spectrometry,laws of photometry,absorption spectroscopy

1. IN 504 Analytical Instruments
Reference Text: R S Khandpur
“Handbook of Analytical Instrumentation”
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Presented by;
Anju Sunny
CUSAT

2. Introduction…
What are Analytical Instruments ?
 Instruments that are used to analyze materials and to
establish the composition.
 Provide,
 qualitative information
 quantitative data
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3. Elements of an
Analytical Instrument
Chemical
Information
Source
Transducer
Signal
Conditioner
Display
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4. 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 of radiation of a
substance is known as Absorption Spectroscopy or
Absorption Spectrophotometer.
 Advantages:
 High speed
 Sensitivity to very small amounts
 Simple operational method
4

5. 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
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6. 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.
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7. 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)
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8. 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
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9. Types of
Absorption Spectrophotometer
 Commonly used Absorption 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.
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10. UV - VIS SPECTROPHOTOMETERS
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11. 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.
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12. 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
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13. 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
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14. 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

15. Absorption Filters
 Used in the visible range.
 Have effective bandwidths from 30 to 250 nm.
 Less expensive than interference filters.
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16. 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.
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17. 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.
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18. 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.
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19. 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.
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20. 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.
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21. 21
Resultant wave
Wave 1
Wave 2
Constructive interference Destructive interference

22. Monochromators
 Principle is based on Refraction.
 Allows only certain wavelengths to be selected and used.
 Types;
 Prism
 Grating
 Diffraction Grating
 Reflection Grating
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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 23

24. Basic Principle
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Polychromatic
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray

25. Basic Setup – Prism Monochromator
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26. Working - How select one particular wavelength?
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28. h1sinq1 = h2sinq2Snell’s Law of 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.

29. Grating Monochromator
Two types:
 Diffraction Grating
 Reflection Grating
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30. Diffraction /Transmission Grating
 Basic Principle: Diffraction
 Diffraction phenomenon is
described as the apparent
bending of waves around
small obstacles and the
spreading out of waves past
small openings.
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Interference pattern from
two-slit diffraction.

31. 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.
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32. Diffraction Grating
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33. Relation between λ, d, and θ
 λ  wavelength of radiation
 d  distance between grooves
 θ  angle at which the radiation is reflected
 m  order of interference
 N  total no. of grooves
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m λ = 2 d sin θ
Resolving Power= mN

34. Diffraction Grating in Monochromator
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35. Reflection Grating
 On contrary to the above, if
the plate is mirrored, we get
reflection grating which
have got vast applications in
spectrophotometry.
 Most commonly used
grooved surface with
reflective coating (Al, Au,
Pt) .
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nl = d(sin b + sin )

36. Effect of Slit Width
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37. Comparison between Grating & Prism
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Type of Dispersion Size Stray Light l range of
use
Grating uniform dispersion vs. l Smaller Higher stray light unlimited
Prism shorter l better separated larger Less of problem Limited
(l ≤ 350 nm)
 Increase size of either prism or grating will give better
dispersion.
 Stray light can be removed with filters.

38. Stray light
 Stray light is light of wavelengths different from those wanted.
 Sources of stray light:
 incompletely removed higher order wavelengths in grating
instrument
 scattering of light by dust
 reflection of light by lens and grating mountings
 Prevention of stray light:
 use a double monochromator
 first monochromator selects wavelengths and passes them to a second
which refines the wavelengths
 paint all components except for reflecting surfaces which are
desired black
 enclose system to keep out dust
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39.  Bandwidth is defined as the frequency band around the
carrier frequency containing 99 percent of the signal
power.
 The bandwidth for an individual AM station is about
10,000 Hz.
 The bandwidth for an individual FM station is about
200,000 Hz.
Signal bandwidth
frequency
bandwidth
fc =carrier frequency
fc+fm=upper sidebandfc+fm=lower sideband

40. Resolution
 It is the smallest amount of input signal change that
the instrument can detect reliably.
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41. 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)
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42. Photosensitive Detectors
 Different types:
 Photovoltaic Cell
 Photo-emissive Cell
 Silicon Diode Detectors
 Photo Diode Array (PDA)
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43. Photovoltaic or Barrier Layer Cell
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44. 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)).
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45. 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)
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46. High vacuum Photo-emissive cell
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47. 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.
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48. 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.
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49. Photomultiplier Tube (PMT)
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photochathode
anode
high voltage
voltage divider network
dynodes
light
electrons
e-

50. 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. 50

51. 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
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52. Silicon Diode Detectors
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 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

53. Silicon Diode Detectors
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 Diagram (Refer Note)

54. Photo Diode Array (PDA)
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 Diagram (Refer Note)

55. 55
Scanning Double Beam Spectrophotometer

56. 56
Scanning Double Beam Spectrophotometer
 In double beam arrangement, the light alternately passes
through the sample and reference (blank), directed by rotating
half-sector mirror (chopper) into and out of the light path.
 When light passes through the sample, the detector measures
the P. When the chopper diverts the beam through the blank
solution, the detector measures P0.
 The electronic circuit at the detector automatically compares P
and P0 to calculate absorbance

57. 57
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).

58. 58
Applications of Ultraviolet/Visible Spectrophotometry
 Molecular spectroscopy based upon UV-Vis radiation is used
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.