Analytical

Instrumentation:
Introduction
ER. FARUK BIN POYEN, Asst. Professor
DEPT. OF AEIE, UIT, BU, BURDWAN, WB, INDIA
faruk.poyen@gmail.com
Contents 2
 Introduction
 Definition
 Selecting an Analytical Method
 Understanding the Measurement Process
 Uses of microcomputer in Analytical Instrumentation
 Beer Lambert Law
 Spectroscopy
 Radiation Sources
 Optical Fibres
 Monochromators
 Detectors
Introduction: 3
 Analytical instruments provide information on the composition of a
sample of matter.
 They are employed to obtain qualitative and quantitative information
about the presence or absence of one or more components of a given
sample.
 It comprises the four basic elements viz. chemical information source,
transducers, signal conditioners and display system.
 The first two elements constitute the characteristic module whereas the
last two constitute the processing module.
 4
 Molecules possess three types of internal energy – electronic, vibrational
and rotational.
 Electronic transitions correspond to the UV and visible regions,
vibrational transitions to the near IR and IR regions and rotational
transitions to the IR , far IR or even microwave regions.
 When a beam of radiant energy strikes a surface, the radiation interacts
with the atoms and molecules of the substance.
 The radiation may be then transmitted, absorbed, scattered, deflected,
refracted or reflected or it may excite fluorescence depending on the
property of the substance.
Definition: 5

 Analytical instrumentation is the study of the separation,

identification, and quantification of the chemical components
of natural and artificial materials. Qualitative analysis gives
an indication of the identity of the chemical species in the
sample and quantitative analysis determines the amount of
certain components in the substance.
Steps of Analytical Instrumentation 6
Define Problem

Select Appropriate Method

Obtain and store sample

Pretreat sample

Perform required measurements

Compare results with standards

Apply necessary statisiical methods

Present results in form understandable to analyst

Present results to customer

Selecting an Analytical Method 7
 The requirements of the analysis determine the best method. In choosing
a method, consideration is given to some or all the following design
criteria: accuracy, precision, sensitivity, selectivity, robustness,
ruggedness, scale of operation, analysis time, availability of equipment,
and cost.
 Accuracy
 Precision
 Sensitivity
 Specificity and Selectivity
 Robustness and Ruggedness
 Scale of Operation
 Equipment, Time, and Cost
 Making the Final Choice
Selecting an Analytical Method 8
 Other Considerations:
1. Speed
2. Ease and Convenience
3. Skill of operator
4. Cost and availability of equipment
5. Per – sample cost
 Other characteristics to be considered for Method Choice.
Analog Domain
Information is encoded as the magnitude of current, voltage, charge or power.
• In analog domains information encoded is continuous in amplitude and time.
• Amplitude is related to property of interest
Selecting an Analytical Method 9
 Time Domain
• Information is encoded in the form of a time dependent fluctuation of a signal.
• Frequency of fluctuation is related to property of interest
• e.g.: in photon counting the rate of arrival of photon at the detector is related to intensity.
 Digital Domain
• Information is encoded in the form of a number.
• Devices used (LED, toggle switch, logic circuit) display two states only behavior (on or
off).
– Lights (on/off)
– Logic levels (HI/LO) (1/0)
• Fundamental unit of information in digital domain
– Bit = each piece of Hi-Lo info. 8 bits = 1 byte
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Understanding the Measurement Process: 13
Signal generator (glass electrode)
↓
Signal transduction (information encoding) (diff. in pH to potential diff.)
↓
Signal modification (information decoding) (voltage to pH)
↓
Signal display

 Information is encoded, processed, transferred, decoded and displayed.

 Steps in the measurements involve transformation and processing of the signal between
the different data domains.
Use of microcomputers in Analytical 14
Instrumentation
1. Control of the instrument
2. Processing data
3. Storing data
4. Displaying data
5. Transferring data
Beer Lambert Law: 15
 Beer Law: This law states that the absorption of light is directly
proportional to both the concentration of the absorbing medium and the
thickness of the medium in the light path.
 𝐴 = 𝜀𝑙𝑐; 𝜀 𝑖𝑠 𝑡ℎ𝑒 𝑚𝑜𝑙𝑎𝑟 𝑎𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑣𝑖𝑡𝑦

 Lambert Law: It states that each layer of equal thickness of an

absorbing medium absorbs an equal fraction of the radiant energy that
traverses it.
 Transmittance, 𝑇 = 𝐼 I = Radiant Energy transmitted; 𝐼0 = Incident radiant energy
𝐼0

 % Transmittance, % T = 100 T
 Absorbance 𝐴 = log10 𝐼0 𝐼, 𝐴 = log10 1 𝑇 = log10 100 %𝑇
Prerequisite of Beer Law 16
 Prerequisites: There are at least six conditions that need to be fulfilled for Beer's law to
be valid. These are:
1. The absorbers must act independently to each other;
2. The absorbing medium must be homogeneous in the interaction volume
3. The absorbing medium must not scatter the radiation – no turbidity
4. The incident radiation should preferably be monochromatic or have at least a width that
is narrower than that of the absorbing transition
5. The incident radiation must consist of parallel rays, each traversing the same length in
the absorbing medium.
6. The incident flux must not influence the atoms or molecules; it should only act as a non
– invasive probe of the species under study. In particular, this implies that the light
should not cause optical saturation or optical pumping, since such effects will deplete
the lower level and possibly give rise to stimulated emission.
If any of these conditions is not fulfilled, there will be deviations from the Beer's law.
Beer Lambert Law 17

 Beer Lambert Law: A combination of the two laws, defines the

relationship between absorbance (A) and transmittance (T). It states that
the concentration of a substance in solutions is directly proportional to
the absorbance (A) of the solution.
 𝐴 = 𝜀𝑏𝑐
𝜀 = molar absorbtivity with units of L/mol/cm
b = path length of the sample
c = concentration of the compound in solution, mol/L
Derivation of Beer Lambert Law: 18
 The Beer Lambert law can be divided from an approximation for the absorption
coefficient for a molecule by approximating the molecule by an opaque disk whose
cross – sectional area σ represents the effective area seen by a photon of frequency ω.
If the frequency of the light is far from resonance, the area is approximately 0, and if ω
is close to resonance, the area is a maximum. Taking an infinitesimal slab, dz of
sample,
Derivation of Beer Lambert Law: 19
 Consider a beam of parallel monochromatic light with intensity I0 striking the sample
at the surface as shown in the figure above, such that it is perpendicular to the surface.
After passing through the path length b of the sample, which
contains N molecules/cm3, the intensity of the light reduces to IT.
Derivation of Beer Lambert Law: 20
 Now, consider a cross-section of the block having an area S and an
infinitesimal thickness dz placed at a distance z from the surface.
 If the sample has N molecules/cm3, the number of molecules present in
the infinitesimal block would be
= N × S × dz
 If each molecule has a cross-sectional area σ, where photons of light get
absorbed, then the fraction of the total area where light gets
absorbed due to each molecule would be (fractional area for each
molecule)
= σ / S.
 The total fractional area for all molecules in the block where light gets
absorbed would therefore be
= (number of molecules) × (fractional area for each molecule)
i.e.= (N × S × dz) x (σ / S) = σ × N × dz
Derivation of Beer Lambert Law: 21
 If Iz is the light entering the infinitesimal block considered, and the light
absorbed due to the absorbing particles is dI, the light exiting the slab
would be Iz – dI.
 The fraction of light absorbed would therefore be = dI/Iz
 Now, since the fractional area is the probability of light striking a
molecule, the fraction of light absorbed in the block = fractional area
occupied by all the molecules in the slab,
dI / Iz = – σ × N × dz.
 Note: The negative sign is placed to indicate absorption of light.
Derivation of Beer Lambert Law: 22
 If we integrate this infinitesimal block for the whole sample from z=0 to z=b,
where b is the path length of the entire solution we get
ln IT – ln I0 = – σ × N × b
OR
– ln ( IT / I0) = σ × N × b
 One can correlate the number of molecules/cm3 of sample to the concentration of the
sample in moles/liter using the relationship:
c = N × 1000/ (6.023 × 1023)
OR
N = (6.023 × 10 23) × c / 1000 = 6.023 × 1020 x c
 where c is the concentration in moles/litre, N is the number of molecules/cm3, and
(6.023 x 1023) is the Avagadro’s number. N × 1000 would be therefore molecules / liter
as 1 liter = 1000 cm3.
Derivation of Beer Lambert Law: 23
 Substituting this term for N in the above equation we get:
– ln (IT / I0 ) = σ × 6.023 × 1020 × c × b
 To simplify this equation further we convert to log using the term 2.303 × log (x) = ln
(x).
Therefore we get,
– log (IT / I0) = (σ × 6.023 × 1020 × c × b) / 2.303
OR
log (I0 / IT) = [(σ × 6.023 × 1020)/2.303 ] × c × b
 The term log (I0 / IT) is nothing but absorbance (A). While the term in the brackets [ ] is
a constant and can be replaced by a constant ε = (σ × 6.023 × 1020)/2.303. Thus we get:
A=ε×c×b
OR
A=ε*b*c
Derivation of Beer Lambert Law: 24
 Since we have used moles/liter as the units of concentration, ε stands for
molar absorptivity (or molar extinction coefficient).
 If the units of concentration were g/liter then the ε would be replaced by
“a” (absorptivity).
 By definition, the molar absorptivity (ε) at a specified wavelength of a
substance in solution is the absorbance at that wavelength of a 1
mole/liter solution of the substance in a cell having a path length of 1
cm.
 The units of molar absorptivity are liter mole-1 cm-1
Limitations of Beer Lambert Law: 25
 The linearity of the Beer Lambert law is limited by chemical and instrumental factors.
Causes of non linearity include
1. Deviations in absorptivity coefficients at high concentrations (> 0.01 M) due to
electrostatic interactions between molecules in close proximity.
2. Scattering of light due to particulates in the sample
3. Fluorescence or phosphorescence of the sample
4. Changes in refractive index at high analyte concentration
5. Shifts in chemical equilibrium as a function of concentration
6. Non – monochromatic radiation, deviations can be minimized by using a relatively
flat part of the absorption spectrum such as the maximum of an absorption band.
7. Stray light.
Deviations from Beer Lambert Law: 26
 The linearity of the Beer Lambert law is limited by chemical and instrumental factors.
The causes of the non linearity include
1. Deviations in absorptivity coefficients at high concentrations (> 0.01 M) due to
electrostatic interactions between molecules in close proximity
2. Scattering of light due to particulates in the sample
3. Fluorescence or phosphoresce of the sample
4. Changes in the refractive index at high analyte concentration.
5. Shifts in chemical equilibrium as a function of concentration.
6. Stray light
7. Non – monochromatic radiation.
Spectroscopy: 27
 Spectroscopy is the use of the absorption, emission, or scattering of electromagnetic
radiation by matter to qualitatively or quantitatively study the matter or to study
physical processes.
 The matter can be atoms, molecules, atomic or molecular ions, or solids. The
interaction of radiation with matter can cause redirection of the radiation and/or
transitions between the energy levels of the atoms or molecules.
 Absorption: A transition from a lower level to a higher level with transfer of energy
from the radiation field to an absorber, atom, molecule, or solid.
 Emission: A transition from a higher level to a lower level with transfer of energy from
the emitter to the radiation field. If no radiation is emitted, the transition from higher to
lower energy levels is called non radiative decay.
 Scattering: Redirection of light due to its interaction with matter. Scattering might or
might not occur with a transfer of energy, i.e., the scattered radiation might or might
not have a slightly different wavelength compared to the light incident on the sample.
Spectroscopy: 28
 Atomic Absorption: Matter can capture electromagnetic radiation and convert
the energy of a photon to internal energy.
 This process is called absorption. Energy is transferred from the radiation field
to the absorbing species.
 We describe the energy change of the absorber as a transition or an excitation
from a lower energy level to a higher energy level.
 Since the energy levels of matter are quantized, only light of energy that can
cause transitions from one level to another will be absorbed.
 Absorption spectroscopy is one way to study the energy levels of the atoms,
molecules, and solids. An absorption spectrum is the absorption of light as a
function of wavelength.
 The spectrum of an atom or molecule depends on its energy-level structure,
making absorption spectra useful for identifying compounds.
Spectroscopy: 29
 Atomic Emission: Atoms, molecules, or solids that are excited to high energy
levels can decay to lower levels by emitting radiation (emission or
luminescence).
 For atoms excited by a high-temperature energy source this light emission is
commonly called atomic or optical emission and for atoms excited with light it
is called atomic fluorescence.
 For molecules it is called fluorescence if the transition is between states of the
same spin and phosphorescence if the transition occurs between states of
different spin.
 Separate documents describe molecular fluorescence, which can be done with
compact instruments, and laser-induced fluorescence.
 The emission intensity of an emitting substance is linearly proportional to
analyte concentration at low concentrations.
 Atomic emission and molecular fluorescence are therefore useful for
quantitating emitting species.
Spectroscopy: 30
 Scattering: When electromagnetic radiation passes through matter, most
of the radiation continues in its original direction but a small fraction is
scattered in other directions.
 Light that is scattered at the same wavelength as the incoming light is
called Rayleigh scattering.
 Light that is scattered in transparent solids due to vibrations (phonons) is
called Brillouin scattering.
 Brillouin scattering is typically shifted by 0.1 to 1 cm-1 from the incident
light.
 Light that is scattered due to vibrations in molecules or optical phonons
in solids is called Raman scattering.
 Raman scattered light is shifted by as much as 4000 cm-1 from the
incident light.
Spectroscopy: 31
Radiation Sources: 32
 The function of the radiation source is to provide sufficient intensity of
light for making a measurement.
 Blackbody Sources: A hot material such as an electrically heated
filament in a light bulb, emits a continuum spectrum of light.
 The spectrum is approximated by Plank’s radiation law for blackbody
radiations.
B = {2hv3/c2}*{1/exp(hv/kT) - 1}
Tungsten filament lamps: 350 nm – 2.5 µm;
Glowbar: 1 – 40 µm;
Nernst glower: 400 nm – 20 µm
Radiation Sources: 33
 Discharge Lamps: Discharge lamps pass an electric current through a
rare gas or metal vapour to produce light.
 The electrons collide with gas atoms, exciting them to higher energy
levels which then decay to lower levels by emitting light.
 Low pressure lamps have a sharp line emission characteristics of the
atoms in the lamp and the high pressure lamps have broadened lines
superimposed on a continuum spectrum.
The common discharge lamps and their wavelength ranges are:
Hydrogen or Deuterium: 160 – 360 nm;
Mercury: 253.7 nm and weaker lines in the near-UV and visible;
Ne, Ar, Kr, Xe discharge lamps: many sharp lines throughout the near-UV
to near-IR; Xenon arc: 300 – 13 nm.
Radiation Sources: 34

 There are two classes of radiation sources used in

spectrometry: continuum sources and line sources.
 The former are usually lamps or heated solid materials that emit a wide
range of wavelengths that must be narrowed greatly using a wavelength
selection element to isolate the wavelength of interest.
 The latter sources include lasers and specialized lamps that are designed
to emit discrete wavelengths specific to the lamp’s material (Skoog).
Radiation Sources: 35
 Electrode lamps are constructed of a sealed, gas-filled chamber that has
one or more electrodes inside.
 Electrical current is passed through the electrode, which causes
excitation of the gas. This excitation produces radiation at a wavelength
or a range of wavelengths, specific to the gas.
 Examples include argon, xenon, hydrogen or deuterium, and tungsten
lamps, which emit radiation in the following ranges.
Radiation Sources: 36
 There are also non-electrode lamps used as line sources that contain a
gas and a piece of metal that will emit narrow radiation at the desired
wavelength.
 Ionization of the gas occurs from radiation (usually in the radio or
microwave frequencies).
 The metal atoms are then excited by a transfer of energy from the gas,
thereby producing radiation at a very specific wavelength (Skoog).
Radiation Sources: 37
 Laser (an acronym for light amplification by stimulated emission of radiation)
sources work by externally activating a lasing material so that photons of a
specific energy are produced and aimed at the material (Skoog).
 This triggers photon production within the material, with more and more
photons being produced as they reflect inside the material.
 As because all the photons are of equal energy they are all in phase with each
other so that energy (and wavelength) is isolated and enhanced.
 The photons are eventually focused into a narrow beam and then directed at
the sample.
Optical Filters: 38

 A filter must meet the following two requirements

High transmittance at the desired wavelength
Low transmittance at other wavelength
 Filters are broadly classified as absorption filters and interference filters.
Optical Filters: 39

 Absorption Filters: The absorption type optical filter usually consists of

colour media including colour glasses, coloured films (gelatin) and
solutions of coloured substances.
 This type of filters has a wide spectral bandwidth which may be 40 – 50
µm in width at one-half the maximum transmittance.
 Their efficiency of transmission is very poor and is of the order of 5 t0
25 %.
Optical Filters: 40
 Interference Filters: this usually consists of two semi-transparent layers of silver, deposited
on glass by evaporation in vacuum and separated by a layer of dielectric (ZnS or MgF2).
 In this arrangement, the semi- transparent layers are held very close together. The spacer
layer is made of a substance which is of a low refractive index.
 The thickness of the dielectric layer determines the wavelength transmitted.
 Constructive interference between different pairs in superposed light rays occurs only when
the path difference is exactly one wavelength or some multiple thereof.
 The relationship expressing a maximum for the transmission of a spectral band is given by
 mλ = 2d(n) sin θ, d = thickness of dielectric spacer, n = refractive index.
 Interference filters allow a much narrower band of wavelength s to pass and are similar to
monochromators in selectivity.
 For efficient transmission, multi-layer transmission filters are often used.
 One type of interference filters is the continuous wedge filter, which permits a continuous
selection of different wavelengths.
Monochromators: 41
 These are optical systems which provide better isolation of spectral energy than optical
filters and are therefore preferred in cases where it is required to isolate narrow bands
of radiant energy.
 These usually incorporate a small glass of quartz prism or a diffraction grating system
as dispersing media.

 Prism Monochromator: The isolation of different wavelengths in a prism

monochromator is based upon the fact that the refractive index of materials is different
for radiation of different wavelengths.
If a parallel beam of radiation falls on a prism, the radiation of two different
wavelengths will be bent through different angles.
Monochromators: 42
 Diffraction Gratings: A diffraction grating consists of a series of parallel grooves
ruled on a highly polished reflecting surface.
 When the grating is put into a parallel radiation beam so that one surface of the grating
is illuminated, this surface acts as a very narrow mirror.
 The reflected radiation from this grooved mirror overlaps the radiation from the
neighbouring grooves.
 The waves would therefore interfere with each other. On the other hand, it could be
that the wavelength of radiation is such that the separation of the grooves in the
direction of the radiation is a whole number of wavelengths.
 Then the waves would be in – phase and the radiation would be reflected undisturbed.
 mλ = 2d sin θ, d = distance separating the grooves and is known as grating constant
and m is the order of interference.
 43
Monochromators:
 Holographic Gratings: These have superior performance in reducing stray light as
compared to diffraction gratings.
 Holographic grating are made by first coating a glass substrate with a layer of photo –
resist, which is then exposed to interference fringes generated by the intersection of
two collimated beams of laser light.
 When the photo – resist is developed, it gives a surface pattern of parallel grooves.
When coated with aluminium, this becomes diffraction gratings.
Detectors 44
 Detectors are transducers that transform the analog output of the
spectrometer into an electrical signal that can be viewed and analyzed
using a computer.
 There are two types:
1. Photon detectors
2. Thermal detectors.
Detectors 45
 Photon detectors work generally by either causing electrons to be emitted or
development of current when photons strike the detector surface.
 Examples include photovoltaic cells, phototubes, and charge-transfer
transducers.
 Photovoltaic cells generate electrical current on a semiconductor material
when photons in the visible region are absorbed.
 Phototubes, however, emit electrons from a photon-sensitive material.
 Charge-transfer transducers (such as photodiodes) develop a charge from
visible region photon-induced electron transfer reactions within a silicon
material (Skoog, Atkins).
 In each case, it is the current, the number of electrons, or the charge that is
actually detected (not the photons themselves) and is then related to the
energy/quantity of photons that caused the change in the material.
Detectors 46

 Thermal detectors detect a temperature change in a material due to photon

absorption.
 Thermocouples work by measuring the difference in temperature between a pair of
junctions (usually the reference against the sample) and are generally used for the
infrared wavelengths (Skoog).
 The temperature difference is related to a potential difference, which is the output
signal.
 Pyroelectric transducers are used in infrared region and utilize a dielectric material
that produces a current when its temperature is changed by radiation absorption.
47
References: 48

 Analytical Instrumentation, Bela G. Liptak

 Principles of Instrumental Analysis 6E, Skoog, Holler and Crouch
 Handbook of Analytical Instruments, 2nd Edition, R S Khandpur