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Satish Pradhan Dnyanasadhana College, Thane Basic Concepts in Instrumental Methods

By Dr. BhushanLangi

Basic Concepts in Instrumental Methods Relation between the Analyte, Stimulus and
measurement of change in the observable property • Analyte: • It is a substance
whose chemical constituents are being identified and measured. • There are highly
efficient and sensitive chromatographic and electrophoretic techniques are used for
the separation of the components of complex mixtures. • An instrument for chemical
analysis converts information about the physical and chemical properties of the
analyte into information which can be easily interpreted. • Thus, an analytical
instrument is a communication device between the analyte and the investigator. • To
get the desired information about the analyte, a stimulus must be provided which is
usually in the form of energy - electromagnetic, electrical, mechanical or nuclear.
• The stimulus produces a response from the analyte which can be measured and
interpreted.

Block Diagram of an Analytical Instrument Source of Energy Analytical Instruments

Analyte Under Study • The analytical instruments measure the response of the
analyte to the radiation. • The response should be proportional to the
concentration of the analyte.

Types of Analytical Instruments:

A. Depending on Optical Properties: • Optical methods of analysis are based on the

interaction of the analyte with radiation. • The components of a sample interact
with radiation in different ways — absorption, emission, scattering etc. •
Spectrophotometry: • Photoelectric Calorimeter, the amount of visible light
absorbed depends on the concentration of the light absorbing component in the
sample. • UV absorption is mostly used for identification of the components. • IR
absorption is used for the elucidation of the structure of the component by
identification of the functional groups present in the component. • In
turbidimetry, the amount of light scattered by the analyte is used to determine its
concentration in solution. • ii. Polarimetry: • Substances with asymmetric carbon
atoms (Chiral carbons) rotate the plane of polarisation of polarised light. • This
property of rotation of light is used in a polarimeter to determine the
concentration of the optically active compound in solution.

e.g. Sucrose rotates the plane of polarisation of polarised light to the right
(dextrorotatory) Glucose is also dextro-rotatory but fructose rotates the plane of
polarisation to the left (laevo-rotatory). • B. Depending on Electrochemical
Properties: • Electrochemical methods of analysis are based on the measurement of
electrical properties of the sample. • i. Potentiometry: • In potentiometric
measurements, the sample solution forms part of a galvanic cell. • The potential
developed in the galvanic cell is related to the concentration of the sample
solution. • In potentiometric titrations, the emf of the electrochemical cell set
up is measured after the addition of successive instalments of the titrant. • The
end point is determined from the graph of emf versus volume of the titrant. • eg.
Acid-Base titrations

ii. Conductometry: • The conductance of the sample solution is measured and related
to the concentration of the sample solution. • In conductometric titrations, the
conductance of the solution is measured after the addition of successive
instalments of the titrant • eg. Acid-Base titrations • iii. Voltammetry: • The
sample under study is made a part of the electrolytic cell and the current passing
through the cell is measured at different potentials. • The current measured is
related to the concentration of the electroactive species present in solution. •
This method again sub classified as • Polarography: • In this voltammetric
technique, a substance is identified by measuring the electrical potential at which
it is electrolytically deposited from its solution. • eg. estimation of metals
present in a solution.

b) Amperometry: • In this voltammetric methods of analysis, the potential applied

is kept constant and current passing through the electrolytic cell is measured
after the addition of successive instalments of the titrant. • The end point is
determined from the graph of current versus volume of titrant. Amperometric
titrations are used for the estimation of several metallic analytes which can be
deposited (reduced) on a mercury drop cathode. • eg. Bi3+ vs EDTA • Mg2+ vs 8-
hydroxyquinoline • Pb2+ vs K2Cr2O7 • iv. Coulometry: • In this technique, the
sample solution is made part of an electrolytic cell and electrolysis is carried
out. • The quantity of electricity passed through the solution is correlated with
the concentration of electrooxidized (or electro reduced) species in solution. •
eg. electrogravimetry.

C. Depending on Thermal Properties: • In thermal analysis, some physical or

chemical property of the sample is measured as a function of temperature. When
matter is heated, it undergoes certain physical or chemical changes or both over a
wide range of temperatures. • By determining the temperatures at which these
changes occur, it is possible to characterise the components present in the sample
and thus identify them. • Thermal methods are further classified as: • i) Thermal
Gravimetric Analysis (TGA) or Thermogravity: • Analysisin which the mass of the
sample is measured as a function of temperature. • ii) Different Thermal Analysis
(DTA): • Analysis in which the difference in temperature between the sample (Ts)
and a suitable reference material (Tr) is measured as a function of temperature. •
The temperature difference, ΔT = Ts — Tr is measured at different temperatures.

iii) Differential Scanning Calorimetry (DSC): • Analysisin which the energy

required to establish a zero temperature difference between the sample and
reference material (ΔT = Ts - Tr = 0) is measured as a function of temperature. •
iv) Thermometric Titrations (TT): • Analysisin which the changes in temperature are
measured during the course of a titration • eg. acid-base titrations or
complexometric titrations.

Spectroscopy: • Spectroscopy is the study of the interaction of matter with

electromagnetic radiation. • When radiation is incident on atoms and molecules, a
part of it is absorbed and the rest is transmitted (emitted). • The radiation
absorbed by a molecule is used for performing different types of motion. • The
amount of radiation absorbed depends on the concentration of the absorbing species
in solution. • This absorption of radiation can be used to determine the chemical
structure of the absorbing species. • Thus, spectroscopy can be considered as an
important tool for the chemical analysis of molecules. • Radiation travels in the
form of waves. • A wave is associated with three important wave properties -
wavelength λ, frequency ν and wave number ν̅ which are correlated by the equation,

Basic Terms involved: • Energy of Light: • Electromagnetic Spectrum: It is used to

indicates all types of electromagnetic radiations

Planck’s Quantum Theory of Radiation According to this theory, the emission and
absorption of energy takes place not in a continuous manner but in discrete
instalments called Quanta or Photon. Even the propagation of energy through space
is discontinuous and takes place in quanta. Each quantum can be considered as a
packet of energy equal to hν, where h is the Planck’s constant, 6.625 x 10-34Js and
ν is the frequency of the energy radiation in Hz (s-1). Thus,

2. Radiant Power P • Radiant power P is the energy of the radiation that reaches a
given area per second. • 3. Intensity of Light • Intensity of light, I is defined
as the number of photons of radiation incident per unit area per unit time. •
Intensity is independent of the frequency or wavelength of the incident radiation.
• Energy is given by the term, hv and hence depends on the frequency and wavelength
of the radiation. • 4. Polychromatic and Monochromatic Light • A polychromatic beam
of light contains radiation with several wavelengths • e.g. white light has several
wavelengths in the range 380 — 780 nm, violet light has wavelengths in the range
380 - 435 nm and a beam of red light has wavelengths in the range 650 - 780 nm. • A
monochromatic beam has radiation of a single wavelength • e.g. a violet beam of
wavelength X = 395 nm, a red beam of wavelength X = 700 nm. Monochromatic beams are
produced by using special devices called monochromators.

5. Absorbance: • It is defined as logarithm of reciprocal transmittance. It is also

known as optical density. • 6. Wavelength of maximum absorption, λmax •
Spectroscopic analysis of a substance is carried out using radiation of a
particular wavelength which is absorbed to the maximum extent i.e. maximally. This
wavelength is called the λmax of the substance. • The constituent structural groups
in a molecule also absorb their own characteristic wavelengths. Thus, a substance
may show strong absorption of several wavelengths depending on its structure. •
Table below indicates the λmax values of important groups

7. Transmittance: • It is the fraction of incident radiation that is transmitted by

the sample. • Beer Lambert’s Law • When a beam of light passes through an absorbing
medium (solution) a part of the light is absorbed and the rest is transmitted. •
The amount of light absorbed depends on the concentration of the solution and the
length traversed by the light through the solution. • The quantitative relation
between the amount of light absorbed, concentration and the length of the absorbing
medium is governed by three laws - Lambert’s law, the Beer’s law and the Beer -
Lambert’s law.

Lambert’s Law: This law state that “Equal fractions of the incident light are
absorbed by successive layers having equal thicknessof the absorbing medium (the
layers have the same thickness)”. Consider a layer of thickness dx absorb a small
amount dI of the incident light. The fraction is proportional to the thickness of
the layer dx. ----------------- (1) ----------------- (2) Where, I = intensity of
the light incident on the layer and K1 = constant The negative sign indicates that
due to absorption, the intensity of light decreases as it passes through the
absorbing medium.

If, Io = initial intensity of the light incident on the absorbing medium of length
l and It = intensity of the transmitted beam then by integrating equation (2)
----------------- (3) Converting to base 10 ----------------- (4)

Beer’s Law: This law state that “Equal fractions of the incident light are absorbed
by successive layers having equal concentration of the absorbing medium”. Consider
a layer of concentration dc absorb a small amount dI of the incident light. The
fraction is proportional to the thickness of the layer dc. ----------------- (5)
----------------- (6) Where, I = intensity of the light incident on the layer and
K2 = constant The negative sign indicates that due to absorption, the intensity of
light decreases as it passes through the absorbing medium having total
concentration c.

If, Io = initial intensity of the light incident on the absorbing medium of

concentration c It = intensity of the transmitted beam then by integrating equation
(6) ----------------- (7) Converting to base 10 ----------------- (8) The above
equation is referred as Beer’s equation for absorption of light.

Beer-Lambert’s Law: A combination of Lambert’s law and Beer’s law results in the
Beer - Lambert’s law which states “Equal fractions of the incident light are
absorbed by successive layers of equal thickness and equal concentration of the
absorbing medium”. From the Labert’s Law, From the Beer’s Law, By combining both
equations,
----------------- (9) Where, a = K1 . K2 = constant = Absorptivity of absorbing
medium c = concentration in mol dm-3 l = length of the absorbing medium in cm
----------------- (10) This is the combined mathematical equation of Beer-Lambert’s
Law.

Validity of Beer—Lambert’s law: The Beer-Lambert’s law is strictly applicable to

dilute solutions whose concentrations are below 10-2 M. Such solutions obey Beer-
Lambert’s Law equation according to which absorbance is proportional to the
concentration of the solution and transmittance is inversely proportional to the
concentration. Beer - Lambert’s plots for dilute solutions Figure (a) showsa
straight-line graph passing through the origin confirms that the absorbing species
obeys Beer-Lambert’s law. The dotted curve I indicates a positive deviation from
the law whereas curve II indicates a negative derivation.

Derivation from the Beer - Lambert’s law: • Deviations from Beer — Lambert’s law
are of two types. • When the observed absorbance is greater than the expected
value, it is called positive deviation and • When the observed absorbance is lesser
than the expected value, it is called negative deviation. • The deviations can be
broadly classified into three categories: • Real deviation • Instrumental
deviations • Chemical deviations • Real Deviations • The linear relationship
between absorbance and concentration of solution is observed only in dilute
solution and does not hold true at concentrations above 10-2 M. • The molar
absorptivity ε depends on the refractive index of the absorbing medium. The
refractive index changes with the concentration of the absorbing solution. At high
concentrations, these changes are considerable but at concentrations below 10-2 M,
they can be neglected.

2. Instrumental Deviations • The light incident on the absorbing medium should be

strictly monochromatic otherwise deviations from Beer-Lambert’s law are observed. •
Monochromators are used to produce monochromatic beams which are absorbed to the
maximum extent by the absorbing medium (λmax). • 3. Chemical deviations • The Beer
Lambert’s law is not obeyed if the absorbing species undergoes dissociation,
association or reaction in solution. • The molecules of the absorbing species
should remain as simple molecules in solution and should not undergo any change in
molecular condition. • Also the absorbing species should not react with the
solvent.

Instrumentation for Absorption Spectroscopy Absorption Spectroscopy Colorimeter

Spectrophotometer • Work in the visible region. • Used for quantitative analysis of
coloured compounds which absorb visible light • Work in UV, Visible and IR regions.
• Used for determination of the structure of colourless as well as coloured
compounds in addition to quantitative analysis Single Beam Colorimeter Double Beam
Colorimeter Single Beam Spectrophotometer Double Beam Spectrophotometer

Single Beam Photoelectric Colorimeter Principle: In a single beam colorimeter, a

narrow band of wavelengths is incident on the absorbing sample. The absorbance (or
percentage transmittance) of the sample is determined after adjusting the
absorbance of the solvent (blank) to 0 (or 100% T) for the same band of
wavelengths. All measurements are done using a single optical path for the beam of
light. Diagram: Filter Holder

Construction: • A photoelectric colorimeter has the following components: • A

source of visible light e.g. an incandescent lamp with a tungsten filament. The
filament on electrical heating emits visible radiation. • The emerging beam is
collimated using a convex lens. • A colour filter which selects a narrow band of
wavelengths which enters a fine slit and is then incident on the sample. • A sample
holder called cuvette. It is a rectangular transparent container of glass or
quartz. Sample holders in test tube form are also widely used. Most sample holders
have a standard path length of 1 cm. • A photocell or transducer which converts the
transmitted beam emerging from the sample into an electrical current. This
electrical pulse is due to the emission of electrons from the photoelectrode
surface caused by the transmitted beam falling on the photoelectrode. • A read-out
system like a dial to directly read A or % T of sample. The read out system is
needed because the electrical current from the photocell has to be converted into
the absorbance or percentage transmittance of the sample.

Working: • The cuvette is first filled with the solvent and the % T is adjusted to
100%(or A is adjusted to 0). • Then the cuvette is filled with the sample solution
and its % T (or A) is determined. • The concentration of the sample solution can
then be found out using Beer—Lambert’s equation.

Details of Major Components: • Colour Filters: • They are of two types: • Glass
filters: A glass filter is a solid sheet of glass which is coloured by dissolving
or dispersing a pigment in the glass. • Interference filters: • An interference
filter consists of two thin films of silver separated by a film of transparent
material of low refractive index. • Light undergoes interference at the silver
surface. • The unwanted wavelengths are removed by selective reflection and only
the required wavelengths pass out of the filter. • Interference filters give narrow
bands of wavelengths as compared to glass filters. • The absorbance of the sample
is first determined using the different colour filters. • The filter giving maximum
absorbance or minimum % T is chosen for use in the colorimeter.

2. Detectors • Photoemissive Cell: A photoemissive cell consists of an evacuated

glass tube fitted with a photosensitive cesium cathode. A metal wire functions as
the collector anode. When light falls on this cathode, electrons are emitted which
pass over to the anode. A potential difference of 100 volts is maintained between
the two electrodes. The electric current produced can be amplified and is
proportional to the amount of light falling on the photocathode. The output of the
amplifier is recorded on a dial on which the absorbance or % T can be directly
read. Colorimeter

b) Photomultiplier Tube (PMT): • Construction: • Photomultiplier tube is an

evacuated tube having photocathode at one end. • The tube having series of
electrodes inside. These electrodes are known as dynodes. • Both photocathode and
dynodes are made up from Arsenic or Antimony. • Dynodes are arranged in such a way
that each dynode having increasing potential than previous dynode.

Working: • When radiation strikes photocathode of PMT electrons are ejected from
the surface of photocathode due to photoelectric effect. • These negatively charged
electrons are travel towards positively charged dynodes. • From one electron 10
secondary electrons are formed at each dynode. Thus, electron from photocathode
incident on 1st dynode form number of secondary electrons at dynode. • The process
is repeated at 2nd dynode and again more number of secondary electrons are form at
2nd dynode. • Thus, as number of dynode increased number of secondary electrons
also increased. • Finally, all electrons collected by collector electrode.

Photovoltaic or Barrier Layer Cell: Photovoltaic cells are most commonly used in
colorimeters. • Construction: • A barrier cell consists of an iron plate (A) on
which a thin layer of a semiconductor like selenium (B) is deposited. • A very thin
layer of silver (C) is sputtered on the selenium layer to serve as the collector
electrode. • The iron plate serves as the second electrode. The silver film is
mechanically strengthened by an iron ring D.

Working: • The transmitted light from the sample emits electrons from the selenium
surface. • These electrons pass through an imaginary or hypothetical barrier or gap
in between the selenium and silver layers and are then collected by the silver
collector electrode. • Thus under the influence of light, a cell is formed with the
iron plate (A) as positive electrode and the metal ring (D) as negative electrode.
• This type of cell generates its own emf and no external power supply is required
for the flow of current. • If the cell is connected to a galvanometer, a current
will flow provided the resistance in the external circuit is small. • The
electrical current is proportional to the amount of light falling on the selenium
surface.

Double Beam Photoelectric Colorimeter: Principle: In such colourimeter beam of

light splits into two identical beams one passing through the sample and the other
passing through the solvent (blank). Diagram:

Construction: A source of visible light e.g. an incandescent lamp with a tungsten

filament. The emerging beam is collimated using a convex lens. A colour filter
which selects a narrow band of wavelengths A mirror splits this narrow band into
two identical beams-one passing through the sample and the other passing through
the solvent (blank). The sample absorbs a part of the beam whereas the solvent
transmits it completely. A sample holder called cuvette. It is a rectangular
transparent container of glass or quartz. Sample solution is held in sample holder.
Blank Cuvette used to hold the solvent. The two beams then fall on the respective
photocells where photoelectrons are emitted.The two electric currents thus produced
pass through the variable resistances - resistance AB for the current coming from
the solvent side and resistance CD for the current coming from the sample side. A
potential gradient is set up across AB and CD depending on the two currents. AB is
calibrated to read % T (or absorbance). A galvanometer connected across the two
resistances serves as the null indicator.

Working: The solvent is first taken in both the cuvettes. Jockey J1 is set at point
A(100% T). Jockey J2 is adjusted along CD till there is no current flow as
indicated by the null in the sensitive galvanometer. The sample is then placed in
the sample cuvette. It absorbs a part of the light and the transmitted beam
emerging from it falls on the photocell. This beam has less intensity than when
only solvent was present in the sample cuvette. Hence there will be a proportionate
decrease in the electric current produced in the photo-cell and the voltage across
CD decreases. As a result, the null in the galvanometer is disturbed. Now Jockey J1
is moved along AB to a lower value so that the null is restored; The %T is directly
read on the scale when the null is restored.

Double beam photometers have several advantages over single beam photometers. •
Changes in the intensity of incident light due to voltage fluctuations in the power
supply are compensated by splitting the incident beam into two identical beams by
the use of a mirror and two balanced photocells. • Any errors due to solvent or
impurity are balanced out as identical beams pass through the blank and the sample
and the absorbance of blank is initially adjusted to zero. • The readings are not
affected by changes in sensitivity of photocells or galvanometer as the null method
is used. • The scale readings of the instrument are linear with the concentration
of the sample solution.

Single Beam Spectrophotometer: Principle: In this instrument, a monochromator i.e.

prism or diffraction grating produces a monochromatic beam which is then made
incident on the sample. The absorbance A (or % T) of the sample solution is
determined for different wavelengths after adjusting the absorbance (or % T) of the
blank to zero (or 100% T) for each of the wavelengths. All measurements are done
using a single optical path for the monochromatic beam of light. Diagram:

Construction: • A single beam spectrophotometer has the following components: •

Sources of Radiation: A tungsten lamp is used for obtaining visible light. U.V.
light is obtained from a hydrogen or deuterium discharge lamp. • Monochromator: A
monochromator, either a prism or diffraction grating disperses the light into its
constituent wavelengths. By rotating the monochromator, the different wavelengths
are focused one by one on the sample. • Sample Holder (Sample cell): Glass cuvettes
are used in the visible region. Quartz cuvettes are used in the U.V. region since
glass absorbs U.V. light. • Photocell: The transmitted beam emerging from the
sample falls on the photocell. An electric current is produced due to the photo-
emission of electrons at the photocathode. If this current is small, it can be
amplified using an electronic amplifier. • Read Out Device: It is a dial on which
absorbance A or % T can be directly read or a digital display.

Working: The sample holder is filled with the solvent (blank). The absorbance A
value (or % T) of the solvent is adjusted to 0 (or 100 % T) for a particular
wavelength, obtained by the rotation of the monochromator. The sample is then taken
in the sample holder and its A value (or % T) is determined for the same
wavelength. The procedure is repeated for different wavelengths obtained by the
rotation of the monochromator. The A (or % T) values can be read either on a dial
or a digital display. The λmax values for the sample can thus be found out.

Details of Major Components: • Monochromators: • Monochromators are dispersing

devices which split a beam of light into its constituent wavelengths. •
Monochromators are of two types viz.Prisms and Diffraction gratings • a) Prism: The
collimated beam is dispersed by the prism into its constituent wavelengths. The
prism is rotated to allow a particular wavelength to fall on a focusing lens and
then emerge out of an exit slit. The slit width controls the band width of thebeam.
It is observed that the dispersion is greater for short wavelengths and smaller for
long wavelengths. Hence the slit width is increased for short wavelengths and
decreased for long wavelengths. Glass prism are used in the visible region whereas
quartz prisms are used in the U.V. region.

b) Diffraction Gratings: A diffraction grating is made by cutting a large number of

perfectly parallel straight lines (grooves) into an aluminium surface. For the U.V.
and visible regions, 15000 - 30000 lines are ruled per inch. When the light is
reflected at the grating surface, dispersion takes place due to the constructive
interference of the reflected rays. The net path difference between two rays is an
integral multiple of the wavelength and is given by the equation; Net path
difference = nλ = d (sini ± sin θ) Where, n = order of interference and is a
positive integer λ = wavelength of the incident light d = distance between two
successive grooves (i.e. grating constant), i = angle of incidence of the beam θ =
angle of reflection of the emergent beam.

The positive sign indicates that the incident and emergent beams are on the same
side of the grating normal and negative sign indicates that the two beams are on
either side of the grating normal. • Angle θ is different for different
wavelengths. Thus, the light is dispersed into its constituent wavelengths at the
grating surface. • Grating monochromators have certain district advantages over
prisms. • A grating gives a much better dispersion of light than a prism. •
Gratings are made of non-corrosive materials like aluminium which are not easily
attacked by moisture. • Gratings can be used over a longer wavelength range as
compared to prisms.

Double Beam Spectrophotometer: Principle: In the double beam spectrophotometer, the

monochromatic beam coming from the diffraction grating monochromator is split into
two beams by means of mirror M1. The reference beam is reflected by mirror M2 into
the blank cell containing the solvent and the sample beam passes into the sample
cell. Diagram:

Construction: An optical attenuator mounted in the path of the reference beam

reduces its intensity to match that of the sample beam i.e. the amount of
transmitted light coming from the blank is decreased so that its intensity is equal
to that of the transmitted light coming from the sample. The attenuator is
connected to a recorder pen which moves across the chart paper wound on a rotating
drum. The two beams are then reflected by mirrors M3 and M4 into a suitable
detector. Photocells are used as detectors in the U.V. and Visible regions. The
detector produces an electrical current which is proportional to the radiation
received by it. The electric current is magnified by use of an electronic amplifier
and the electric signal is sent to the recorder pen. A motor synchronizes the speed
of the rotating monochromator with that of the rotating drum carrying the chart
paper. As a result, the wavelength marked on the chart is identical with that
received by the detector.

Working: The monochromator is rotated to allow different wavelengths to fall on the

blank and sample. The process is called scanning. The recorder pen plots a graph
ofabsorbance or % T versus wavelength (λ), frequency (ν) or wave number ( ν̅ ) on
the rotating drum. The wavelengths at which maximum absorption occurs are noted
from the respective spectra and the groups responsible for these absorptions or
transmissions are identified. Thus the structure of the sample can be elucidated
from spectrum analysis.

Comparison between Photometers and Spectrophotometer • Photometers usually work in

the visible range while spectrophotometers can be used in U.V., visible and IR
regions. • Photometers use colour filters which allow a band of wavelengths of the
complementary colour of the sample to be incident on the sample. Spectrophotometers
employ monochromators (prisms or diffracting gratings) which produce perfectly
monochromatic beams hence spectrophotometric analysis is more accurate as the band
width of the light beam is minimum. Further, the Beer Lambert's law is strictly
obeyed for the monochromatic beams used in spectrophotometers. • Photometers are
generally used for quantitative analysis of coloured compounds whereas
spectrophotometers are used for structure determination of colourless as well as
coloured compounds in addition to quantitative analysis. • Spectrophotometers are
much more costly than photometers.

Applications of UV - Visible Spectrophotometry • Identification of Structural

Groups in Molecules Spectroscopic analysis of a substance is carried out using
radiation of a particular wavelength which is absorbed to the maximum extent. This
wavelength is called the λmax of the substance. Spectrum of the unknown compound is
matched with the known. If each end every peak of the two spectra match, then the
compound can be identified. U.V. visible spectra alone is very rarely used for
identification of a compound. However, it is used for the detection of functional
groups.