagen content
and indirectly interfere with food chain.
• Since slight concentrations of organochlorine pesticides affect
reproduction in fishes, there is every possibility that these pesticidal
pollutants may adversely affect local fishery.
• Varieties of crustacea that make up the most valuable marine harvest
are badly affected by pesticides. The oysters might be susceptible
because of their tendency to concentrate and store trace chemicals
from the surrounding environment.
• Pesticides-induced mortality pattern of marine molluscs, crustaceans
and teleosts are also measurably related to various physico-chemical
environmental parameters like concentration of pesticide and
duration of its exposure.
• PCBs are reported to be concentrated in the food chain of marine
ecosystems.
• Pesticidal pollutants ranked second to all other industrial wastes.
Effects on Grains.
• Many vegetables, fruits, rice, cereals and grains such as wheat, maize,
grams and barley have been found to be contaminated with significant
amounts of DDT and BHC. The level of DDT residue vary from 1·6 to
17·4 ppm in wheat, 0·8 to 16·4 ppm in rice, 3 to 17 ppm in pulses, 3
to 19 ppm in ground nuts, upto 5 ppm in vegetables and 68·5 ppm in
potatoes.
Effects of Pesticides on Soil.
Pesticides not only pose potential hazards to man, animal, livestock, wild
life and fish but they seriously interfere with the desired use of soil and water
as follows:
• The most dangerous characteristic of pesticides, particularly
organochlorines, is their long persistence in the soil which causes
numerous adverse effects on grain quality.
• Even the accepted dosages of pesticides create deleterious effects in
the long run in the soiL
• Pesticide retained in the soil also concentrate in some crops,
vegetables, cereals and fruits which taint them to such an extent that
they are not usable.
WATER POLLUTANTS AND THEIR EFFECTS 145
15. FERTILIZERS
Modem agricultural techniques have introduced NPK fertilizers into
water systems. The excess of fertilizers not consumed by crops are washed
away from land by rain water and pollute water bodies. These fertilizers are
generally retained in the soil by crops but due to negligence in their
application, some nitrates are liable to be washed out during rainy season. If
phosphate and nitrate concentration exceeds one part and thirty parts per
hundred million part of water respectively, it can cause eutrophication and
the whole stretch of water may become chocked. The less DO content may
result in the death of aquatic biota.
o
7
ANALYSIS OF WATER POLLUTANTS
(148)
ANALYSIS OF WATER POLLUTANTS 149
Preconcentration Methods.
1. Carbon Adsorption Method. A large volume of collected water, say
1000 gallons, is allowed to pass over activated charcoal. The organic matter
adsorbed on activated carbon is extracted from the dried charcoal with CHC13
followed by alcohol. The solvents are evaporated and the weights of the
extracts are expressed as Ilg/L of carbon-chloroform extract (CCE) and
carbon-alcohol extract (CAE). Organic pollutants, such as phenols and oils,
which impart tastes and odour to water are usually present in CCE.
Compounds such as phenols, fulvic acid, pesticides, polycyclic aromatic
hydrocarbons, carboxylic acids, sulphonic acids etc., can be characterised from
these extracts by making use of a number of separation techniques. The carbon
adsorption method, though, widely been used due to its speed, efficiency and
simplicity, but it has a number of disadvantages. For example:
• There is a possibility of a chemical reaction on the surface of activated
charcoal.
• During evaporation of solvents, some organic material may also
evaporate.
2. Freeze Concentration Method. In this process, the water sample
is allowed to freeze, as a result of which pure crystals of ice are formed and
water soluble impurities are left behind in the liquid phase of much reduced
volume. The process of freezing, also minimizes the loss of volatile
constituents in the sample.
3. Solvent Extraction Method. The liquid extraction is a technique
in which a substance dissolved in one phase (usually an aqueous phase) is
more or less completely transferred to the second phase, essentially
immiscible with the first, i.e., an organic liquid such as benzene, ether,
chloroform etc. Thus in solvent extraction, organi~ matter soluble in water is
separated by using a water insoluble organic solvent. Parts per billion
(llgiL) amounts of various metals such as Co (II), Fe (III), Pb (II), Zn (II),
Ni (II) etc., in saline water can be determined by solvent extraction method
in which metal complexes of these metals with ammonium pyrrolidine
dithiocarbamate are first extracted with methyl isobutyl ketone solvent and
ANALYSIS OF WATER POLLUTANTS 151
TURBIDITY
Turbidity in water is due to suspended matter like silt, clay, organic or
inorganic matter, planktons and other-micro-organisms. It is an expression of
optical property (Tyndall effect) of water which causes light to be
scattered and absorbed rather than transmitted. Turbidity of suspension
may vary due to different optical properties, refractive indices and particle
size of matter.
MEASUREMENT
1.·By Jackson Candle Turbidimeter.
Although standard method but Jackson model permits measurement of
turbidity from 25 to 1000 JU. Lower turbidity values (100 NTU) can be
measured by Nephelometer.
2. By Nephelometer.
Standard Turbid Suspension. Dissolve 1 g of hydrazine sulphate in
100 mL of distilled water. Also dissolve 10 g of hexamethylene tetramine in
100 mL of water. Mix 5 mL of each solution in 100 mL volumetric flask and
dilute it with distilled water to the mark. This suspension gives a turbidity of
400 NTU.
Method .
• Set the nephelometer at 100 using 40 NTU (10 mL ofthe above stock
solution in 100 mL water) standard suspension. Every percent of the
scale will be equal to 0·4 NTU.
• Shake the sample thoroughly. Take the sample in nephelometer tube
and read the value on the scale. If the sample has turbidity more than
40 NTU, dilute it, so that turbidity can be read on the same scale.
Calculation.
Turbidity (NTU) = Nephelometer reading x 0·4 x Dilution factor
CONDUCTIVITY
Conductivity of water varies directly with the temperature and is
proportional to its dissolved mineral matter content. Specific conductance
can be measured by conductivity meter using dip-type cell. The instrument
and cell are calibrated with 0·005 M KCI solution (conductivity = 654
Il mho cm- l ).
1 A
Specific conductance, K=-'-
R I
where R is the observed resistance of a column of electrolyte, 1 cm long and
cross-section area A cm2 .
Electrical Conductivity (EC).
EC is a measure of water's capacity to convey electric current. EC is
directly proportional to area (a) and inversely proportional to length (I).
EC oc all or EC = K all
where K is proportionality constant called specific conductance.
ANALYSIS OF WATER POLLUTANTS 153
TOTAL SOLIDS
The term solid refers to the matter that remains as residue upon
evaporation. Total solids include both dissolved solids and suspended solids.
Potable waters contain mineral matters in dissolved conditions whereas
industrial eftluents and sewage contain huge amount of undissolved matter.
Suspended Solids.
500 mL of a sample is taken exactly in a volumetric flask and allowed
to filter through a dried and weighed Gooch crucible containing an asbestos
mat. The suspended solids retained in the crucible are washed with distilled
water to remove chloride. The crucible is finally dried, cooled in a desiccator
and weighed. The increase in the weight of the crucible is equivalent to the
154 ANALYTICAL CHEMISTRY
suspended impurities present. The total solid contents of 500 mL sample can
also be calculated by evaporating it to dryness on a steam bath and drying at
about 100-HODC in an oven for about one hour. From this, substract the
dissolved solids to get the quantity of suspended solids.
Dissolved Solids.
Filter 500 mL sample in a Gooch crucible to free it from suspended
matter and evaporate to about 50 mL. It should be noted that any deposit on
the walls of the beaker due to evaporation of water should not touch the flame
of the burner. The 50 mL liquid is carefully transferred to a weighed platinum
dish with the help of a policeman and wash with distilled water. Evaporate
the solution to dryness on steam bath and dry the dish in an oven at about
lOO-HODC for about an hour. Cool it in a desiccator and weigh.
6
Weight of solids x ~go = ppm. dissolved solids.
Results. Disposal of industrial effluents and sewage contribute
suspended solids to the water bodies. The lSI has specified a maximum limit
of 30 mg/L for suspended solids discharged into river. Solid determination is
particularly useful in the analysis of sewage and other waste waters.
ACIDITY
Acidity is a measure of the effects of combination of compounds and
conditions in water. It is the power of water to neutralise hydroxyl ions and
is expressed in terms of calcium carbonate. Water attain acidity from
industrial effluents, acid mine drainage, pickling liquors and from humic acid.
Measurement of Acidity by Titration Method.
Principle.
Acidity of water can be determined by titration with sodium hydroxide
solution. The amount of sodium hydroxide required for the sample (pH below
4·5) to reach pH 4·5 (methyl orange end point) is a measure of mineral acidity
while the amount of sodium hydroxide to reach pH 8·3 (phenolphthalein end
point) is a measure of total acidity. Samples containing acidic wastes (pH
below 4·5) correspond to both mineral and CO2 acidity.
Procedure.
Mineral Acidity.
Take 50 mL or suitable dechlorinated aliquot of the sample in a 250 mL
conical flask. Add 2 drops of methyl orange indicator and titrate with 0·02 N
NaOH solution till faint orange colour.
Total Acidity at Boiling Temperature.
To 50 mL of the sample, add 5 drops of phenolphthalein indicator. Heat
to boil for 2 minutes. Titrate with 0·02 N NaOH solution to pink colour.
Calculation.
A .dit C CO IL =mL titrant (NaOH) x 1 x 1000
CI Y as a 3' mg mL sample taken for titration
ANALYSIS OF WATER POLLUTANTS 155
ALKALINITY
Alkalinity of water is due to the presence of carbonate, bicarbonate and
hydroxide ions.
Determination of Alkalinity by TItrimetric Method.
Principle. Alkalinity is determined by titration with 0-02 N H 2S04
using methyl orange and phenolphthalein as indicators.
Reactions.
2CaCOg + H 2S04 ~ CaS04 + Ca(HCOg)2
Ca(HCOg)2 + H 2S04 ~ CaS04 + 2C02i + 2H20
Ca(OH)2 + H 2S04 ~ CaS04 + 2H20
Reagents. :
(i) Sulphuric acid 0-02 N. Dilute 20 mL of IN-H2S04 to 1000 mL with
distilled water.
(ii) Sodium carbonate solution. Dissolve 13-25 g Na2COg in distilled
water to 250 mL.
(iii) Phenolphthalein indicator solution. Dissolve 500 mg
phenolphthalein in 50 mL alcohol and 50 mL distilled water. Add 0-02 N
NaOH solution till light pink colour appears.
Procedure. Take 50 mL of the sample in a 250 mL conical flask. Add
2 drops of phenolphthalein indicator. Titrate the pink colour with
0-02 N H 2S04 till it becomes colourless. If the sample contains waste waters,
then remove the suspended matter by filtration or centrifugation and then
determine alkalinity_
Calculations. Phenolphthalein alkalinity (as CaCOg) mg/L
mL 0-02 N H 2S04 for phenolphthalein end point x 1 x 1000
=
mL sample taken for titration
156 ANALYTICAL CHEMISTRY
HARDNESS
Hardness indicates water quality, mainly in terms of Ca2+ and M~+
expressed as CaCOa.
Temporary hardness is due to the presence of bicarbonates of Ca2+
and M~+. It may be removed by boiling the water.
Permanent hardness is due to the presence of sulphates and chlorides
ofCa2+ and M~+.
Complexometric Titration.
Principle. During titration with EDTA (Na2H2Y)' Ca2+ first reacts to
form stable Cay2- followed by M~+ to give Mgy2- complex (indicator!
wine-red) releasing free indicator (blue). The colour changes from wine-red to
blue at the end point.
ANALYSIS OF WATER POLLUTANTS 157
Reactions.
Ca2+ + H2y2- ~ Cay2- + 2H+
M~+ + H2y2- ~ Mgy2- + 2H+
MgD-(red) + H 2y2- ~ Mgy2- + HD-(blue) + H+
Procedure for Ca2+ and Mg2+ Hardness.
Take 20 to 50 mL sample of hard water in a conical flask. In presence
of organic matter in waste waters, add 2 to 4 drops of 30% H 20 2.
• Adjust the pH to 8, boil for 15 minutes and cool.
• Add 5 mL buffer of pH 10 (142 mL conc. NH3 + 17·5 g NH4CI diluted
to 250 mL with deionised water) and warm the solution.
• Add two drops of 1·4% Eriochrome black T in triethanolamine.
• Titrate with 0·01 M EDTA solution till the colour changes from,
wine-red to blue.
Calculations.
1 mL 0·01 M EDTA == 1·0 mg CaC03
H d (fL) Vol. of EDTA used x 0·01 M x 1000
ar ness mg = mL of Sample taken
This gives total Ca2+ and M~+.
Procedure for Ca2+ Hardness.
• To a 100 mL sample add 20% KOH solution to bring the pH to 12 and
precipitate M~+ as Mg(OH)2'
• Add 5 to 10 drops of calcon carboxylic acid indicator (0·4% in
methanol).
• Titrate with 0·05 M EDTA under magnetic stirring till the colour
changes from wine-red to pale blue.
Alternatively, add drops of mureoxide indicator (0·1 g stirred with 2·5
mL deionised water and filtered). Titrate with 0·05 M EDTA solution till the
colour changes from orange to violet.
Calculation. 1 mL of 0·01 M EDTA == 0·4008 mg Ca.
CHLORIDE
Chloride in drinking water is harmless if present below 250 ppm but its
higher content harms metallic pipes and crops.
Mohr's Method.
Chloride is determined by titration with AgN0 3 solution using ~Cr04
as an indicator. The end point is indicated by the appearance of reddish tinge.
The method is valid for 0·15 to 10 mg cr and electrical conductivity of water
is greater than 1 dS/m at 25°C.
Procedure.
• Take 100 mL sample in 250 mL conical flask. Adjust pH from 7 to 10
with H 2S04 or NaOH (0·5 g Na2B407 will keep the pH at 9).
• Add 1 mL of 5% ~Cr04 indicator with stirring.
158 ANALYTICAL CHEMISTRY
SULPHATE
Sulphate usually occurs in natural waters. Mine drainage wastes also
contain high content of sulphate by virtue of pyrite oxidation. The presence
of Na2S04 and MgS04 in drinking water beyond the prescribed limits may
cause cathartic action.
Gravimetric Method.
In gravimetric procedure sulphate is precipitated as BaS04 in acidic
(HCI) medium using BaCl2 solution. The precipitate of BaS04 is digested,
filtered and washed with hot water to remove Cl- ions, ignited and weighed.
Volumetric Method.
100 mL of the sample water is taken in a conical flask and add 10 mL
of benzidine hydrochloride solution (solution of benzidine in dilute HCl
containing 4 g. of the diamine base per litre of the solution). The precipitate
of benzidine sulphate is filtered and washed free of acid with minimum
amount of distilled water. The precipitate and filter paper are taken in a
conical flask and 50 mL of distilled water are added to it. Now few drops of
phenolphthalein are also added.The conical flask is well shaken to dissolve
the precipitate and the solution so obtained is titrated against N17 NaOH
until pink colour is obtained at the end point.
Calculation.
Sulphate (as N~S04) ppm = No. ofmL ofNI7 NaOH x 100.
Na2S04 ppm can be converted into CaC03 ppm by multiplying with a
factor of 0·705.
FLUORIDE
Fluoride occurs in all natural water supplies and in chemical wastes
from industries. Fluorides, if present in small concentration up to 1 ppm, are
generally considered to be beneficial in water. Excessive fluorides in drinking
water may cause mottling of teeth or dental fluorosis, which results in
discolouration of enamel, chipping of teeth in children in severe cases. Bone
fluorosis or crippling effects are observed in case the concentration of
fluorides exceeds 1·0 mg/L.
ANALYSIS OF WATER POLLUTANTS 159
Spectrophotometric Method.
Alizarin-S Visual Method. Fluoride reacts with Zr Alizarin-S lake to
form colourless ZrF~- and the dye. The colour of the dye lake becomes
progressively weak with increase in amount of F-.
~r~o
~S03Na+
o
Zr-Alizarin red-S lake
To a 100 mL sample add 1 drop of NaAs0 2 solution (5g/L) to remove
residual CI, if any. Add 5 mL acid-zirconyl-alizarin reagent (300 mg
ZrOC1 2 ·8H20/50 mL + 70 mg alizarin red S/50 mL + 800 mL of 1·5 N
HCl-1.2 N H 2S04 made up to 1 litre). Mix thoroughly and compare the
samples and standards after 1 hour.
Spadns Method.
Spadns method is preferred to the Alizarin visual method as the latter
takes 1 hour for colour development. [Spadns is Sodium 2-(para sulphoph-
enylazo)-l, 8-dihydroxy-3, 6 naphthalene disulphonatel.
The reaction rate between F" and Zr02+ is influenced greatly by the
acidity of the reaction mixture. By increasing the proportion of acid in the
reagent, the reaction can be made practically instantaneous.
Procedure.
• Prepare a series of standard solutions ofF-, i.e., 0·5,1·0 and 2·0 mg/L.
• Add 1 drop of NaAs0 2 solution (0·5%) to remove any residual chlorine.
• Dilute sample to 50 mL. Add 5 mL of SPADNS (1·9 giL) and 5 mL of
zirconyl acid reagent (0·26 g ZrOCI2·8H20 and 700 mL of 50% cone.
HClIL).
Mix well and read absorbance at 570 nm against standard immediately.
Calculate the concentration of fluorine from a standard curve.
lon-Selective Electrode Method.
This is the most convenient method for the estimation of F-, down to
10-5 M (0·2 mg/L). It is based on potentiometric measurements with a
membrane electrode consisting of a single crystal of europium doped
lanthanum fluoride LaF3' The purpose of Eu doping is to improve electrical
conductivity. The membrane is cut as a 1-mm thick disc, a few mm in
diameter. The disc is sealed into the end of a rigid plastic tube, filled with an
equimolar solution of KCI and NaF, into which dips a AgCI electrode. A
reference electrode (saturated calomel electrode) is inserted into the test
solution along with the fluoride electrode. The potential difference is
measured.
Cell Ag/AgCI, CQO·3 M), F (0·001 M) ILaF31 test solution/reference
electrode
RT
Emeas = Const + -F log ar E = 0·058 log [F"] + constant
n SCE
160 ANALYTICAL CHEMISTRY
SILICA
Silica is not a water pollutant but excess of silica is undesirable. It is
generally removed by the use of strongly basic anion-exchange resin in the
deionization process or by distillation. The silica content of natural water is
usually 1 to 30 mg/L. Brackish waters and brines may contain as high as 1000
mg/L of silica. The gravimetric method is useful for 20 mg/L or more of
Si0 2 and spectrophotometric method, for 0·4 to 25 mg/L of Si02.
Gravimetric Method.
Silicates and dissolved Si02 are decomposed by HCI giving silicic acids
during evaporation. Ignition completes dehydration of Si02 which is weighed
and then volatilised as SiF4 , leaving impurities as non volatile residue. The
residue is weighed and the difference gives Si02 lost on volatilisation.
Na2SiOa(Si02) + 2HCI ~ 2NaCI + H 2SiOa
H 2SiOa ~ Si02 + H 20
Si02 + 4HF + 2H2S04 ~ SiF4i + 2H20 + 2H2S04
Procedure.
1. Take a clear sample (10 mg Si02) in a 200 mL platinum dish. Add
5 mL 6N HCI and evaporate repeatedly with addition of HCI to
dryness on a water bath.
2. Bake the residue on a hot plate for half an hour.
3. Leach with 5 mL 6N HCI, warm and add 50 mL hot distilled water.
While hot, filter through an ashless filter paper. Wash the dish and
residue with hot 0·2 N HCI and then with small volume of distilled
water till the filtrate is chloride free.
4. Evaporate the filtrate and washings from the above step to dryness
in the original platinum dish and repeat steps 2 and 3 above.
5. Transfer the two filter papers and residues thus obtained to a
covered and weighed platinum crucible, dry at no°c and finally
ignite at 1200°C to constant weight.
6. Moisten the residue in the crucible with distilled water. Add 4
drops of 18 N H 2S04 and then 10 mL HF. Slowly evaporate to
dryness over a hot plate in a hood. Ignite the crucible at 1200°C to
constant weight.
ANALYSIS OF WATER POlLUTANTS 161
.Procedure •.
1. Place 50 mL sample in a 100 mL platinum dish and 200 mg
NaHC03 and digest on a steam bath for 1 hour. Cool and add slowly
with stirring 2·5 mL H2S04,
(Note : The digestion step is necessary to convert any molybdate
unreactive silica to the reactive form). Make the volume to 50 mL and
transfer to a 100 mL separatory funnel.
2. To 50 mL treated sample, add quickly 1 mL of 6 N HCI and 2 mL
ammonium molybdate. Shake and allow to stand for 10 minutes.
3. Add 1·5 mL oxalic acid (10 gin 100 mL distilled water) and shake
vigorously. Measure the absorbance at 650 nm against a reagent
blank.
PHOSPHATE
Industriai efiluents, domestic sewage and agricultural run off are the
major contributor of phosphorus in water. Phosphates are largely used for
laundry purposes and treatment of boiler waters. Phosphorus occurs both in
inorganic and organic (85%) forms.
Spectrophotometric Method.
Principle. Orthophosphates form heteropoly acid when they react with
ammonium molybdate and potassium antimony tartrate in acid medium. The
phosphomolybdic acid reduces to molybdenum blue by ascorbic acid.
Reagents.
Standard Phosphate Solutions.
Dissolve 2·194 g of anhydrous potassium hydrogen phosphates in
deionised water and make up the vol:ume to 500 mL. Take 10 mL of this
solution and add deionised water to make 1 L of stock solution containing
1 mg PIL.
Prepare standard phosphorus solutions of various strengths from 0·0 to
1·1 mg PIL at intervals of 0·1 mg PIL by diluting the stock solution with
distilled water.
Reagent A. Take 1 g of ammonium molyb4ate and 0·02 g of potassium
antimony tartrate in 1000 mL volumetric flask. Add 16 mL of concentrated
H 2S04 slowly. Dilute with distilled water to the mark.
162 ANALYTICAL CHEMISTRY
a
3NO + BAl + 50ft + 2H20 ~ BAl02+ 3NHa
• Take 500 mL sample in NHa distillation apparatus. Add 50 mL of 10%
NaOH and evaporate to about 200 mL.
, Cool the solution, add 3 g Devarda's alloy (20 mesh) and then 30 mL
of 10% NaOH and immediately connect the flask with a vertical
condenser whose outlet dips into a receiver containing 200 mL of
0·2NH2S04·
• Distil at 50 to BO°C for one hour.
• Disconnect the receiver. Make up the volume of the solution in the
receiver to 250 mL. Take 5 to 10 mL aliquot in a 50 mL volumetric
flask and neutralise to pH 4·5.
164 ANALYTICAL CHEMISTRY
Procedure.
• Take 200 mL of the sample in a dish and evaporate to dryness.
• Add 4 mL of digestion mix~ure to the residue and dissolve it in 20 mL
of distilled water.
• Heat the solution for 15 mi~utes. Cool and transfer the digest to
micro-Kjeldahl distillation assembly. Add 5 mL of hypo solution.
• Take 5 mL of 1% boric acid solution containing 2 drops of mixed
indicator in a conical flask.
ANALYSIS OF WATER POLLUTANTS 165
• Place the flask below the condenser so that the tip of outlet of the
condenser is dropped in contents of the flask.
• Heat the Kjeldahl flask. Continue distillation for 10 minutes. Remove
the conical flask having distillate.
• Titrate the distillate against 0·01 N HCI till.the colour changes from
blue to pink.
• Run a blank using distilled water in a similar manner.
Calculation.
(8 - B) x 0·01 N x 1000 x At. wt. ofN2 (14)
TON (mg/L) = Volume of sample
where 8 = Volume of HCI used against sample.
B = Volume of HCI used against blank.
5. DISSOLVED ORGANIC NITROGEN (DON).
Take the sample and filter through millipore filter paper, Employ the
above procedure of TON. Express the result of dissolved organic nitrogen in
mg/L.
6. PARTICULATE ORGANIC NITROGEN (PON).
Determine the dissolved organic nitrogen and total organic nitrogen as
described above and calculate particulate organic nitrogen.
PON (mg/L) = TON - DON.
Interferences. Iron, nitrite and microbial mass are the chief sources of
interference in this method. The interference due to nitrite can be eliminated
by adding sodium azide.
2NaN3 + H 2S04 ~ 2HN3 + Na2S04
Sodium azide Hycirazoic acid
HN02 + HN3 ~ N 20 + N2 + H 20
Reagents Required.
• Standard N120 KtCr207 (Mol. wt. 294·21; Eq. wt. 49·035). Dissolve
2·4518 g of dry A.R. ~Cr207 powder in distilled water and make up
to 1000 mL in a measuring flask. For more accurate work, the
KaCr207 powder should be dried at 140°C for 1 hour in an electric
oven and cooled in a desiccator before weighing.
• N/20 Sodium thiosulphate solution. Dissolve 12·41 g of A.R.
sodium thiosulphate pentahydrate (Na2S203·5H20) in 1 L of distilled
water. The solution may be preserved by adding 3 drops of chloroform.
• Cone. H 2S04•
• Manganese sulphate. Dissolve 400 g of MnS04 or 480 g of
MnS04·4H20 in distilled water and make up to 1 L. This solution
should not give colour with acidified potassium iodide and starch.
• Alkaline iodide-azide reagent. Dissolve 500 g of NaOH and 150 g
of KI in distilled water. Add JO g of NaN3 in 40 mL distilled water.
Dilute to 1 L. This solution should not give colour with starch on
dilution and acidification
• Starch solution. Prepare a fresh paste of 0·5 g of starch with
distilled water and pour this into 100 ml of boiling distilled water
while stirring.
• Solid KI free from iodate, NaHC03 and cone. HCl.
Procedure.
(A) Standardization of hypo solution: Transfer 100 mL of 90iled,
cooled distilled water in a 250 mL iodine flask. Add about 2 g of KI, '2 g of
N aHC0 3 and shake until the salts are dissolved. Add 6 mL of conc. HCI
slowly while gently rotating the flask. Add 25 mL of N/20 KaCr207 solution
and mix the solutions well. Wash the inner sides of the flask with a littl~
distilled water and stopper the flask. Allow to stand for 5 minutes for the
completion of the reaction.
Cr20~- + 6r- + 14H+ ~ 2Cr3+ + 312 + 7H20
Rinse the stopper with distilled water and titrate the liberated iodine
with the hypo solution from the burette. When most of the iodine has reacted,
the solution acquires a greenish-yellow colour. At this stage, add 1 mL of
starch solution when the solution turns blue due to the formation of starch
iodine complex. Continue the titration until the greenish blue colour changes
to light green by the addition of a single drop of hypo at the end-point.
Note. If KI is not iodine free, a blank experiment has to be performed
and the corresponding correction should be made to the titre value.
ANALYSIS OF WATER POLLUTANTS 167
(B) Titration with the water sample.· For the. 1;>0 determination, the
sample should be collected from below the surface at ,a known depth and
without aeration. Generally the sample is collected in a BOD bottle with a DO
sampler.
For labl)ratory practice, collect the water sample in a 300 m BOD bottle,
avoiding contact with air. Add with the help of a pipette 2 mL of MnS04
followed by 2 mL of the alkaline iodide-azide reagent in such a way that the
tip of the pipette should dip below the liquid surface while adding the
reagents. Stopper the bottle immediately an<~ mbnvell by inverting the bottle
3 to 4 times. Allow to stand for 2 minutes, add 1 mL of conc. H 2S04, insert
the stopper and mix well till the precipitate goes into solution. Allow to stand
for 5 minutes. Take 203 mL of the clear solution into a conical flask and
titrate against hypo solution using starch as indicator.
Note. When 2 mL of MnS04 + 2 mL of alkali iodide-azide reagent are
added into full BOD bottle, from below the liquid surface, 4 mL of the original
water sample is lost. Hence 203 mL now taken for titration will correspond
to 200 mL of the original sample.
200 x 300 =·203 L
(300-4) m
Model Calculations.
20 mL of N/20 ~Cr207 == 20 mL of N/20 hypo
Normality of hypo = 20 ;0 ;0
x x N =~
2. Polarographic Method.
02 can be reduced at various electrodes in aqueous solutions. if a small
negative voltage is applied. The magnitude of the current which -flows is
determined by the rate at which 02 can diffuse to the electrode.
168 ANALYTICAL CHEMISTRY
-
sample and reagents
_~=======::::::::;::===~~::::::::::;~ Integrating CO2
CO detector
2
Sample
Oxidiser chamber
t
Gas for sparging
unoxidised sample Gas for sparging
~===~ -::::::::()()::=:Jo.-- oxidised sample
PESTICIDE ANALYSIS
ANALYTICAL METHODS.
Methods employed widely for the analysis of pesticides, their residues
and degradation products include :
1. Chromatographic methods are TLC, GLC and HPLC. Organo-
chlorines and carbamates are best analysed by TLC. Organophosphates are
accurately detected by GLC using flame ionisation detector.
2. Polarographic methods are used to determine pesticides containing
oxidisable or reducible groups.
3. Spectroscopic methods. JR, UV, NMR and GC-MS help to identify
metabolites and degradation products of pesticides.
[Refer to Unit 6 for pesticide pollutants, their classification and effects].
ANALYSIS OF WATER POLLUTANTS 173
15 10 5 o
Fig. 2. Gas chromatogram of organochlorine pesticides. Column packing 5% av.
Glass column 180 cm x 4 mm i.d. Solid support Gas-chrom Q (100/128 mesh).
174 ANALYTICAL CHEMISTRY
and diluted with hexane to 5 mL. An aliquot of this extract is injected into
the gas chromatographic column (180°C) with a micro syringe using Ar/CH4
as the carrier gas at 70 mL I minute. The pesticides are vapourised. They
move through the column at different rates and detected by electron capture
detector (Fig. 2).
Organophosphorus pesticides can be analysed by GC using
dichloromethane as the extractant and flame ionisation detector.
Phenoxy herbicides (2,4-D and 2, 4, 5-T) can be analysed by
derivatisation GC using electron capture detector. The herbicides are
extracted with ether and after hydrolytic precipitation, methyl ester is
analysed by gas chromatography.
ANALYSIS OF INSECTICIDES BY HPLC.
Dolphin and coworkers used automated HPLC pesticide analyser for the
analysis of chlorinated insecticides (Fig. 3) and fat in milk by injecting raw
milk onto a short silica precolumn where the fat was retained and the
pesticides eluted with hexane. The less polar fat eluting pesticides such as
pp' DDT, pp' DDE and (X-BHC were not separated from each other by the
precolumn and stored at the top of the column. Pesticides such as I3-BHC,
'Y-BHC and dieldrin which eluted latter were resolved from each other and
passed directly into an electron capture detector by means of a computer
controlled pnematically operated surtching valve.
§
c.
~
BenomyVMBC
I
c
0.1 cm 3 Benomyl/MBC
injected (0.05 ppm)
0.1 cm 3
injected
\
o 10 20 30 o 10 20 30
Time (min) Time (min)
Fig. 4. HPLC analysis of benomyl in cucumber extracts.
Column packing of Zipax SCX pellicular strong cation exchanger.
Column dimension. 1 m x 2·1 mm i.d.
Eluent. 0·025 N tetramethyl ammonium nitrate/0·025 N HN03 .
Pressure. 300 psi. Flow rate 0·5 cm3 min-I, column temperature 60°C.
UV detection is carried out at 254 nm.
ANALYSIS OF HERBICIDES BY HPLC.
Herbicides such as 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4-dichloro-
phenoxy butyric acid (2,4-DB), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T),
2-(2, 4, 5-trichlorophenoxy propionic acid) (silvex) and their corresponding
methyl esters are separated by using gradient elution system of mobile phase.
The eluate is monitored by a UV detector at 280 nm.
176 ANALYTICAL CHEMISTRY
Experimental Technique.
One litre of sample is quantitatively transferred to a 2 L separatory
funnel and acidified to pH 2 with H 2S04 , Water sample is then partitioned
with 60 niL of acetone by shaking for 2 minutes. Water layer is collected in
the original water sample container and the organic phase is transferred in a
500 mL Kudema Danish flask.
The extraction is repeated twice with 50 mL each of dichloromethane
and hexane. Solvent extract volume is reduced to 0·5 mL under low pressure.
Few microlitres of this concentrate is injected by stop flow injector to HPLC
for the analysis of samples. Same procedure is adopted for the analysis of
methyl esters of 2,4-D, 2,4-DB, 2,4,5-T and silvex.
The linearity of the UV detector is determined at 280 nm. The plots of
peak height versus concentration for the chlorophenoxy acids and their esters
are found to be linear at low microgram levels (0·5 to 1·0 ~g/L) and from them
the concentration of unknown herbicide calculated.
Lowrence detected linuron herbicide at 200 ppb level by using
(i) HPLC with UV detection at 254 nm.
(ii) GLC with electrical conductivity detector and
(iii) GLC with electron capture detector.
(vi) The Water (Prevention and Control of Pollution) Cess Act, 1977
(Amended in 1991).
(vii) The Environment (Protection) Act, 1986.
(viii) The Merchant Shipping Act, 1987.
2. State Enactments. Acts passed by State Governments are :
(i) The Orissa River Pollution Prevention Act, 1953.
(ii) The Maharashtra Prevention of Water Pollution Act, 1969.
The Water (Prevention and Control of Pollution) Act, 1974
(Amended in 1988).
• The Act defined terms like pollution, sewage effluent, trade effluent,
stream and boards. The Act also assigns the functions to be carried
out by the Central and State Boards.
• The Water Boards have power to obtain information, to take samples
of effluents from any industry and to make survey of any area and
gauge and keep record of the volume and other characteristics of any
stream or well.
• A person empowered by the Board has the right to enter, inspect and
examine any plant, record register, document or any other material
object, or for conducting a search of any place where he has reason to
believe that no offence of water pollution is comm~tted.
• The Board has wide powers to prohibit the use of any stream or well
for discharging any pollutant in it. The Board has powers to
restructure the outlets for dumping pollutants.
• The Act prohibits disposal of any poisonous, noxious or polluting
matter to the flow of water in a stream. However, dumping of any
material into a stream for the purpose of reclamation of land is not
considered an offence. The Act provides for severe and deterrent
punishments for violation of the Act which includes fine and
imprisonment.
The Water (Prevention and Control of Pollution) Cess Act, 1977
(Amended in 1991).
• The Act empowers the Central Water Board to collect cess on water
consumed by persons carrying on certain scheduled industries and by
local Authorities responsible for supplying water.
• The cess and the consent fees form the major sources of revenue to
run the Central and State Water Boards.
• The Act has been amended in 1991 with a view to augment the
resources of the Boards by removing the lacunae in the Act and to
provide rebate to the industries for complying with the consumption
and effluent quality standard.
Technical Difficulties in Controlling Water Pollution.
There are, however, several enforcement problems. Although the Water
Cess Act was passed to meet the expenses of the Central and State Boards yet
the Water Board has no power to take direct action against the erring party.
The court procedures are time consuming and delays often prevent quick and
preventive action thereby defeating the sole purpose of the Act. Because of the
problems inherent in the implementation of the Act, amendments were
proposed for strengthening the working of the State Boards. Inspite of the
178 ANALYTICAL CHEMISTRY
legislative measures, the pollution of our water ways continues unabated. This
is due to the lack of civic sense among people and due to the lack of necessary
infra structure for enforcing implementation of the laws efficiently.
Al As Be B Cr Co Cu Pb Li Mn Ni Zn V
1·0 1·0 0·5 0·75 5·0 0·2 0·2 5·0 5·0 2·0 0·5 5·0 10·0
o
8
HEAVY METAL POLLUTION
METAL TOXICITY.
Out of 112 elements discovered so far, over 65 are metals. Almost all
metals are toxic at high concentrations and some are extremely lethal even
at low levels. Toxic metals are added in aquatic system from industrial
effluents, domestic sewage discharges, agricultural run off, street dust and
fossil fuel burning. Traces of heavy metals such as Hg, Cd, Pb, As, Co, Cr, Mn,
Fe and Se have been identified as cumulative poisons causing deleterious
effects in living organisms.
The toxicity due to metals is mainly determined by
• Its solubility, stability, biological reactivity, physical form at the site
of action and the presence or absence of a homeostatic mechanism.
• The delivery of the metal to the cell to attain a critical concentration
at the active site.
• The cellular biochemical defence mechanisms.
The actual toxic action of each metal is different but most of them bind
to the metabolically active groups such as carboxyl, phosphoryl, amino, imino,
phenolic, sulphydryl or imadazole group.
Factors Responsible for Heavy Metal Toxicity.
• Solubility, stability and reactivity of heavy metal or its complexes.
• Oxidation state and electrochemical character of the metal.
• The rate of transport of heavy metal complex in blood, its distribution
and retention in the body tissues.
• The ability of the heavy metal to chelate with various ligands in the
body tissues and reactivity of the chelate thus formed.
• The efficiency of the enzymatic and homeostatic mechanism which
controls the absorption, distribution, retention and excretion of the
heavy metal ions or complexes.
Reasons for the Adverse Effects of Metal Toxicity in Biological
Systems.
• Interaction of the metal with protein leading to denaturation.
• Interaction with DNA causing mutation.
• Effect on cell membranes and regulatory enzymes.
The adverse effects in mammals may manifest in the following
disorders.
• Pathological changes.
• Retardation of growth.
• Symptoms of chronic disease.
(182)
HEAVY METAL POLLUTION 183
• Morbidity.
• Decrease in longevity.
• Detrimental changes in the reproductive cycle causing mortality in
the offspring.
CHROMIUM
Occurrence. Chromium is distributed in the earth crust at about 200
ppm levels and in sea water at 3 ppb. In nature, Cr occurs as chromium iron
ore (FeO . Cr203)' The Cr concentration in man's blood is found to be 0·5 to
5 JlgL-1, in urine 5 to 10 JlgL-1 and in hair over 500 ng g-l.
Uses and Pollution Sources. Chromium is an ingredient of stainless
steel. Cr203 is used in chrome plating, copper stripping, photography and as
a corrosion inhibitor. Chromium sulphate is used as a mordant in textile
manufacture, in leather tanning, paints, varnishes, inks and glazes for
porcelain. Wastes from industrial units contain soluble chromate salts which
is a source of water pollution.
Health Effects of Chromium.
• Chromium in its trivalent form is essential to man since it plays a
vital role in insulin metabolism as the glucose tolerance factor.
Supplementation of chromium helps to improve glucose tolerance in
diabetics, older people and malnourished. Most mammalian species
tolerate about 100 times their normal body burden of Cr(lll) without
experiencing any adverse effect.
• Chromium deficiency is characterised by impaired growth and by
disturbances in glucose, lipid and protein metabolism.
• Orally administered Cr(IlI) is poorly absorbed (1% only) than Cr(VI).
Absorbed anionic Cr(VI) readily passes through the membrane of red
blood cells and gets bound to globin fraction of haemoglobin. Cationic
Cr(IlI) is unable to pass through the membrane. It combines with
~-globulin fraction of the plasma protein and transported to the
tissues bound to siderophilin.
• The chromium entering the tissues is distributed among the
subcellular fractions and is mobilized from the body. stores to glucose
administration.
Toxic Effects of Cr(lll). Chronic exposure to chromate dust has been
correlated with increased incidence of lung cancer. Oral administration of
excessive levels (50 ppm) has been associated with liver, kidney damage and
depression.
Toxic Effects of Cr(VI).
• Cr (VI) is 100 times more toxic than Cr(III). Exposure to Cr(VI) causes
allergic skin irritations, dermatitis, conjuctiva, gastro intestinal ulcers
and irritation to mucous membranes.
• Cr(VI), a teratogen, is also mutagenic and carcinogenic inducing
bronchogenic cancer.
HEAVY METAL POLLUTION 185
• Higher levels of Cr(VI) causes chrome holes that is, penetrating ulcers
occur around finger nails, joints, eyelids and on forearms. Lesions on
the nasal mucosa may lead to perforation of the septum. Alkali
biochromates can be absorbed through skin lesions to cause renal
damage.
COPPER
Occurrence. Copper, an essential chalcophile, is ubiquitous in earth
crust as sulphide deposits along with Pb, Cd and Zn. Soluble copper levels in
contaminated water ranges from 0·5 to 2 j.1gL-l .
Uses and Pollution Sources. Copper is also used in the manufacture
of alloys, paints, ceramics and pesticides. Contamination of air with copper
occurs near industrial smelters, fertilizer industry, iron and steel industry.
Water pollution by copper results from the discharge of mine tailings, flyash,
disposal of municipal and industrial wastes.
Health Effects of Copper.
• Copper is necessary for the normal biological activities of amine
oxidase and tyrosinase enzymes. The former enzyme is involved in the
formation of elastin and collagen. Elastin is the major protein
constituent of the walls of large blood vessels while collagen is the"
proteinaceous component of tendons and bones.
• The Cu-containing enzyme, tyrosinase, is required for the catalytic
conversion of tyrosine to melanin, the pigment located beneath the
skin protecting it from radiation injuries. Tissues lacking tyrosinase
cannot produce melanin and thus would extremely be sensitive to
sunlight and probably be prone to early death.
• Defects in bone formation, pigmentation, reproduction, myelination of
the spinal cord, cardiac function, connective tissue formation, defects
in growth and hematopoiesis were found to be the manifestations of
copper deficiency.
• Cytochrome oxidase, ascorbic acid oxidase, laccase, tyrosinase,
urinase, amine oxidase, aminolevulinic acid dehydrase, dopamine
hydroxylase are all copper enzymes.
A daily dietary intake of 2 to 3 mg of Cu is recommended for human
adults. Copper absorption and retention depends on factors such as the
chemical form in which the metal is ingested, dietary levels of other
minerals and the acidity of the intestinal contents in the absorptive area.
Toxic Effects of Copper.
• Inhalation of air borne copper causes irritation of the respiratory tract
and metal fume fever.
• Workers involved in the spraying of vineyards with Bordeaux mixture
(a fungicidal preparation containing about 2% of copper sulphate)
develop a respiratory disorder known as vineyard sprayer's lung
disease. This condition is characterised by the development of
interstitial pulmonary lesions and nodular fibro-hyaline scars
186 ANALYTICAL CHEMISTRY
Porphobilinogen
HEAVY METAL POLLUTION 187
Lead inhibits ALA-D enzyme (I) so that it cannot form PBG(II). The
overall effect is the disruption of the synthesis of haemoglobin and other
respiratory pigments. such as cytochromes, which require heme. Finally, Pb
does not permit utilisation of 02 and glucose for life sustaining energy
production.
• Lead can even disturb the structure-function relationships of DNA
molecules. It can combine with specific biochemical residues, like
sulphydryl, carboxyl and imidazol groups.
• Lead inhibits acetylcholinesterase, ATP, cytochrome oxidase, carbonic
anhydrase and alkaline phosphate etc.
• Elevated lead levels « 0·8 ppm) in the blood cause anaemia, facial
pallor, gradual paralysis of muscles, atrophy, insomnia, tremor,
dizziness, arthralgia, arteriosclerosis, cardiorenal manifestation.
• Chronic lead poisoning results in vertigo, dyspepsia, reticulocystosis,
porphyrinuria, sterility, anorexia, vomiting and brain damage.
• Due to chemical analogy of Pb2+ with Ca2+, bones act as
repositories for Pb. This Pb may be remobilised along with phosphates
from the bones which exert toxic effects to soft tissues.
Toxic Effects of Tetraethyl Lead (TEL).
• TEL is an inert compound and does not exert its toxic effects as such.
But its toxic metabolite R3Pb+, biotransformed by oxidases enzyme
system in the liver, is 100 times more toxic than inorganic lead.
• Brain is the target organ in TEL poisoning. The organo lead
compounds, being lipophilic, are selectively localised in the nervous
tissues resulting in CNS toxicity.
• Prolonged TEL exposure is associated with stippled RBCs and WBCs,
lead line in gums, hallucination and encephalopathy.
Lead poisoning can be treated bl giving CaNa2 EDTA intravenously,
when lead is chelated (Pb2+ displaces Ca + from the chelate) and excreted via
urine. But a person suffering from renal disorder should not be treated with
this chelating agent. In such cases d-penicillamine may be used orally.
Sodium citrate or British antilewisite (BAL) i.e., 2, 3-dimercaptopropanol can-
also be prescribed.
ZINC
Occurrence. Zinc is an essential metal occurring as zinc blende (ZnS),
zincite (ZnO), willemite (Zn2Si04) and smithsonite (ZnC03 ). It occurs as
0·004% in earth crust and 20 ppb in oceans. Zinc content in milk lies between
3 to 5 IlgmL-1, in wheat germ and bran from 40 to 120 ppm. Oysters, the
richest source of zinc contain 1000 ppm of zinc. Human body contains 300 mg
zinc distributed in muscles (65%), bones (20%), plasma (6%), RBCs (2·8%) and
liver (53%).
Uses and Pollution Sources. Zinc is used in dry batteries, pigments,
protective coatings on iron, printing processes and in construction materials.
Smelting of ores and agricultural use of pesticides like zineb and ziram are
the pollution sources of zinc. Zinc salts being astringent and antiseptic have
limited use in medicine.
188 ANALYTICAL CHEMISTRY
MANGANESE
Occurrence. Manganese is an essential element but it is toxic at
higher concentrations. Pyrolusite, braunite, magnatite, tephroite are the
minerals of Mn. Human beings get their daily quota of Mn from vegetables,
fruits, nuts and germinal portions of grains. Mn is found in sea water at one
ppb level.
Uses and Pollution Sources. Manganese is used in dry battery cells,
electrical coils, ceramics, alloys and in glass and steel manufacturing
industries.
Health Effects of Mn.
• Mn at low concentrations (2-5 to 5·0 mg) is essential for cell
development.
• It acts as a cofactor in a number of enzymatic reactions, particularly
those involved in phosphorylation, cholesterol and fatty acid
synthesis.
HEAVY METAL POLLUTION 189
ARSENIC
Occurrence. Arsenic occurs in earth crust (2 ppm), sea water (5 ppb),
soils (1 to 40 ppm), in human body tissues (18 mg) and blood (25 mg).
Uses and Pollution Sources. Arsenic oxide, known as white arsenic
is formed as a by-product in smelting of Pb, Cu, Ag ores. As 20 3 is used in
insecticides, as a preservatives and mordant in textile industry. Paris green
(copper acetoarsenite) is used as a feed additive. Arsenites are used as weed
killers. Elemental arsenic is employed in the manufacture of infrared
transmittance glass, semiconductors and oil cloth.
Toxic Effects of Arsenic.
Arsenic is a general protoplasmic poison and it affects all the systems in
man. The order of toxicity of arsenic compound is : Arsines [As(II!)] > arsenite
[As(III)] > arsenate [As(V)] > arsenic organic acids fAs(v)].
1. Complexation of As with coenzymes. As(III) exerts its toxic
action by attacking 8H group of an enzyme thereby inhibiting enzyme action.
The enzymes which generate cellular energy in the citric acid cycle are
adversely affected. The inhibitory action is based on inactivation of pyruvate
dehydrogenase by complexation with As(Il1) whereby generation of ATP is
prevented.
H8-CH2 S-CH2
/
0- I /1
"I
-
-O-As + CH2 O=As CH2
""-0- I
Arsenite HS-CH S-CH
1 1
(CH2)4 (CH2)4
1 I
C=O C=O
I 1
Protein Protein
Dihydrolipoic acid protein Inactivated protein complex with As(III)
CH20PO~
I Additional
Phosphate H-C-OH process Glyceraldehyde
I leading to
)
3-phosphate
C=O formation of ATP
I
OPO~- As033-
1, 3-Diphospho- Arsenite
glycerate
1-Arseno-3
phosphoglycerate
Reagents.
1. DTPA Solution (0·005 M). Take 1·967 g of DTPA in 1 L volumetric
flask and make up the volume to the mark with deionised water.
2. Stock Standard Solution. Dissolve 0·1 g of the metal foil in dilute
HCI (1 : 1) and make the volume to one litre with deionised water to obtain
100 ).lg/mL (i.e., mg/L or ppm) of solution of every metal ion. Alternatively,
dissolve separate
0·3928 g of CuS04·5H20 0·4964 g of FeS04·7H20
0·3076 g of MnS04·H20 0·4398 g ofZnS04·7H20
in 1 L of deionised water to prepare 100 ).lg / mL of stock solutions of Cu, Fe,
Mn and Zn respectively. Add 5 mL of H 2S04 to the solutions.
3. Working Standard Solutions. Transfer 0, 1, 2, 4, 6 and 8 mL of
stock solution (100).lg M2+/mL or 100 ppm of M2+, where M2+ = Cu2+, Fe2+,
Mn2+, Zn2+) to a series of 100 mL volumetric flasks. Add DTPA solution and
make up the volume to the mark. This will give standard solutions having
metal ion concentration of 0, 1, 2,4,6 and 8 ).lg/mL (or ppm).
Procedure.
1. For the determination of Cu2+, Fe2+, Zn2+ and Mn 2+ in aqueous
sample, set the atomic absorption spectrophotometer to zero using
blank solution.
2. Feed standards of the metals to be determined to AAS to standardise
the instrument to read absorbance or concentration in the sample
having the given metal within the standardised range.
3. Feed the DTPA extract and record the absorbance or concentration of
the metal.
4. Repeat the above steps for every metal.
5. In case the AAS shows a sign of over for some metal in a particular
sample indicating thereby that the sample has a concentration out of
the range for which the instrument has been standardised then make
further dilution of the sample 2-5 times. Feed the sample and record
absorbance or concentration.
Calculations.
Most of the modern AAS are calibrated to display the concentration of
a metal in ppm directly in the sample. In such cases, the concentration of a
given metal in the sample is calculated by multiplying the displayed reading
by the dilution factor, 2.
If the AAS displays the reading in absorbance, then a standard curve
has to be prepared for the known standards on a graph paper. Convert the
absorbance readings into concentration ().lg/mL) from the standard curve. The
amount of given metal is then calculated as follows :
Volumelweight of the sample taken= 10 mL
Volume ofDTPA extractant added = 20 mL
Dilution = 2 times
Absorbance shown by AAS = A
Concentration of the metal as read from the standard curve against
A = C ).lg/mL (or mg/kg = ppm).
Content of the metal cation in the sample = C x 2 mg/kg or ppm.
HEAVY METAL POLLUTION 195
Precautions.
• Apparatus (glasslpolythene) to be used for analysis must be
thoroughly washed with acidified water and deionised water.
• Turbid solutions should not be used for feeding since they may block
the capillary of AAS.
ANALYSIS OF COPPER
1. Spectrophotometric Method.
(i) Cuprethol method. Cupric ions yield a yellow chelate when react
with cuprethol (bis-2-hydroxy ethyl) dithiocarbamates at a pH of 5 which is
maintained by HCI and CHaCOONa solution. The interference from Fe is
removed by adding pyrophosphate. The yellow colour is measured
spectrophotometrically.
(ii) Neocuproine method. Copper reacts with
~) {~
neocuproine (2, 9-dimethyl 1, 10-phenanthroline) in
weakly acidic solution (pH 3 to 8) to form a complex
which can be extracted as a yellow coloured solution
into a mixture of CHCla and CHaOH. The yellow HaC CHa
coloured solution is measured spectrophotometrically at Neocuproine
457 nm.
Procedure. To 100 mL sample in a 250 mL beaker add 1 mL conc.
H 2S04 and 5 mL conc. HNOa. Evaporate to white dense fumes of SOa. Add 5
mL conc. HNOa and heat to fumes until the solution becomes colourless. Cool
the solution, add 70-80 mL of distilled water and again boil. Cool and filter
the solution in a 100 mL ~olumetric flask and make the volume to 100 mL by
distilled water.
Transfer 50 mL of this solution into a 150 mL separatory funnel and
dilute it with 50 mL of distilled water. Add 5 mL of 1% hydroxyl amine
hydrochloride, 10 mL of 40% sodium citrate and 10 mL of neocuproine reagent
(100 mg/100 mL alcohol, CHaOH), maintaining the pH between 4 to 6. Extract
the solution with CHCla and repeat the extraction of aqueous layer with 10
mL chloroform. Transfer the CHCla extract in a volumetric flask containing
CHCla extract. Dilute the combined extract to 25 mL with methanol. Measure
the absorbance of the coloured solution at 457 nm against a reagent blank.
2. By Salicylaldoxime. Dissolve 1 g salicylaldoxime in 5 mL of alcohol
and dilute to 100 mL with water. The neutral solution OH
of the sample reacts with a drop of 10% acetic acid and r(3Y
a drop of salicylaldoxime, yielding green precipitate. ~ CH - NOH
The sensitivity is 0·0005 mg of Cu in 10 ppm -
concentration. Salicylaldoxime
3. Atomic Absorption Method. Aspirate Cu2+ solution into an
air-acetylene flame of an atomic absorption spectrophotometer and measure
the absorbance at 325 nm.
Differential Pulse Polarography (DPP) for the Determination of Copper
and Zinc in Tap Water.
Most tap water mains contain sufficient Cu and Zn for them to be
determined directly using DPP. Since the sample contains very less
196 ANALYTICAL CHEMISTRY
DPPTRACE
iii
c:
Cl
u;
1
O.21lA
f
INTRODUCTION
Soil is a dynamic natural body developed as a result of pedogenic
processes during weathering of rocks. The soil is in fact the very heart of the
life layer called biosphere. It consists of mineral and organic constituents,
exhibits definite physical, chemical and biological properties, has variable
depth over the surface of earth and provides a suitable medium for plant
growth. Soil mainly consists of 50% pore space (air and water) and 50% solid
phase. The solid phase is broadly composed of 45% mineral matter and 5%
organic constituents. The liquid phase of the soil (40 to 50%) is an aqueous
solution of salts. The soil acts as a reservoir for supplying water to plants.
The gaseous phase of soil consists of same nitrogen and oxygen contents as
of air but carbon dioxide concentration is much higher (0·1%). All these phases
of the soil system have a definite role to play. The solid phase provides
mechanical support and nutrients to the plants. The liquid phase supply
dissolved nutrients to plant roots. The gaseous phase satisfies the aeration
needs of plants. These three phases share the soil's responsibility to sustain
the plant growth.
COMPONENTS OF SOIL
1. Inorganic Components (Mineral matter) of the Soil (45%).
The soil is essentially a silicate mineral. The composition of common
elements in the soil is ; ° 46·6%, Si 27·7%, Fe 5%, Al 8·1%, Ca 3·6%, Na
2·8%, K 2·6%, Mg 2%. Finely divided quartz, Si02, commonly occurs in soil.
Among the silicates, orthoclase KAlSigOS, albite NaAISigOS and epidote
4CaO·3(AlFe)20g. 6Si02·H20 are common components of soil minerals. In
some soils, iron oxides FeO(OH), magnetite Feg04, Mn20g, TiO and CaCO g
are relatively abundant. The clay minerals in soil are secondary minerals,
essentially hydrated aluminium and iron silicates, which serve to bind cations
such as Na+, K+, Ca2+, Mg2+ and NHt. These elements are not leached by
water and are available as plant nutrients. Rocks which form the earth crust
(Petrology i.e., science of rocks) are made up of minerals.
2. Organic Components of the Soil (5%).
The organic matter content of soil varies from 3·5% by weight in a top
soil. In addition to the partly decayed plant and animal residues, soil organic
matter contains microbially synthesized compounds. Organic matter
functions as a granulator mineral particles. It is responsible for the loose,
easily managed conditions of productive soils.
(199)
200 ANALYTICAL CHEMISTRY
Deficiency of Micronutrients.
(i) Growth of plant is hampered.
(ii) Rate of photosynthesis get reduced thereby decreasing crop yield.
(iii) It may cause shedding of flowers, improper utilisation, poor seed
setting and attack of diseases.
Macronutrients.
The essential macronutrients are C, H, 0, N, P, S, Ca, Mg, K. The
atmosphere and water are the sources of C, H, O. Nitrogen may be obtained
by some plants, directly from the atmosphere, through N-fixing bacteria. N,
P, K are commonly added to soil as fertilizers. Mg is made available to plants
through ion-exchange of organic matter or clays which hold magnesium
strongly.
Nutrient Functions.
Nitrogen. The nitrogen content in soil is mostly of organic nature
(90%) resulting from the decay of dead plants (biomass) and animals, plant
residues and excreta of animals etc. Nitrogen fixing bacteria, rhizobium
convert atmospheric N to organic form that can be used by host plant.
When nitrogen is applied to soils as NH! (fertilizer), nitrifying bacteria
convert it in to NOg for use by plants. Certain leguminous plants, e.g., alfalfa,
clover, soyabeans, ground nut, pulses and guar etc., exhibit the unique ability
to fix atmospheric N through nitrogen fixing bacteria on their nodules.
Legumes can add considerable quantities of N to soil.
Functions of Nitrogen.
(i) Nitrogen is not only an essential constituent of protein and
chlorophyll but it is also involved in photosynthesis, respiration and
protein synthesis.
SOIL ANALYSIS 203
ANALYSIS OF SOIL
Soil analysis is essential for getting the maximum yield as it provides
the knowledge of soil components, nutrients and their deficiency in a
particular part of soil.
SOIL MOISTURE MEASUREMENT.
Soil moisture can be measured in a variety of ways.
1. Gravimetric Method. In this method soil is taken in a container,
weighed in the moist condition, oven dried and weighed again after cooling.
The drying in the oven is carried out at 110°C to constant weight. Drying
takes about 2 hours for small samples, but as much as 24 hours for bulky
clayey soil samples.
The mass water percentage is calculated on the basis of dry soil weight.
Weight of moist soil = W 1 g.
Weight of oven dried soil =W 2 g.
Weight or mass of moisture =(Wl - W2 ) g.
(Wl - W2 ) x 100
Percent moisture = W
2
2. Electrical Conductivity Method. This method is based on the
change in electrical conductivity with the variation in soil moisture. In this
method, a gypsum block, which is provided with two electrodes at a definite
distance, is used. The gypsum block requires calibration for uniformity before
use. The blocks are buried in the soil at desired depth and the conductivity is
measured with modified wheatstone bridge.
The electrical conductivity between two electrodes set at a fixed distance
apart inside a small block is an indirect measure of soil moisture from the
field capacity to the wilting point. The blocks may be made of nylon, fibre
SOIL ANALYSIS 207
glass or porous material. It can be buried in the soil at any desired depth with
wires from each block extending to the surface of the soil for easy reading of
conductivity with the modified Bouyoucos wheatstone bridge. However,
electrical conductivity method is unsuitable for soils containing high salt
contents.
3. Tensitometric Method. Tensitometer can effectively measure the
moisture content of sandy soils because of their high matric potentials. In
tensitometer, a porous cup is attached to a glass tube which is connected to a
mercury manometer. The tube and cup are filled with water and cup is inserted
in the soil. As the soil dries, water moves out through the porous cup, creating
a suction or vacuum on the water column. Thus water flows through the porous
cup into the soil until equilibrium is maintained. These tension readings
(suction or vacuum readings) in the manometer, expressed in terms of cm or
atmosphere, measure the tension or suction of the soil. If the soil is drier, water
moves through the porous cup, setting up a negative tension (Fig. 1).
Stopper
r----,.- Water
- ---- - -+-Unbalanced
- column
Water
Mercury
Soil surface
DETERMINATION OF SOIL pH
The pH of soil suspension can be determined by electric pH meter and
colorimetric methods.
208 ANALYTICAL CHEMISTRY
• The second buffer is removed and the electrodes are again flushed
with water.
• The electrodes are then immersed in a beaker containing soil paste or
soil suspension. Read the pH and record it.
2. BDH universal indicator is used to obtain approximate pH. For
this, take some soil and a little BaS04 in a flat dish and knead the mixture
with a few drops of the indicator. Tilt the dish and note the colour of the
supernatant liquid. It will show the approximate pH of the soil.
3. Lovibond comparator instrument is also used for pH measurement.
It has an opal glass screen and two glass tubes, one containing the sample
and the other containing the sample as well as the indicator. A circular disc,
having a set of nine standard coloured glasses, fits into the front portion and
can be easily rotated in order to bring each coloured glass in front. The disc
is rotated so that the colour matches with the standard from where the pH is
noted.
Procedure.
Take 10 g of soil which should pass through a 20 mesh sieve. Add 50 mL
of sulphuric-salicylic acid and shake. Add 5 g of sodium thiosulphate and heat
the contents gently for 5 minutes. Cool and then add 10 g of sulphate mixture
and digest in Kjeldahl flask at full heat. Cool and then add 300 mL of distilled
water and fit in the distillation apparatus. Add 100 mL of 45% NaOH and a
large piece of zinc. Connect to distillation head, and distil off 150 mL into 50
mL of 2% aqueous boric acid solution. Add few drops of the bromocresol
green-methyl red indicator. Titrate to the faint pink colour with standard
solution of 0·1 N H 2S04 . Repeat the titration with the blank.
Calculations.
Weight of soil sample = 10 g.
Volume of 0·1 N H 2S04 used in titration = V mL
Weight of nitrogen in 10 g soil = V x 0·1 x 0·014 = 0·0014 V
. 0·0014 x V x 100
Percent mtrogen = 10 = 0·014 x V percent
Milliequivalent of nitrogen in 10 g soil = V x 0·1 = 0·1 V
Milliequivalent percent of nitrogen in soil= O·lV x 10 0 = V
10
"t " "I Percent nitrogen x 106
ppm 0 f m rogen m sOl = 100
Place the sample of the soil in an oven at 70°C for 4 hours. Grind and
pass it through a 2 mm sieve. Take 50g of soil in a 500 mL flask and add 250
mL of distilled water containing 5 mL of 1 N copper sulphate solution and
shake for 10 minutes. If the soil is not very acidic and does not give a coloured
extract, add 0·4 g of Ca(OH)2 and 1·0 g of MgC03 to the soil suspension and
shake for 5 minutes to precipitate copper. Filter on a dry filter paper and
discard the first 20 mL of filtrate.
If the soil gives a coloured extract, allow it to settle. Decant 125 mL
of the supernatant liquid into a flask. Add 0·2 g of Ca(OH)2 and 0·5 g of
MgC03. Shake the contents well and filter. Pipette out 10 mL of the filtrate
in dish and evaporate to dryness. Cool and rapidly add 2 mL of
phenoldisulphonic acid directly to the centre of the evaporating dish. Rotate
the dish in such a way that the reagent comes in contact with all the residue.
Allow it to stand for 15 minutes and then add about 15 mL of cold water and
stir with a glass rod until the residue dissolves to form a solution. Add
NH40H to make the solution slightly alkaline. Transfer this solution to a
Nessler tube, dilute and compare with standard solution containing 1 ppm
nitrogen as nitrate using a colorimeter or Nessler tube.
Note. (i) If the soil contains more than 15 ppm of chloride, the latter is
precipitated by adding 10 mL AgN03 solution in the 250 mL of test solution.
This will remove 80 ppm of chloride calculated on the basis of dry soil. If the
soil contains high concentration of chloride, add solid Ag2S04 to the soil
suspension before shaking.
lit
212 ANALYTICAL CHEMISTRY
(ii) In the case of highly coloured soil extract, add 1 g of chloride free
carbon black to 100 mL of the supernatant liquid and shake for 15 minutes
before adding Ca(OH)2 and MgC0 3 to the solution. If the soil is calcareous,
add 5 mL of IN copper sulphate also to the soil extract with the carbon black
in order to ensure complete removal of colloidal carbon with copper hydroxide.
Note that copper sulphate is used for the clarification and
decolourisation of the soil extract. MgC0 3 is used to remove the excess of
Ca(OH)2'
DETERMINATION OF TOTAL PHOSPHORUS
Phosphorus in soil ranges from 0·01 to 0·3% and occurs in the form of
apatite and hydroxy phosphates of Fe, AI, Mn, Ca and Hg.
Olsen Method.
Principle. Phosphorus in the form of phosphate is extracted from the
soil by NaHC03 (pH 8·5). PO~- ion reacts with ammonium molybdate in acidic
medium to form molybdophosphoric acid which is reduced to blue coloured
complex by ascorbic acid.
H 3P04 + 12H2Mo04 ~ H3P(M03010)4 + 12H20
Molybdophosphoric acid
Absorbance readings are taken on spectrophotometer at 730 nm. A
standard curve between absorbance and concentrations of standard
phosphorus solutions helps to deduce the total phosphorus content of the
sample.
Reagents.
1. NaHCOa (0·5 M) solution. Dissolve 42 g NaHC03 in 1 L distilled
water. Adjust the pH to 8·5 with 1 M NaOH solution.
2. Ammonium molybdate solution. Dissolve 40 g ammonium
molybdate in 1 L of distilled water.
3. Darco G-60 or phosphorus free charcoal. 80 g Charcoal or Darco
G-60 is slurried in distilled water. Keep it over night with 1 L of 6 M HCl in
60 mm diameter column (250 mL capacity). Add deionised water till the
leachate is chloride free. Dry at 110°C.
4. Ascorbic acid solution. Dissolve 26·4 g of L-ascorbic acid in 500 mL
distilled water.
5. Antimony potassium tartrate solution. 1.454 g of antimony
potassium tartrate is dissolved in 500 mL distilled water.
6. H 2 S04 (2·5 M). Add 140 mL concentrated H 2S04 in 1000 mL distilled
water.
7. Murphy-Riley developing solution. It consists of 250 mL of
2·5 M H 2 S04, 75 mL ammonium molybdate solution, 50 mL ascorbic acid, 25
mL antimony potassium tartrate solution and 100 mL distilled water.
s. Standard stock phosphorus solution. Dissolve 0·439 g KH 2P04
in 500 mL distilled water. Add 25 mL of 7 N H 2S04 and make the volume to
one litre. It forms 100 ppm stock solution of P. From this take 5 mL solution
in a 100 mL flask to obtain 5 ppm of P solution .
•
SOIL ANALYSIS 213
Method.
• Take 2·5 g air dried soil sample in a 125 mL flask. Add a little
phosphorus free Darco G-60.
• Add 50 mL NaHC03 solution at 25°C and shake for 30 minutes.
• Similarly run a blank without soil.
• Filter the extract using Whatman No. 40 filter paper.
• Pipette 10 mL aliquot in 50 mL volumetric flask. Add 10 mL distilled
water and one drop of p-nitrophenol indicator. Acidify the content to
pH 5·0 by adding 2·5 M H 2S04 till the colour disappears.
• Add 8 mL of Murphy-Riley solution and make the volume to 50 mL
with water.
• Wait for 10 minutes and record the absorbance on spectrophotometer
or colorimeter at 730 nm.
• Process the standard phosphorus solution of different concentration in
a similar manner.
• Plot a standard curve between absorbance and concentration of
standard phosphorus solutions.
• Deduce the total phosphorus content of the soil sample by
comparing its absorbance with standard curve.
Calculations.
Weight of soil taken = 2·5 g
Volume of NaHC03 extractant added = 50 mL
Volume of extract taken for colour development= 10 mL
Reading of spectrophotometer/colorimeter = x
Concentration of P in 50 mL extract = ClIO Ilg mL-1.
Concentration of P in 1 g of soil
C 50 -1
= 10 x 2.5 Ilg mL
P043- - P (mgll't) PS x V
1 re = 1000 x W
DETERMINATION OF SILICA
Silicon, the most abundant element, is present in nearly all the
minerals. Most of the minerals in igneous rocks occur as silicates, hence soil
is largely composed of silicates. Si is required for the growth of rice, barly and
cucumber etc.
SOIL ANALYSIS 215
Total Silicon.
• Soil is digested with perchloric-nitric acid and heated for 15 minutes.
• Filter the silica from the diluted digest through Whatman No. 41
filter paper. Wash the residue with warm dilute HCI (1: 20) and then
with distilled water.
• Evaporate the combined filtrate and washings to dryness. Heat the
residue at 110°C for an hour to recover traces of silicic acid from the
solution.
• Extract the residue with dilute HCI, filter and wash as before.
• Ignite the combined residue and filter paper to a constant weight in
a platinum crucible using a muffle furnace and record the result as
crude silica.
• Crude Si02 can be converted to Si by the factor 0·4675.
Water Soluble Silicon.
Molybdosilicate Method.
Principle. Ammonium molybdate at pH 1·2 reacts with silicon and any
phosphate present to produce heteropoly acids. Oxalic acid is added to destroy
molybdophosphoric acid, HsPMo12040. The remaining molybdosilicic acid,
H4SiMo12040 is measured at 650 nm.
Reagents.
1. Standard silicon solution.
2. Ammonium molybdate solution (10% w/v).
3. Sodium sulphite (17 gL-1) solution.
Procedure.
• Extract the soil with water and filter.
• Pipette 25 mL of the clear filtrate into a 150 mL conical flask and add
15 mL dilute HCI.
• Add 15 mL ammonium molybdate solution. After 5 minutes, add 2 mL
oxalic acid and 25 mL sodium sulphite solution.
• Read the optical density at 650 nm exactly one minute after adding
the reductant.
• The standard curve should cover the range 0·1 to 5·0 Ilg per mL Si.
DETERMINATION OF LIME
Lime is mainly used for the correction of soil acidity, to increase the soil
fertility and to supply nutritional calcium to crops. Agricultural liming
includes CaO, Ca(OH)2 and CaCOs . These compounds are generally derived
from chalk or limestone e.g., calcite CaCOs and dolomite [CaMg(COs)21. Lime
reactions in soil depend upon the nature and the fineness of the liming
material. Lime or lime stone should not contain silica. Burnt lime or
quicklime is commercially available with a known content of CaO.
Principle.
The lime sample for agricultural U3e generally contains CaO, CaSiOs,
MgCOs , Fe20s and Al20 S' This sample is finely ground, suspended in water
216 ANALYTICAL CHEMISTRY
and treated with a solution of cane sugar. As a result, soluble calcium sucrate,
C12H220U·CaO is obtained, whereas CaC03 and CaSi03 remain insoluble.
The solution containing calcium sucrate is titrated as CaO against a standard
solution of HCl using phenolphthalein as an indicator.
Reagents. (i) 0·05 N HCl (ii) 10% cane sugar solution.
Procedure.
Accurately weigh 5·0 g of the powdered sample and transfer it to a 500
mL flask. In order to prevent the possibility of caking, moisten it with 10 mL
of alcohol. Add 10 mL of 10% solution of cane sugar and make up the solution
to the mark with water. Immediately stopper the flask and shake the flask
atleast for 4 hours. Filter the solution through a filter paper into a dry beaker.
Titrate 50 mL of the filtrate with 0·05 N HCI using phenolphthalein as an
indicator.
1 mL of 0·05 N HCI= 0·0014 g. CaO.
Calculations.
. X x 0·0014 x 500 x 100
Percent of CaO In the sample = 50 x 5
where X is the volume of 0·05 N HCI used to titrate 50 mL of calcium sucrate.
DETERMINATION OF MAGNESIUM
In soils, magnesium occurs in the clay minerals, micas, vermiculites and
chlorites. Mg, a macronutrient, is essential for the plant growth. It affects
translocation of phosphorus and helps to increase sugar, starch, vitamin and
inulin in root crops.
Mg can be determined by titrimetric method, atomic absorption
spectrophotometric method and gravimetric method after removing calcium
salts.
Titrimetric Method.
Principle. Mg in solution can be titrated with 0·01 N EDTA using
Eriochrome black T dye as indicator at pH 10 in presence of
NH4 CI-NH40H buffer. At the end poipt, colour changes from wine red to
blue.
Reagents.
1. 0·01 N EDTA.
2. NH4 CI-NH40H buffer. Dissolve 67·5 g NH4CI in 570 mL NH3
solution and make it to one litre.
3. Eriochrome black T indicator.
Method.
• Pipette out 25 mL aliquot containing not more than 0·1
milliequivalent (me) of Ca and Mg.
• Add 2 to 5 crystals of carbamate and 5 mL NH4CI-NH40H buffer.
Add 3 drops of Eriochrome black T indicator.
• Titrate this solution with 0·01 N EDTA solution till colour changes
from wine red to blue or green.
SOIL ANALYSIS 217
DETERMINATION OF SULPHUR
Sulphur occurs in soil in both organic and inorganic forms but only a
fraction of it is available for crop growth. Direct uptake of sulphur by plants
occurs largely as inorganic sulphate. Sulphate may be present in soil solution.
adsorbed on soil surface or as insoluble compounds such as gypsum or
SOIL ANALYSIS 219
Method.
• Pipette out 5 mL BaCr04 solution into 100 mL volumetric flask. Add
1·2 mL of 5 N NH 40H in it to reduce free acidity to about 0·05 N.
• Measured aliquot of soil extract (5 to 10 mL) is poured into the flask
maintaining sulphate-sulphur content below 2000 Ilg. Shake and
allow to stand for half an hour.
• Add 1 mL NH 40H to precipitate unreacted BaCr04 and make up the
volume to 100 mL with distilled water.
• The flask is stoppered and inverted twice for thorough mixing. Filter
through Whatman No. 42 filter paper.
• The intensity of the yellow coloured filtrate is measured III
photoelectric colorimeter using blue (420 nm) filter.
• Concentration of S is calculated from the standard curve.
Note. The test solution should have a pH around 2 or 3.
Preparation of Standard Curve.
Take 0, 4, 8, 16 and 20 mL of working solution of sulphate-sulphur (of
100 ppm) in 100 mL volumetric flask. Make up the volume to 100 mL with
the distilled water. This gives standard solution having 0, 4, 8, 16 and 20
ppm S concentration. Prepare a blank sample without soil. Measure colour
intensity by photoelectric colorimeter using blue filter.
FUELS
Fuels may be defined as naturally occurring or manufactured
combustible organic substances which on burning supply heat energy for
practical applications without the formation of objectionable byproducts.
According to the modern concept, a fuel is any fissionable material or
reactant which produces energy in a form that can be used for producing
power. Fuels contain carbon as the main constituent associated with small
amounts of N, S, H, 0, moisture and mineral matter. Coal, wood, petrol,
diesel, kerosene, oil gas etc. are some common fuels.
Characteristics of a Good Fuel.
• A good fuel should have a high calorific value.
• It should have a low non-combustible matter content.
• Its ignition temperature should be low so that it can burn smoothly.
• Moisture content should be low because it reduces the combustion
process of a fuel.
• It should be safe, cheap, readily available, easy to handle and
transport.
• The products of.combustion should be non-polluting.
• Solid fuels should have low ash and uniform size to achieve regular
combustion.
• Liquid fuels should have correct surface tension, boiling range,
volatility and free from objectionable odour and acid components.
• A good fuel for internal combustion engines should have high octane
number, no ash and sulphur and high antiknock characteristics.
CLASSIFICATION OF FUELS
Fuels may be classified into two categories :
1. Primary or Natural Fuels. These fuels occur freely on earth's crust
and may be solid (wood, coal, peat, dung), liquid (crude oil, petroleum) or
gaseous (natural gas) fuels.
2. Secondary or Derived Fuels. The fuels are manufactured from
primary fuels and may also be solid (coke, charcoal), liquid (kerosene, petrol,
gasoline, LPG) and gaseous (oil gas, water gas, bio gas) fuels.
SOLID FUELS
Solid fuels consist of combustible organic matter and incombustible
matter. Combustible matter contains C, H, S whereas incombustible matter
contains moisture and minerals such as carbonates, phosphates, silicates,
(224)
FUEL ANALYSIS 225
sulphides of Ca, Fe, Mg, AI, K, Na etc. After burning, solid fuels form ash.
Solid fuels are classified into natural, artificial and industrial solid fuels.
1. Natural Solid Fuels.
(a) Wood. Wood derived from plants contains lignocellulose which
consists of three primary precursors viz.
(i) Cellulose, a polysaccharide, (C6HlQ05)n,
(ii) Hemicellulose and
(iii) Polyphenolic lignin (C19HlS05)x
Wood also contains resins and proteins. Ultimate analysis of dry wood
shows C = 49·65%, H = 6·25%, N = 0·92% and 0=43·20%. Calorific values
varies from 4000 BTU to 6480 BTU. On destructive distillation, wood yields
charcoal (23%), wood tar (5·3%), pyroligneous acid (44·3%) and gases (26·9%).
Wood is a convenient fuel because of its low ignition temperature and low ash
(0·3%) content.
(b) Bagasse. It is crushed sugarcane residue.
(c) Coal. Coal is a primary fuel formed from fossil remains of
terrestrial plants by the combined action of high temperature and pressure.
It consists of cellulose, lignin and small amounts of resins, fat and water. The
progressive transition from .
Vegetation ~ Peat ~ Lignite ~ Bituminous ~ Anthracite ~ Graphite
is accompanied by increase in aromatisation, calorific value, hardness and
decrease in ° content, reactivity and moisture. C 13 NMR and X-ray studies
have shown coal to be composed of macromolecules (mol. wt. 100,000) which
are predominantly aromatic and some hetrocyclic rings. The ring
macromolecules are linked together by methylene bridges or longer aliphatic
chains which may contain oxygenated functional groups. Typical composition
of coal is C 100 H S5 S 2 .1 N 1.5 09.5'
Grading of Coal.
Coal can be graded on the basis of heating values, coking properties,
geological age, chemical composition and commercial applications etc.
However, the most accepted system is used by ASMT based on the ranks
which signifies the degree of coalification, maturation or metamorphism
(change in form and structure under the influence of heat, pressure and
water). Coal is graded in the following ranks.
(i) Peat (Precoal).· Peat is formed by gradual decomposition of
vegetable matter in moist places. It is the initial transformation stage during
the formation of coal. It is not an economic fuel but can be used as fertilizer.
It has low calorific value (7700 BTU per pound).
(ii) Lignite. Lignite is obtained from finely divided plant tissues. There
are two types of lignite. Brown coal lignite is unconsolidated while black
lignite is consolidated. It has high moisture content but sometimes it may
spontaneously ignite by absorbing oxygen. Its calorific value is 6000-9900
BTU. Ash content is about 4%.
,(iii) Sub-bituminous Coal or Black Lignite. It ignites very easily.
Calorific value is 7000-15000 BTU and moisture content 20%.
226 ANALYTICAL CHEMISTRY
GASEOUS FUELS
Advantages of Gaseous Fuels.
Gaseous fuels have distinct advantages over solid and liquid fuels. For
instance,
(i) Many gaseous fuels have high calorific value.
(ii) Smoke and ash are eliminated.
(iii) Rate of combustion is high.
(iv) Temperature can be easily controlled.
(v) Much higher fuel economy because of regenerative heating.
(vi) Uniform heating is possible.
(vii) Beside industrial and domestic use, they can be used in internal
combustion engines for direct production of power.
Classification of Gaseous Fuels.
(i) Producer gas (ii) Water gas (iii) Coal gas
(iv) Coke oven gas (v) Ethylene gas (vi) Acetylene gas
(vii) Natural gas
PRODUCER GAS
Producer gas is a mixture of CO + H2 and CO2 + N 2 . Producer gas is a
cheap industrial fuel because it can be obtained from low grade coal. Bagasse,
peat, lignite, anthracite, highly volatile bituminous coals, saw dust and coke
can be used for its production.
Gas Producer for the Manufacture of Producer Gas.
Producer gas can be prepared by passing a mixture of air and steam over
red hot coal or coke in a special type of furnace, called gas producer. A
typk:al producer (Fig. 1) consists of a refractory-lined cylindrical vessel (3 m
diameter, 4 m height) made of steel. The solid fuel enters the producer at the
top, through cup and cone feeder and is burnt on an iron grate.
Producer Gas Production Reaction Zones.
1. Ash Zone.
It is the lowest zone containing a layer of ash (0·5 m thick) on the grate.
It helps to
228 ANALYTICAL CHEMISTRY
Reduction
Pyrolysis zone =111111~illll~~= Refractory brick lining
Combustion zone Coke at 1000°C
is enough hot, steam is passed for 1-5 minutes. This period is known as cold
blow. In modern plants, attempts have been made to decrease the hot blow
period and to increase the run period for the generation of much water gas.
Average composition of water gas is : CO = 41%, H2 = 51%, N2 = 4%
and CO2 = 4%. Calorific value of water gas is 2800 kcallm 3 .
Carburetted Water Gas.
To enhance calorific value of blue water gas, the latter is carburetted by
adding gaseous hydrocarbons obtained by cracking petroleum oils.
Plant producing carburetted water gas consists of a generator,
carburettor and a superheater along with purifiers and scrubbers (Fig. 2).
Chimney :~-++--Cooler
Carburettor
- ------
~=:~:~~=~=~=~
Water box
Coal
Superheater
Steam ~~
NATURAL GAS
Natural gas is a mixture oflower alkanes (C l to C5 ), CO2 and N2 etc. It
accumulates in under ground reservoirs either with or without petroleum oil.
Natural gas obtained from oil wells is of two types.
1. Dry variety. When there is no oil but only natural gas exist in a
petroleum well, it is said to be dry. A typical dry gas contains 83·5% CH4 ,
12·5% C2H 6 , 0·2% CO2 and 3·8% N2. Calorific value is 1000 BTU/cu. ft.
2. Wet variety. When natural gas occurs along with petroleum in oil
wells, it is called wet gas. It consists of a mixture of methane along with
n-propane, n-butane, isobutane and isopentane etc. Wet gas is suitably
treated to remove propane, propene, butane, butene and is used as LPG. It
gives out low boiling gasoline known as casing head gasoline.
Helium is also found in natural gas. The calorific value of natural gas
lies between 950 and 1100 BTU/cu. ft. The gas obtained by fermentation of
organic matter in sewage (or cowdung) is also called natural gas. It contains
70% CH4 , 30% CO2 and has calorific value 62·5 BTU/cu. ft.
Properties and uses. Natural gas can be liquefied by pressure and
cooling to -121°C. It is used
(i) as a clean domestic and industrial fuel
(ii) in the manufacture of carbon black.
Beckman
Oxygen
valve
. _ - - - - - Electrically
operated stirrer
Ebonite cover
Electrodes to which --I~II~t5
a ring is attached j+---r.=+--Copper calorimeter
Mg fuse wire -+=i---M~=t>t-+-.
=::=::=::-=::=::=::=::=::=::=::=E=::=::=::=::=::=====::=::=::===::=::=::=::=
. Water jacket
Peat 20 50 02 27 7700
Lignite black 16 42 12 32 10200
Bitwninous 02 35 13 49 12500
Semibitwninous 01 35 04 84 15000
Anthracite 01 08 03 88 15000
Significance of Proximate Analysis.
Each constituent determined under proximate analysis has its own
importance in the assessment of the coal quality.
1. Moisture. Moisture decreases the calorific value of coal. A
considerable amount of heat is wasted in evaporating the moisture during
combustion. However, moisture content upto 10% produces a more uniform
fuel bed and less fly ash.
2. Volatile Matter. The volatile matter percentage gives some idea
about coking property of coal and denotes the proportion of the coal which will
be converted into gas and tar products by heat. High volatile matter coals
burns with a smoky flame and have low calorific values. For the manufacture
of metallurgical coke, a coal with low volatile matter and fixed carbon is
preferred. High volatile matter content is desirable in coal gas manufacture
and in carbonisation plants.
3. Ash. Ash reduces the heating value of coal. There is a heat loss of
about 1·5% for each 1% ash present in coal. Ash consists of Si02 (50%),
Al 20 3 (30%), Fe203, CaO, Ti02, MgO etc. Clinkers (lumps of ash) cause
uneven temperature on the grates and reduce air supply.
Fused particles of ash may stick to the boiler tubes and affect heat
transfer. Ash with low melting point forms molten slag which is absorbed in
the pores of the refractory lining of boiler furnace. Difference in the
coefficients of expansion and contraction of the slag (ash) and refractory
material causes spalling of the refractory lining thereby reducing its life.
Thus coals used in boilers should have high ash fusion temperature.
4. Fixed carbon. It is the fixed carbon which burns in the solid state.
Higher percentage of fixed carbon increases the calorific value of fuel from
lignite (low ranking coals) to anthracite (high ranking coals).
Stirrer
crucible containing the levelled layer of sand (Fig. 7). It is provided with a lid
having a small opening for the escape of vapours. The whole assembly of the
crucibles is covered with the hood in Bridge
order to distribute the heat •. - - - - (Flame
uniformly. The outer iron crucible is height guide)
heated strongly till the smoke
appears above the chimney. When
the vapours cease to burn and no
further smoke is observed, the
Iron crucible
bottom of the crucible is heated to
Skidmore
redness. After 10 minutes, the crucible
burner is put off and the whole Silica
apparatus is allowed to cool until no :~f--I-- crucible
smoke is seen. The silica crucible is Asbestos
transferred to a desiccator, cooled insulation
and weighed. From the weight of )+---\-\-- Meker burner
carbon deposited in the crucible, the
percentage of carbon residue in the
oil can be calculated. Fig. 7. Conradson apparatus.
Calculations. Weight of the crucible + beads = WIg
Weight of the crucible + beads + oil = w2fJ
Weight of the crucible with carbon residue = wgg
Weight of the oil taken = (w2 - WI) g
Weight of the carbon residue obtained = (w3 - WI) g
w3- w l
Percentage of carbon residue in the oil = x 100
w2- w l
2. Ramsbottom Method.
1 to 4 g ofthe oil sample is taken in a stainless steel bulb which is placed
in a sheath consisting of an iron tube having a flat closed end. The sheath is
immersed in a bath of molten lead which is heated to 550°C for 20 minutes.
The bulb is taken out, cooled and weighed. The result is reported as 2%
carbon residue to the weight of oil taken for the experiment.
Calculations. Weight of the oil taken = WIg
Weight of the carbon residue obtained = w2fJ
COMPOSITION OF BLOOD
Blood is a fluid that circulates in a closed system of blood vessels. Whole
blood can be divided into two main components as follows :
1. Plasma (55%).
Blood plasma, the non-cellular fraction, contains about 92% water and
8% solids. Solid portion consists of following constituents.
(A) Organic Substances (7% to 8%). Organic substances include
(i) Proteins (7%). Proteins are the main constituents among the
non-diffusible compounds. Proteins present in blood include
haemoglobin, albumin, globulin, fibrinogen and
prothrombin.
These plasma proteins help in regulating blood viscosity and blood
volume and can exert an osmotic pressure of 25-30 mm. Fibrinogen
helps in blood clotting.
(ii) Non-Protein Nitrogenous Substances. These are the products
of tissue activity and are transported from tissues to kidneys or
skin for excretion. Non-protein substances include urea, uric acid,
creatinine, ammonia, free amino acids, bilirubin, xanthine, choline,
thyroxin, ATP, neutral fats such as glucose, pentoses, cholesterol
and phospholipids.
(iii) Hormones. Hormones or internal secretions are secreted by
endocrine glands and act as chemical messangers.
(iv) Antibodies. Antibodies maintain blood viscosity and are
important in immunity reactions.
(v) Enzymes. Enzymes act as catalysts and include lipase, amylase
and sucrase etc.
(vi) Organic Acids. Traces of organic acids such as lactic acid, pyruvic
acid, malic acid, citric acid, acetoacetic acid, ~-hydroxybutyric acid
have also been found in blood.
(B) Inorganic Substances (0.99%). The diffusible electrolytes present
in plasma include Na+, K+, Ca2+, Mg2+, cr, SO~-, PO~- and HCOg etc.
(C) Respiratory GaSes. 02 and CO2 are also the main constituents of
plasma.
Note. Plasma is thus the liquid portion of circulating blood. The cells
are separated from the plasma by centrifuging whole blood. If blood is
allowed to clot, the fibrinogen is removed with the cells, leaving serum. The
(247)
248 ANALYTICAL CHEMISTRY
FUNCTIONS OF BLOOD
Blood transports oxygen from the lungs to the tissues and CO2 from the
tissues to the lungs. Blood is thus responsible for respiration.
• Blood performs nutritive function by transporting absorbed dietary
materials to all the body tissues.
• Blood also performs excretory function by transporting metabolic
wastes to kidneys, lungs, skin and intestine for removal.
• It provides the temperature regulating and defensive mechanism. The
ability of the body to maintain constant temperature is mainly due to
high specific heat of water.
• The efficient buffer system present in blood helps in maintaining a
constant pH in and around tissues.
• Along with the lymph and tissue fluids, blood provides the connecting
link between individual cells of organs and tissues.
not be frozen because the red cells will be ruptured. Plasma and serum
samples fractionate into layers of different composition when frozen and so
should be shaken gently after thawing. Samples are more stable if a
protein-free filtrate (PFF) is prepared. Glucose is stable in PFF because the
glycolytic enzymes are removed.
CLINICAL ANALYSIS
SERUM ELECTROLYTE.
Physiologically serum electrolytes (ionic solutes) include Na+, K:, Ca2+,
s.
M~+, pot and HCO All higher life forms require a subtle and complex
electrolyte balance between intracellular and extracellular millieu. The
maintenance of precise osmotic gradients of electrolytes is extremely
important to regulate blood pH, hydration of the body and for nerve and
muscle functions. Electrolyte drinks containing Na+, K+ salts and Gatorate
(sports drink) are used to replenish dehydration caused by exercise,
starvation, diarrhoea and diaphoresis etc.
. Electrolyte balance is regulated by hormones, generally by the kidneys
flushing out excretory products. In humans, hoemostatis is regulated by
antidiuretic hormone, aldosterone and parathyroid hormones. Serious
electrolyte disturbances may lead to cardiac and neurological
complications.
Clinical analysis of serum electrolytes is performed by blood
testing, estimating blood chloride, Ca2+, Na+, K+, RCO) and urine analysis.
centrifuge and drain. Add 2 mL of 1 N H 2S04 and shake. Keep the tube in
boiling water bath until the precipitate dissolves. '
Titrate the hot solution with 0·01 N KMn04 until a pink colour develops.
Let the volume of 0.01 N KMn04 solution is X mL. For blank, take 2 mL of
1 N H 2S04 and titrate it with 0·01 N KMn04' Suppose the volume of 0·01 N
KMn04 used is Y mL.
Calculations.
0·2
Serum Ca (mg/dL) = (X - y) x 2 x 100
=(X - y) x 10
Clinical Interpretation. Serum calcium ranges from 9-11 mg/dL in
healthy persons. The product of serum Ca and serum P is about 40 in adults
and 50 in children. Excess of serum calcium causes hyperparathyroidism,
multiple myeloma, sarcoidosis, hypervitaminosis, idiopathic infantile
hypercalcemia, alkali syndrome and polycythaemia. Decrease in serum
calcium results in hypoparathyroidism, osteomalacia, nephrotic syndrome,
renal failure, acute pancreatitis, starvation and rickets.
Estimation of Calcium in Urine. Dilute urine in water (1 : 10) and
determine Ca as described above. Multiply the result by 10.
Excess
Reagents.
(i) 1% NaCI (saline solution) in CO2 free water.
(ii) Phenol red solution, 0·1% in 0·003 M NaOH.
(iii) Antifoam A.
(iv) Prepare and standardise 0·1 M HCI and 0·1 M NaOH.
(v) Freshly prepare 1 L of 0·01 M HCI and 0·01 M NaOH solutions by
diluting 100 mL of 0·1 M solutions to 1000 mL with saline solution.
Saline helps in volatilisation of CO 2 from the acidified solution by
decreasing its solubility.
Preparation of the Sample.
To 10 mL of the fresh blood sample, add NaF to prevent glycolysis of
glucose which can change the pH. Keep the sample tube anaerobically.
Preparation of Comparison Solution.
• Take 6 mL of 1% saline solution in a 25 mL flask and add 0·10 mL of
serum.
CLINICAL CHEMISTRY 255
\
• Add 2 drops of phenol red indicator and shake the contents vigorously.
• Because of the buffering action of the blood, yellow to red colour
change occurs from pH 8·4 to 6· 7.
Procedure.
• The pooled serum or plasma sample should be prepared by touching
the end of a stirrer to Antifoam A and rotating it in the pooled sample.
This will prevent excess foaming when the sample is swirled.
• Place 0·1 mL serum or plasma in a 25 mL Erlenmeyer flask and add
1 mL of 0·01 M HCI and 4 mL of 1% saline. Swirl the flask to escape
CO2 ,
• Add 2 drops of phenol red and titrate with 0·01 M NaOH dropwise
until pink colour appears.
Calculations. Since 0·1 mL of serum was taken for analysis, it should
consume 0·26 mL of 0·01 M HCI. Hence 0·74 mL of HCI should remain
unreacted. Back titration should consume 0·7 mL of 0·01 M NaOH. Normal
s
value of HCO in blood is 26 meqlL.
5. Enzymatic Method.
Enzymatic determination of glucose is an established method. The
enzyme glucose oxidase catalyses the aerobic oxidation of glucose to gluconic
acid and H 2 0 2 .
Glucose
C6 H 120 6 + 02 + H 20 --~
Oxidase
Actually, this enzyme shows almost complete specificity for ~-D-glucose,
a-D-glucose reacts at a rate 0·64 relative to 100 for the ~-form. In the ~-form,
all the hydrogens are axial and the hydroxyl groups are equatorial, allowing
the molecule to lie down flat on the enzyme active site and form the
enzyme-substrate complex. The a-form can not lie flat on the enzyme. Thus
the aerobic conversion of glucose (36% a, 64% ~) depends on the mutarotation
of the a-form to ~-form. Mutarotation is shifted as the ~-form is removed,
thereby analysing the glucose in blood sample.
6. Biosensor Autoanalysers to Estimate Blood Glucose.
The diabetic simply places a small drop of blood (from a sterile
lancet-prick) on a test strip. Glucose oxidase on the strip catalyses the
oxidation of glucose to produce H 2 0 2 , which is measured either
electrochemically or photometrically.
Clinical Interpretation. Blood glucose is increased in hyperthyro-
idism, hyperadrenalism, hyperpituitorism, diabetic mellitus and decreased in
Addison's disease, glycogen storage and excessive insulin secretion.
Estimation of blood sugar helps to detect cases of border line-frank diabetes
and to prescribe doses of antidiabetic drugs used to treat diabetic patient.
Blood sugar can generally be estimated as
(i) Fasting blood sugar,
(ii) Post prandial blood sugar (two hours after meal) and
(iii) Glucose tolerance test.
Here the blood samples are collected at half an hour interval for 2·5
hours after administration of 75 g of glucose orally and analysed for sugar.
Examination of urine sample is also conducted simultaneously for 2·5 hours.
Note that glucose disappears from blood on standing due to glycolysis
at the rate of 15 mg/100 mUhour at 35°C. An antiglycolytic agent such as NaF
must be present in blood samples to be used for glucose estimation.
2. Crocker Method.
Principle. Urea reacts with diacetyl monoxime (DAM) and
thiosemicarbazide (TSC) in presence of Fe3+ ions to form pink complex which
is measured colorimetrically at 520 nm.
Reagents.
(i) 0·1 % Benzoic acid.
(ii) 10% H 2 S04 .
(iii) Stock diacetyl monoxime. Dissolve 6·25 g DAM in 250 mL of
distilled water.
(iv) Stock thiosemicarbazide. Dissolve 1·25 g TSC in 250 mL of
distilled water.
(v) Stock FeCla.HaP04 reagent. Dissolve 3·34 g FeCI3.6H2 0 in
65 mL H 3P04 (85%). Make up the volume to 100 mL.
(vi) Working acid FeCls • Dilute 1·3 mL stock FeCl3 to 1 L with
10% H 2S04 .
(vii) Working DAM. Mix 67 mL of stock DAM with 67 mL stock
thiosemicarbazide and dilute it to 500 mL with distilled water.
(viii) Stock urea nitrogen standard (1·0 mg/mL). Dissolve 0·2143
g pure urea in 100 mL of 0·1% benzoic acid.
(ix) Working urea nitrogen standard (1 mg/dL). Dilute 1 mL of
stock standard to 100 mL with 0·1% benzoic acid.
Procedure. Dilute 0·25 mL serum/plasma to 5 mL with 0·1% benzoic
acid. Set the experiment as illustrated below.
Reagent Test Standard Blank
Diluted serum/plasma 1mL - -
Working urea nitrogen standard - 1mL -
Mix the contents and place all the tubes in boiling water bath for 10
minutes. Cool the tubes and read the absorbance of the tubes against blank
at 520 nm.
Calculations.
ODT
mg of Urea Ntrube (0·05 mL blood) = ODs x 0·01 (conc. of std.ltube)
OD = Optical density
ODT 0.01
Blood urea nitrogen (BUN) (mg/dL) = ODs x 0.05 x 100
ODT
Conc. of urea N = OD x 20
s
Clinical Interpretation. Protein contents in diet influence urea level.
It is lower in people with low protein diet. In elder persons, urea level may
CLINICAL CHEMISTRY 259
ODT 0·04
Serum uric acid (mg/dL) = ODs x 0.2 x 100
ODT
=--x20
ODs
Normal value of uric acid in serum is between 2 to 7 mg/dL.
CLINICAL CHEMISTRY 261
Procedure. Set the blank (B), test (T) and standard (S) tubes as
follows:
Reagent (mL) Blank Tl T2 SI S2 S3
Serum (1 : 10 diluted) - 1·0 1·0 - - -
Standard protein solution - - - 1·0 1·5 2·0
Distilled water 3·0 2·0 2·0 2·0 1·5 1·0
Biuret reagent working - - - - - 3·0
Mix and keep in water bath at 3TC for 10 minutes. Measure absorbance
at 550 nm using green filter.
Calculation. Plot graph between absorbance and concentration of
protein. Calculate the total protein content in serum by measuring the
absorbance of sample from the standard curve.
Normal Values. Total serum protein = 6-8 g percent
Albumin = 3·5-5·5 g percent
Globulin = 1·5-3·0 g percent
Clinical Interpretation. Total protein content may be altered by
changes in plasma volume without altering the albumin/globulin ratio. An
increase in protein concentration may be due to dehydration and a decrease
due to excess intake of water. Hyperproteinemia occurs due to excess
synthesis of globulin, chronic liver disease, acute infections like kalazar and
multiple myeloma. Hypoproteinemia is due to low protein intake,
malnutrition, starvation, diabetes and nephrotic syndrome.
IMMUNOASSAY
Immunoassay techniques are important for the specific determin.ation of
drugs, vitamins, hormones and other compounds at nanogram and smaller
levels. These techniques involve a competitive reaction between an analyte
antigen and a specific antibody to form a complex.
CLINICAL CHEMISTRY 267
Radioimmunoassay Procedures.
All RIA procedures are based on the original discovery by Rosalyn
Yalow and Berson (awarded Nobel Prize in Physiology, 1977), that low
concentrations of the antigen hormone insulin could be detected
radiochemically by their ability to bind radiolabeled (1131) insulin. The
determination of unknown concentration of antigen is based on the fact that
radiolabeled antigen and unlabeled antigen (from the sample of standard)
compete physiochemically for the binding sites on the antibodies (Fig. 1).
Ab Ag*-Ab Ag*-Ab
(antibody) and Ag-Ab and Ag-Ab
bound bound
Ag* Incubation, Separation,
(radiolabeled
antigen) Ag* and Ag
(free)
Ag (antigen
in sample or
standard)
+
Ag*-Ag
(free)
Specificity of Radioimmunoassay.
No antiserum used in RIA is completely specific for a particular antigen.
The specificity is influenced by
(i) heterogeneity of the antibody,
(ii) cross-section with other antigens and
(iii) possible interferences of the antigen-antibody reaction from low
molecular weight substances that may alter the environment of the
reaction.
A given antigen induces the formation of multiple antibodies. It can
combine with multiple antibodies to various degrees depending on the
respective equilibrium constants.
The problem of heterogeneity has been diminished with the
development of improved techniques for antisera purification. Also, the
synthetic production of monoclonal antibodies provides high specificity.
Non-specific factors that may modify the rate of antigen-antibody
reaction include high temperature, pH, ionic strength, composition of the
incubating medium, buffer, urea, heparin and high bilirubin concentrations.
Antigen standards and unknown should be prepared in antigen-free plasma
to swamp out differences in composition.
Risks are very low when the test is done correctly. There may be
bruising or delayed bleeding from the site. Very rarely, there may be a
problem associated with circulation in the punctured area.
Blood Gas Analyser. Blood gas analysers automatically or manually
measure pH, Po and Peo of blood. The oxygen is measured with a
conventional amperometric 2 membrane oxygen electrode. The CO2 is
measured by a pH glass electrode covered with a plastic membrane that
allows diffusion of only gases. Chemical sensors have been developed for blood
gases, electrolytes and glucose.
DETERMINATION OF BLOOD pH
Measurement of pH of blood gives a ratio of acids to bases. The
respiratory response to changes in blood pH is instantaneous. In acidosis,
CO2 is retained and pH decreases and it stimulates ventilation. In alkalosis,
CO2 is blown off and pH rises.
Procedure.
1. Direct Method. Arterial blood sample is introduced into a blood gas
analyser and pH is measured.
2. Indirect Method. Henderson-Hassel batch equation is used to
determine pH of blood.
CLINICAL CHEMISTRY 273
CALCIUM
Calcium Content. Calcium, the most abundant element in the body,
constitutes about 2% of the total body weight. About 90% Ca is present in
bones and teeth. Ca content in human serum, tissues and urine is 90-100
ppm, 60-90 ppm and 96-100 mg/day respectively.
Dietary sources of Ca are milk, cheese, cabbage, lentils, nuts, egg yolk
etc.
Calcium Requirement. Adult 800 mg, children 1·2 g, infants 300-500
mg/day.
Biochemical Functions.
• Activation of enzymes. Ca2+ ions are required for the direct
activation of lipase, adenosine triphosphate, ATPase and succinate
dehydrogenase enzymes.
• Release of hormones. Ca2+ ions facilitate the release of insulin,
calcitonin and parathyroid hormones.
• Calcium as intracellular messenger. There are certain hormones
which exert their action through the mediation of Ca2+ ions. Ca is
regarded as a second messenger for the hormonal action of
epinephrine in liver glucogenolysis and third messenger for
antidiuretic hormone through AMP.
• Regulates secretory processes. Ca2+ regulates microfilament and
microtubular mediated processes like endocytosis, exocytosis and cell
mortality.
• Calmodulin mediated action. Calmodulin is a calcium binding
regulatory protein. Ca-calmodulin complex activates adenylate cyclase
enzyme .
., Development of bones and teeth. Ca and P are the two
non-protein body building elements. Ca exists in bones as
CaC03.2Ca3(P04)2' Calcium along with phosphate is required for the
274 ANALYTICAL CHEMISTRY
MAGNESIUM
Magnesium Content. Adults contain 20 g of Mg, 70% of which is
found in bones in combination with Ca and P. Remaining 30% is present in
soft tissues and body fluids. Mg content in human serum, tissues and urine
is 22 ppm, 300-500 ppm and 60-120 mg/day respectively. About 75% of serum
Mg is diffusible and the rest is bound to plasma protein.
Dietary Sources. Main sources of Mg are nuts, meat, cereals, fruits,
milk, cauliflower and cabbage.
Magnesium Requirement. Adult man requires 350 mg/dL and
woman 300 mg/dL of mg.
Biochemical Functions.
• Cofactor and activator of enzymes. M~+ ions serve as a cofator
for various enzymes requiring ATP, e.g., hexokinase, glucokinase,
phosphofructokinase. It acts as an activator for enolase, phosphory-
lase, peptidase, RNA and DNA pol:tmerase.
• Neuromuscular functions. M~+ ion is required for proper
neuromuscular functions. In the body, Mg and Ca act as antagonists
to one another. For example, the depression of central peripheral
nervous system due to hypermagnesium can be reversed by
intravenous administration of Ca.
• Mg is required for the development of bones and teeth.
• Mg is absorbed by the intestinal cells through a specific carrier
system. Its absorption decreases by the consumption oflarge amounts
of Ca2+, PO~- and alcohol.
CLINICAL CHEMISTRY 275
SODIUM
Sodium Content. About 50% of body sodium is present in bones, 40%
in extracellular fluid and 10% in soft tissues. Na content in human serum,
tissues and urine is 3200 ppm, 0·07 g/gN and 1000-5000 mg/day respectively.
Dietary Sources. Common salt (NaCl), nuts, whole grains, bread,
milk, eggs and vegetables.
Sodium Requirement. Sodium requirement for an adult is 4-5 g/day
and for a patient of hypertension about 1 g/day.
Biochemical Functions.
• Sodium is required to maintain the osmotic pressure inside the cell,
to prevent its collapse and also to balance the electrical charges
associated with negatively charged organic macromolecules in the
cell.
• Na+ ion produces electrical potential across cell membrane which is
essential for the smooth functioning of nerve and muscle cells.
• The movement of glucose into cells is associated with Na+ ions.
Hypernatremia is characterised by an increase in serum sodium level.
It may occur because of hyperactivity of adrenal cortex, cortisone and sex
hormones or by dehydration. In pregnancy steroid and placental hormones
cause N a and water retention in the body. Hypertension and blood volume
also increases.
Hyponatremia is due to decrease in serum sodium level by
diarrhoea, vomiting, renal diseases and adrenocortical deficiency (Addison
disease). The manifestations of hyponatremia include reduced blood pressure,
retarded growth, nausea, loss of appetite, headache and muscular cramps.
POTASSIUM
Potassium Content. Potassium content in human serum and tissues
is 120-214 ppm and 20-200 ppm (dry) respectively.
Dietary sources are grains, cereals, milk, vegetables, banana, orange,
beans, potato, coffee, fish, chicken and liver etc.
Potassium requirement for an adult man is 3 g/day.
Biochemical Functions. K and Na salts form the chief buffer system
which play vital roles in the regulation of pH of body fluids.
• Potassium is required for the transmission of nerve impulse.
Extracellular K+ ion influences cardiac muscle activity.
• Potassium is necessary for the biosynthesis of proteins and ribosomes.
• The optimal activity of enzyme pyruvate kinase of glycolysis depends
on ~ ions.
Increased level of serum potassium is observed in adrenocortical
insufficiency (Addison disease), renal failure, diabetic coma and severe
dehydration.
276 ANALYTICAL CHEMISTRY
ZINC
Zinc content in human serum, tissues and urine is 1-2 ppm, 12-100
ppm and 0·3-0·6 mg/day respectively.
Dietary sources include cereals, nuts, oil seeds, grains, soyabeans,
wheat, peas, potatoes, onion, almonds etc.
Zinc requirement for an adult is 10-15 mg/day.
Biochemical Functions.
• Zinc is an essential constituent of various enzymes.
• Zinc is a crucial nutrient for immune and brain function, nervous
system, blood sugar and optimal health.
• Zinc guards against infection and is required for healthy skin and
hair.
• Zinc is needed to transport vitamin A to the retina and this improves
vision.
• Gusten, a zinc containing protein is important for taste sensation.
• The absorption of zinc depends on a transport protein
metallothionein. Its absorption is hampered by fibre, Ca, Cu,
CLINICAL CHEMISTRY 277
MANGANESE
Mn Content. Mn content in human serum, tissues and urine is 0·02
ppm, 0·2-1·7 ppm and 0·05 mg/day respectively. Total body content of Mn is
15 mg. Mn is mainly concentrated in liver, muscles, bones and kidneys.
Sources of Mn. Nuts, cereals, fruits, tea, leafy vegetables.
Mn requirement for an adult is 2-9 mg/day.
Biochemical Functions. Mn acts as a cofactor of several enzymes
such as organiase, pyruvate carboxylase, isocitrate dehydrogenase, dismutase
and peptidase.
• Mn as Mn2+ activates liver arginase, choline esterase, mitochondrial
respiratory enzymes.
• Liver arginase converts nitrogenous wastes into urea in the
ornithine-argininecitrulline cycle which is excreted in urine.
• Mn is necessary for cholesterol biosynthesis and also for the synthesis
of glycoproteins and mucopolysaccharides.
• Mn in the serum is bound to a carrier protein transmagnanin (a
p-globulin).
• Mn Deficiency. Mn deficiency causes retarded growth, impaired
haemoglobin regeneration, accumulation of fat in liver and testicular
degeneration etc.
IRON
Iron Content. Average iron content is 1·25 ppm in human serum and
0.1-0.3 mg/day in urine.
Dietary Sources. Liver, meat, fish, poultry are rich sources of heme
iron. Cereals, legumes, nuts, oil seeds, dry fruits, leafy vegetables constitute
non-heme iron.
Iron requirement for an adult man and pregnant woman are 10-14
mg/day and 40 mg/day. An adult body contains about 3 g of iron About 70%
of the total iron present in adult body occurs in erythrocytes of blood and
5% in myoglobin of muscles. Rest is stored in ferretin.
278 ANALYTICAL CHEMISTRY
Biochemical Functions.
• Heme is the important constituent of several proteins and enzymes.
Haemoproteins include haemoglobin, myoglobin, cytochromes,
catalase, xanthine oxidase, tryptophan. Proteins like transferrin,
ferritin and hemosiderin contain non-heme iron.
• Haemoglobin and myoglobin are required for transport of 02 and
CO2 , Muscles store O2 in combination with myoglobin which contains
iron.
• Cytochromes and certain non-heme proteins are necessary for
electron transport chain and oxidative phosphorylation.
• Iron is associated with effective immunocompetence of the body.
• Peroxidase, the lysosomal enzyme is required for phagocytosis by
neutrophils.
• Absorption of iron is promoted by ascorbic acid, small peptides,
amino acids and decreased by tea and eggs.
• Iron, when present in excess, can actually stimulate free radical
formation.
• In hemosiderosis, excessive iron is deposited in ferritin and
haemosiderin.
• In hemochromatosis, iron is directly deposited in tissues (liver, spleen,
pancreas) causing bronze diabetes.
• Deficiency. Iron deficiency causes anaemia which may also be due
to chronic blood loss, defective absorption of iron and hook worm
infection.
IODINE
Iodine Content. Human body contains about 20 mg of 12 , About 80%
12 is stored in the form of iodothyroglobulin (a glycoprotein) in the thyroid
gland.
Sources of 12 , Richest sources are sea foods, shell fish, fish oil. Others
are fruits, vegetables, cereals, meat, milk, eggs, iodised salt.
Requirement of 12 for adults, women and children are 100-150 Ilg, 150
Ilg and 80 Ilg respectively.
Functions. 12 is required for the synthesis of thyroid hormones such as
thyroxin and tri-iodothyroxine. Thyroxin controls metabolism, utilisation of
sugars, regulates energy production and aids growth. It improves cognition
and makes skin, hair and teeth healthier.
Deficiency of 12 may cause cretinism in children. Dwarf child is
mentally retarded with enlarged thyroid gland.
In adults, thyroxine production may be hampered and cause
myxoedema. Symptoms of the disease are slower rate of metabolism, loss of
hair and enlarged thyroid glands.
o
12
DRUG ANALYSIS
INTRODUCTION
The drug, derived from drogue (dry herb), is defined as any substance
used in medicine to diagnose, cure and prevent the occurrence of diseases and
disorders and prolong the lives of patients suffering from serious or incurable
diseases. WHO defines drug as any substance or product which is used to
modifY or explore physiological system or pathological states for the benefit of
the recipient.
Pharmaceutical chemistry is the study of the chemical and physical
properties of drugs, their behaviour, preparation, composition, structure, their
influence on an organism, conditions of their storage, shelf life preservation,
identification and their therapeutic use.
SOURCES OF DRUGS
Majority of the drugs used in therapeutic action are synthetic but many
plant products also provide important therapeutic drugs.
• Plants yield morphine, atropine, quinine, reserpine, streptomycin etc.
• Synthetic drugs are aspirin, procaine, sulphonamides.
• Micro-organisms produce penicillin, bacitracin etc.
• Animals yield insulin, heparin etc.
• Genetically engineered drugs include human growth hormones,
human insulin etc.
NARCOTICS
Narcotic, that refers to opium or opioid, is a drug which produces
stupor, insensibility or sleep. It may be natural, semi-synthetic and synthetic
that behave pharmacologically. Narcotics can be administered orally,
transdermally or by intravenous injections.
Early Symptoms of Narcotics. The addict may suffer from running
nose, watery eyes, irritability, restlessness, yawning, loss of appetite, severe
sneezing, tremors, hypertension, vomiting, depression, pain in muscles and
bones etc. However, administration of a suitable narcotic can dramatically
reverse these early symptoms.
Effects of Narcotics.
Effects of narcotics depend mainly on dose, route of administration,
previous exposure to the drug and expectation of the user. Following effects
are observed : Nausea, vomiting, apathy, drowsiness, constriction of pupils,
dialation of the subcutaneous blood vessels causing flushing of the face and
(279)
280 ANALYTICAL CHEMISTRY
• Lysergic acid diethylamide (LSD) can be extracted into CH2 Cl2 after
solubilizing with a carbonate buffer.
• Mascaline can be extracted with ethanol.
• Serum or urine is adjusted to pH 4 to 7·5 to extract many of the drugs.
• Extractions at pH 3 and 9 yield acidic or basic components of the
drugs.
• Barbiturates (in micro samples 0·01 to 0·05 Ilg) may be methylated
with dimethyl sulphate before extraction into hexane.
• Drugs in powder or pill form are generally dissolved in aqueous
KOH or HCI followed by extraction at appropriate pH.
• Drugs in tissues are usually protein bound. So it is necessary to
precipitate protein before extraction. Macerate (grind) the tissue and
treat it with sodium tungstate followed by hydrolysis with hot aCid. It
forms tungstic acid which is a protein precipitating agent.
Methodology.
Detector. Flame ionization detector is mostly used in drug screening.
Column. OV-17 column (phenyl methyl silicone fluid).
Other columns are :
• PPE 20 (medium polarity)
• Carbowax 20 M (high polarity).
• Glass column with direct column injection or glass injection port.
General Screening of Pills and Powder for a Mixture of Drugs.
To separate, screen and analyse the mixture of amphetamines (basic),
barbiturates (acidic) and alkaloids ( basic), three separate chromatograms are
run using different required temperatures. It is also possible to analyse a
combined extract with a single chromatogram by using temperature
programming to reduce the measurement time from 45 minutes to 15
minutes. The peaks are then identified by comparing with those of known
standards of drugs.
Screening of Methadone Drug in a Urine Sample.
The urine sample of a local methadone treated person was worked up by
the Dole technique (a combined ion-exchange-solvent extraction isolation)
and subjected to gas chromatography (Fig. 1).
Column Conditions. Glass column, medium polarity OV-17 on 80/100
mesh. High performance chromosorb W at 215°C.
Identification of Drugs.
Several drugs identifiable in the sample are methadone, cocaine,
morphine, monoacetyl morphine and quinine. The peaks are positively
identified by comparing with those of known standards of the drugs.
Head Space Technique for Drug Screening.
In head space technique, the drug sample is contained in a closed
container and the volatile constituents are allowed to equilibrate with the
atmosphere. An aliquot of the atmosphere is taken with a syringe, injected
into the gas chromatograph and peaks identified.
DRUG ANALYSIS 285
2
5
L -__~~-L~~~~~~~~~
12 4 6 8 10 12 14 16 18
Retention time (minutes)
Fig. 1. Gas chromatogram from a urine sample containing methadone.
The gas chromatogram showed two peaks (Fig. 2a). The retention time for
peak 1 corresponds to the retention time of glutethimide. The sample was
then subjected to GC-MS analysis. The peak at mass to charge ratio of 217
also matched the ratio for the glutethimide molecular ion and was identical
with the peak 1 from a known sample of glutethimide (Fig. 2b).
The retention time for peak 2 did not match with any known drug. The
GC-MS of peak 2 however showed a molecular ion peak at rn/z ratio of 233
(Fig. 2c).
~
'iii Peak 2
c
CD
~
Iii
c
Cl
en
Time
(a)
~ ~ 233
en 'iii
c c
.Sl .Sl
£ .!:
is obtained from the total ion current (TIC) monitoring mode (Fig. 3). The
peak at 11·5 minutes corresponds to the retention time expected for cocaine.
(I)
c: 6.0E6 -
0
as
"0
c: 4.0E6 -
::l
.0
« 2.0E6 -
I I I L J I I
0 4 6 8 10 12 14 16
Time (min.)
(a)
1.2E6 - 82 Scan 459 (11.541 min.)
182
1.0E6 -
(I)
0
8.0E5 -
c:
as
"0
c: 6.0E5 -
::l
.0
« 94
4.0E5 -
303
2.0E5 - 199
~ ,ll,~"
152 212
111,111.1,,, ~l. Jl, I II I III
o 100 150 200 250 300
Mass/Charge
(b)
82
1.2E6
1.0E6
~ 182
~ 8.0E5
"0
c:
::l
~ 6.0E5
94
4.0E5
303
2.0E5
~RO-C~O
o ..........
CR g
$ NR-C~
0
'CR,
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Aspirin Phenacetin Caffeine
Reagents required. Methylene chloride, NaHC0 3 , 4% (w/v),
1 M H 2S04 and HCl.
Separation of Drugs by Solvent Extraction.
Powdered tablet is dissolved in CH2 CI 2 . Aspirin is separated from
phenacetin and caffeine by extracting it into aqueous NaHC03 solution. The
aqueous layer is acidified and aspirin is separated by back extraction into
methylene chloride. It is measured spectrophotometrically at 277 nm.
Phenacetin and caffeine, which are remaining in the original methylene
chloride layer are determined in the mixture.
Preparation of Standard Solutions.
Dissolve 100 mg/L aspirin, 20 mg/L phenacetin and 10 mglL caffeine in
methylene chloride. Weigh 25 mg of each drug in a flask and make up the
volume to 100 mL with methylene chloride. Since aspirin decomposes in
solution, so analysis should be performed as early as possible.
Procedure.
A tablet may contain 220 mg aspirin, 160 mg phenacetin and 30 mg
caffeine. Grind quarter (114) part of the tablet to a fine powder.
• Add 20 mL methylene chloride into the powder. with constant stirring.
Transfer this mixture to a 60 mL separatory funnel.
• Extract aspirin from methylene chloride solution with two 10 mL
portions of cold 4% NaHC0 3 containing two drops of HCl and then
with 5 mL portion of water.
• Wash the combined aqueous extracts with three 10 mL portions of
CH2CI2 . Add this wash solutions to the original methylene chloride
solution.
• Leave the aqueous extract in the separatory funnel.
• Filter methylene chloride solution into a 50 mL volumetric flask and
dilute to the mark )Vith methylene chloride.
• Again dilute 1 mL aliquot of this solution to 50 mL with methylene
chloride in a volumetric flask.
• AcidifY aqueous bicarbonate solution with 6 mL of 1 M H2S04 to pH 1
to 2 in the separatory funnel to prevent hydrolysis of aspirin.
• Extract the acidified solution with eight separate 10 mL portions of
CH2CI2 , filter into a 100 mL volumetric flask and make up the volume
to 100 mL.
• Dilute further a 5 mL portion of this solution to 25 mL with CH2 Cl 2
in a volumetric flask.
292 ANALYTICAL CHEMISTRY
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