Analytical Instruments

Reference Text: R S Khandpur

“Handbook of Analytical
Instrumentation”

1
 Introduction…

What are Analytical Instruments ?

 Instruments that are used to analyze materials and to
establish the composition.
 Provide,
 qualitative information
 quantitative data

2
 Elements of an
Analytical Instrument

Chemical
Informatio Signal
Transduce Display
n Source Conditioner
r

3
 Absorption Spectroscopy
 Most of the instrumental analysis methods are based on
the absorption of electromagnetic radiation in the visible,
ultraviolet and infrared ranges.

 The method based on absorption radiation of a

substance is of Spectroscopy or
Absorption Spectrophotometer.
known as Absorption

 Advantages:
 High speed
 Sensitivity to very small
amounts
 Simple operational method 4
 Laws of Photometry
1) Lambert’s Law
 States that each layer of equal thickness of an absorbing
medium absorbs an equal fraction of the radiant energy
that traverses it.
 Lambert’s law is expressed as:
Transmittance T= I / I0
Absorbance = Log 10 (1/T)
where I0  incident radiant
energy
I  energy which is
transmitted
5
 Laws of Photometry
2) Beer’s Law
 States that absorption of light is directly proportional to
both concentration of the absorbing medium and the
thickness of the medium in the light path.
 Based on this, for a fixed path length, Absorption
spectroscopy can be used to determine the concentration
of the absorber in a solution.

6
 Laws of Photometry
3) Beer - Lambert Law
 Defines relationship between Absorbance (A)
and Transmittance (T).
 States that the concentration of a substance in
solution is directly proportional to the Absorbance, A of
the solution.
 Absorbance, A = ε c b
where A  measured absorbance, in Absorbance Units (AU)
ε  constant known as the molar absorptivity (function
of wavelength) (dm3 mol-1 cm-1)
c  concentration of the absorbing species (mol dm-3 ) b 
path length through the sample (cm)
7
Limitations of Beer - Lambert Law
 Only applicable to monochromatic radiations.
 Non-linearity arises at high concentration.
Chemical & Instrumental factors which causes non-linearity
 Deviations in absorpitivity coefficient at high concentration
 Scattering of light due to particulates in sample
 Fluorescence or phosphorosence of the sample
 Changes in the refractive index
 Shifts in chemical equilibrium
 Non-monochromatic radiation
 Stray light

8
 Types of Absorption
Spectrophotometer
 Commonly usedAbsorption Spectrophotometer is :

UV –Vis - NIR Spectrophotometers

(Ultra Violet – Visible – Near Infra Red Spectrophotometer)

 This means it uses light in the UV, visible and near-infrared

(NIR)
ranges.

9
UV - VIS SPECTROPHOTOMETERS

10
 RADIATION SOURCES
 Provide sufficient intensity of light for making a measurement.

 Blackbody Sources :- A hot material like electrically heated

filament which emits a continuous spectrum of light.
 Discharge lamps :- When electric current pass through a rare gas
or metal vapour, the electrons collide with gas atoms, exciting
them to higher energy levels and then decay to lower levels by
emitting light.
 Lasers :- Laser beam is highly directional, monochromatic and
provide high density energy which can be finely focused. 11
Blackbody sources
 Tungsten filament lamps (350 to 2500 nm)  visible

 Glowbar lamps (1 to 40 μm)  infrared

 Nernst glower lamps (400 nm to 20 μm)  infrared

12
Discharge lamps
 Hydrogen or deuterium lamps (160 to 380 nm)  ultraviolet

 Mercury Lamps ( 253.7 nm)  visible and near UV

 Ne, Ar, Kr, Xe discharge lamps (300 – 13 nm)  near UV to

near IR

13
Wavelength Selectors or Filtering Arrangement
 For selection of a narrow band of radiant energy.
 Requirements of filters:-
-- High transmittance at desired wavelength
-- Low transmittance at other wavelength
 It can be;
 Optical Filters
 Absorption filter
 Interference filter
 Monochromators
 Prism monochromators
 Diffraction grating
 Reflection Gratings 14
Absorption Filters
 Used in the visible range.

 Have effective bandwidths from 30 to 250 nm.

 Less expensive than interference filters.

15
Three types of absorption filters:
1)Coloured glass :- The filters
absorb all wavelengths of light
except for particular wavelengths
which they pass.

2)Dyed gelatin :- do not last long

and must be frequently replaced.

3)Sharp cutoff (band pass filter) :-

consists of two filters put together.
16
 Interference Filters
 Use optical interference to
provide narrow bandwidths of
radiation.
 Consists of a dielectric
insulator like MgF2 or CaF2
which is sandwiched between
two semitransparent metallic
films.
 These three layers are then
sandwiched between two
plates of glass or transparent
materials.
17
 Interference Filters - working
 Thickness of the dielectric layer determines the wavelength
of the transmitted radiation.
 When the beam of radiation strikes this filter, some of the
radiation passes through the first metallic layer while the
rest is reflected. The remaining light then strikes the second
metallic layer and some is passed while the rest is reflected.

18
Interference Filters - working
 If the reflected light from the second layer is of the proper
wavelength, it is partially reflected from the inside surface of
the first layer in phase with incoming radiation of the same
wavelength. The result is that the desired wavelength is
reinforced while the others wavelengths, being out of phase,
undergo destructive interference.
 Interference filters are used throughout the ultraviolet and
visible regions and about 14 μm into the IR region.
19
 Interference
 Interference is a phenomenon in which two waves
superimpose to form a resultant wave of greater or lower
amplitude.

 Two types ;
 Constructive interference :- Constructive interference occurs
when the phase difference between the waves is a multiple of
2π.
 Destructive interference :- Destructive interference occurs
when the difference is an odd multiple of π.

20
Resultant wave

Wave 1

Wave 2

Constructive Destructive
interference interference
21
 Monochromators
 Principle is based on Refraction.

 Allows only certain wavelengths to be selected and used.

 Types;

 Prism

 Grating

 Diffraction Grating

23
 Prism Monochromator
 Basic principle : The isolation of different wavelengths in a
prism monochromator is based on refractive index of
materials is different for radiation of different wave lengths.
 Optical elements:
 Entrance slit
 Collimating lens
 Prism or grating
 Focussing element
 Exit slit

24
 Basic Principle
Infrared
m
o
n
o
c
Yellow
h SLIT
Polychromatic r
PRISM Green
Ray Blue o
Violet m
a
t
i
Ultraviolet c
R
a
Polychromatic Ray Monochromatic Ray y

Red 25
Basic Setup – Prism Monochromator

26
Working - How select one particular wavelength?

27
 Snell’s Law of 1sin1  sin2
Refraction :

Also, remember that no refraction occurs if light at normal or θ1 = 0

So, light must hit prism at an angle.
Most common is a 60o prism of glass or quartz.
High resolution prism: mixture of Silicon dioxide, Sodium
chloride and Potassium bromide.
 Grating Monochromator
 Diffraction Grating

30
 Diffraction Grating
 A diffraction grating consists of a series of parallel grooves or
slits on a highly polished reflecting surface.
 When light is incident on a diffraction grating, diffractive and

mutual interference effects occur, and light is reflected or

transmitted in discrete directions.
 The separation of grooves in the direction of radiation is a
whole number of wavelength, then the waves would be in-
phase and radiation would be reflected undistributed.
31
Diffraction Grating in Monochromator

32
 Diffraction Grating

Most commonly used

grooved surface
reflective coating (Al, with
Au,
Pt) .

n = d(sin  + sin )
33
Diffraction grating proof
Diffraction grating proof
Detectors: Photosensitive Detectors
 Purpose is quantitative measure of radiation intensities.
 In photosensitive detector, the light energy is converted into
electrical energy.
 Electric current produced by this can be measured with
a sensitive galvanometer.

Requirements of a good detector

 High sensitivity
 Linear response over the wavelength range of interest
 Fast response
 Little or no signal in absence of light (dark current)

36
 Photosensitive Detectors
 Different types:
 Photovoltaic Cell

 Photo-emissive Cell

 Silicon Diode Detectors

 Photo Diode Array

(PDA)

37
Photovoltaic or Barrier Layer Cell

38
 Photovoltaic Cell
 Advantages:
 Robust in construction
 Need no external power source
 Good for portable instrument
 Sensitive to almost the range of wavelength of the spectrum.

 Disadvantage:
 Shows fatigue (decrease in response with
continued illumination),
 Difficult to amplify signal, since small internal resistance of
selenium (Ohm’s law: I=(V/R)).
39
 Photo-emissive Cell
 Requires an external power supply to facilitate flow
of electrons.
 Amplifier circuits are employed for the amplification of
the current.
 Three types;
 High vacuum Photo-emissive cell
 Gas-filled Photo cell
 Photomultiplier Tube (PMT)

40
High vacuum Photo-emissive cell

41
High vacuum Photo-emissive cell
 The spectral response depends upon the nature of the substance

coating at the cathode.

 Cesium-Silver oxide cell  sensitive to Near
InfraRed wavelength.
 Potassium-Silver oxide/ Cesium-Antimony 
sensitive to
visible & UV wavelength.
 Current  number of photons.

 Smaller current than photovoltaic cell, but can be amplified.

42
 Gas-filled Photo cell
 Sometimes inert gas like Ar, is present in the tube. As
e- collide with gas, more e- and ions produce results in
an increase in current.
 Presence of the small quantities of the gas prevent the
phenomenon of saturation current, when higher
potential difference are applied between the cathode
& anode.
43
 Photomultiplier Tube (PMT)
light
dynodes

anode
electrons

e-

photochathode

voltage divider
network

high voltage

44
 Photomultiplier Tube - Working
 It is a very sensitive device in which electrons emitted from the
photosensitive cathode strike a second surface called dynode
which is positive with respect to the original cathode.
 Additional electrons are generated at each dynode.

 If the above process is repeated several times, so more than 105

to 107 electrons are finally collected for each photon striking the
first cathode.
 The amplification depends upon the number of dynodes and
accelerating voltage. 45
 Photomultiplier Tube
 Advantages:

 very sensitive to low intensity.

 very fast response.

 Disadvantages:

 need a stabilized high voltage power supply.

 intense light causes damages.

 Large & expensive

 Dark current 46
 Silicon Diode Detectors
 Silicon diode/Photo diode can be powered from a low voltage
source. And signal can be amplified by a low noise op-amp.
 This type is not as sensitive as PMTs, but are small and
robust.
 Commonly used semiconducting materials;
 Si

 Ge

 InAs – Indium Arsenide

 InSb – Indium Antimonide 47

 Silicon Diode Detectors
•Robust and less power
•Useful for 0.4-1.05µm
•Not so sensitive as PMTs

48
 Types of UV-Visible spectrometers

1.Single Beam Spectrophotometer

2.Double Beam Spectrophotometer
3.Multichannel Spectrophotometer
Single Beam Spectrophotometer
Double beam (In-Space) Spectrophotometer
Double-beam (In Time) Spectrophotometer
Multichannel Spectrophotometer
 Double Beam Spectrophotometers
Ratio Recording Spectrometer:
Principle:
 Double Beam Spectrometers

Optical Null Spectrometer:

Principle:
 Advantages of double beam instruments

1. Automatic correction for changes of the source intensity

and changes in the detector response with time or
wavelength because the two beams are compared and
measured at the same time.

2. Automatic scanning and continuous recording of spectrum

(absorbance versus wavelength).
56
 Applications of Ultraviolet/Visible Spectrophotometry

 Molecular spectroscopy based upon UV-Vis radiation is useful

for identification and estimation of inorganic, organic and
biomedical species.

 Molecular UV-Vis absorption spectrophotometry is employed

primarily for quantitative analysis.

 UV/Vis spectrophotometry is probably more widely used in

chemical and clinical laboratories throughout the world than any
other single method.
57
Applications of Ultraviolet/Visible
Spectrophotometry
• Detection of impurities
•Molecular weight determination
•Chemical kinetics
A+B C+D
Sample Absorption spectrum (Chlorophyll)
 Absorption Methods
1. Quantitative Methodology:
• First step is to choose an “absorption band” (filter) at which
absorbance measurements are to be made.
• Draw the Analytical curve/Calibration curve:
(Concentration vs Absorbance)
• A series of standard solutions is prepared with blank.
• Using one filter at a time, calibration curves are plotted in
terms of absorbance vs concentration.
• A filter which provides the closest adherence to linearity
over the widest absorbance interval and which yields the
largest slope with a zero intercept, will constitute the best
choice for analysis.
 Absorption Spectrum/Analytical Curve

Absorption spectrum Analytical Curve

3
A
A
2
1

Wavelength 1 2 3
λA
Concentration
 Solvents
•Solvent chosen must dissolve the sample, yet be
compatible with cuvette material.
•Relatively transparent in the spectral region of interest
•To avoid poor resolution and difficulties in spectral
region of in spectral interpretation, a solvent should not
be used for measurements near or below uv-cutoff, i.e.,
the wavelength at which absorbance for the solvent
alone approaches one absorbance unit.
•Solvents especially purified and certified for
spectrophotometric use are available from suppliers.
 Absorption Methods
2. Difference Spectroscopy:
In difference spectroscopy two samples are used.
After finding the absorbance spectra for these two samples,
difference is found.
Common features in the two spectra cancel.

A PH =10.3 A PH =10
Difference

λ λ
 Absorption Methods

•Usually concentration of absorbing material in the two

samples is identical but some solution parameter such
as pH is different.
•This method is used in toxicology laboratories for
analysis of drugs.
 Absorption Methods
3. Differential Or Expanded Scale Spectroscopy:
•Ordinary Calibration:
0%T with occluder
100%T with Pure solvent.
•Expanded Scale Spectroscopy:
0%T with occluder is same. But
100%T with more dilute sample solution instead of pure solvent.
Ex: Let ref solution has 36%T and sample solution has 20%T.
0 10 20 30 40 50 60 70 80 90 100%

0 10 20 30 40 50 60 70 80 90 100%

55.6
4. Simultaneous Spectrophotometric Determinations:
Used when no region can be found free from overlapping spectra
of two components

ϵ
Molar
ϵ2
Absorptivity

ϵ1

λ1 λ2 λ
 Absorption Methods

4. Simultaneous Spectrophotometric Determinations:

Using Beer’s Law

C 1 (  1 )  1  C 2 (  2 )  1  A 1

C 1 (  1 )  2  C 2 (  2 )  2  A 2

These equations are solved for determining the concentration

of each component.
 Molecular energies
Molecules possess three types of internal energy.
They are:
1. Electronic Energy
2. Vibrational Energy
3. Rotational Energy
The amount of energy required to cause any change in any
one of the above energy states would generally
correspond to specific regions of electromagnetic
spectrum.
Electronic transistions: UV-Vis Regions
Vibrational transistions: Near-IR and IR regions
Rotational transitions: Microwave regions
•The method based on the absorption of radiation of a substance
is known as ‘Absorption Spectroscopy’.
•Advantages of spectrometric methods: Speed, Sensitivity to
small amounts, Simple operation.
•Electromagnetic radiation:
either represented as a wave or particles (Photons).
•Energy of photon, E=hυ
h= 6.625x10-27 ergs-sec (Planck’s constant)
υ=Frequency (Cycles/sec or Hz)
E=Energy in ergs
c=υλ
c=Velocity of light (3x108 m.sec-1)
λ=Wavelength (nm or µm or A0(Angstrom unit))
 Interaction of radiation with matter
Radiation may be:
Transmitted, Scattered, Absorbed or Reflected.

Incident radiation Transmitted radiation

Absorption

Reflection Scattered radiation

•Absorption spectroscopy is usually concerned with absorption and

transmission.
•Conditions under which the sample is examined are selected to keep
reflection and scattering minimum.
 IR Spectroscopy (Introduction)
Ideally suited for quantitative and qualitative analyses,
particularly of organic compounds. (They are soluble in organic
solvents like Toluene, Xylene, turpentine etc).

Applications to inorganic compounds is limited, because strong

absorption of IR radiation by water. ( As they are soluble in
water).

IR region extends from 0.8 to 200µ in the electromagnetic

spectrum.

Commercial instruments are available in the region from 0.8 to

50µ.
 IR Spectroscopy (Introduction)
•The position of absorption bands in IR spectrum is
expressed in both wavelength (λ) and wave number (ν).
ν( in cm-1)= 104/λ(in µ).
•IR spectrophotometer with prisms: Spectrum is spread
linearly with λ.
•IR spectrophotometer with gratings: Spectrum is spread
linearly with ν.
•Results are preferably reported in wave numbers in
either case,
because: ν α frequency, energy of molecules.
 IR Spectroscopy (Introduction)
•Molecules have Vibrational motion ( Frequency and
Amplitude)
•By absorbing radiation the amplitude of radiation
increases.
•Frequency of absorbed radiation= Vibrational frequency
of molecules.
•Most molecular vibrations have frequency 1014 sec-1.
•λ= c/f= 3x1010cmsec-1/1014 =3x10-6m=3μ is required to
cause transition.
•This wavelength is in IR region.
 IR Spectroscopy (Introduction)

•IR spectroscopy deals with transitions in

Vibrational energies of molecules. (Vibrational
spectroscopy)
•IR spectrum: λ/ν Vs %T.
•IR Spectroscopy is used for analysis of Solids,
Liquids and Gases.
•Interpretation of IR Spectra is highly empirical and
requires huge libraries of reference spectra.
•Used to monitor atmospheric pollutants (Ozone,
Oxides of Nitrogen, CO, SO2 etc)
 Basic Components of IR
Spectrophotometers

Same as those of UV-Vis instruments:

•Sources of Radiation
•Monochromator for dispersing the radiation
•Detector
 Sources of infrared radiation

Globar Rod:
•Silicon Carbide rod with +ve temp coefficient of
resistance.
•Length= 5cm, Diameter=0.5cm
•Heated to 1500K
•Water cooling is required to prevent burnout.
•As Temp increases resistance increases. Hence
voltage should also be increased. So variable
transformer is used.
 Sources of infrared radiation
Nernst Lamp: (ZrO2, Y2O3)
Small rod of oxides of Zirconium and Yttrium.
•-ve temp coefficient.
•Cylindrical in shape.
•Length= 20-30mm, d=1 to 2mm.
•Pt leads are sealed at the ends for passage of
current.
•Temp 1500 to 20000C
•Must be heated externally to dull-red hot
because it is non-conducting when cold.
 Sources of infrared radiation

•Nichrome Strip:
•Less energy than Globar or Nernst filament.
•Simple and reliable in operation
•Tightly wound Nichrome wire heated by passing
current.
•Temp: 800 to 9000C
•Requires no water cooling.