Common spectrometric Instrumentation

1. INSTRUMENTAL ANALYSIS
Instrumentation and Applications of
Spectrophotometry
Mr.S.S.Dipke,
(Assistant Professor)
Department of Chemistry,

2. ATOMIC
ABSORPTION
MOLECULAR
IR UV VISIBLE
FLAME
EMISSION SPARC
ICP
FLUORESCENCE
SPECTROSCOPY
ABSORPTION EMISSION

3. Making Electromagnetic Waves
When an electric charge vibrates, the electric field
around it changes creating a changing magnetic field.

4. The magnetic and electric fields create each other again and
again
An EM wave travels in all directions. The figure only shows
a wave traveling in one direction
The electric and magnetic fields vibrate at right angles to
the direction the wave travels so it is a transverse wave

5. What is the speed of EM waves?
All EM waves travel
300,000 km/sec in space
(speed of light-nature’s
limit!)
EM waves usually travel
slowest in solids and
fastest in gases
Material Speed
(km/s)
Vacuum 300,000
Air <300,000
Water 226,000
Glass 200,000
Diamond 124,000

6. Properties of EM Waves
1. Waves
Wavelength= distance from crest to crest
Frequency= number of wavelengths that pass a given point in 1 s (Hertz or
cycles/sec or sec-1)
As frequency increases, wavelength becomes smaller
The velocity of
light, C, is given
by the equation:

7. Photons/Particles
The energy E of photon depends upon the frequency of the radiation
E = hn
 E = hC/l
Energy is inversely proportional to wavelength
Where, h = Planck’s constant (6.626 x 10-34 J s)
n = frequency of the radiation (common units = cm-1)
Properties of EM Waves

8. The Electromagnetic Spectrum
Human eyes are only
able to process
information from the
visible part of the
spectrum
Toward longer
wavelengths, the
spectrum includes
infrared light,
microwaves, and radio
Toward shorter
wavelengths, the
spectrum includes
ultraviolet light, X-rays,
and gamma rays
All of these are forms of
electromagnetic
radiation

9.  Visible light is a small portion of this spectrum. This is
the only part of this energy range that our eyes can
detect. What we see is a rainbow of colors
Red Orange Yellow Green Blue Indigo Violet

10. Radiation Interactions with Matter
Emission – release of electromagnetic waves
Absorption – receiving of electromagnetic waves
Scattering – deflection of electromagnetic waves in all
directions
Reflection – deflection of electromagnetic waves into the
backwards direction
Energy Transfer
• Conduction –molecule to molecule within a substance
• Convection (and advection) –mass movement of a fluid
• Radiation –absorption of electromagnetic waves

11. The absorption process
 How does matter absorb radiation?
 When polychromatic light (white light/whole spectrum) is
passed though an object it absorbs certain of the
wavelengths, leaving the unabsorbed wavelengths to be
transmitted.
 These residual transmitted wavelengths will be seen as a
color. This color is complementary to the absorbed colors.

12. The colors we see in objects are the colors that are
reflected, all other colors are absorbed.
Red T-shirt Red- Red is reflected
 All wavelengths
are absorbed-
Black (Black also,
isn’t really a color)
 When all colors are being reflected we see white light
(white isn’t really a color)

13.  Absorption is a process in which chemical species (atom,
ion or molecule) in a transparent medium selectively
attenuate certain frequencies of EMR
 Absorption spectrum is a plot of the amount of light
absorbed by a sample as a function of wavelength.
 At room temperature most substance are in their lowest
energy or ground state (G.S.)
 When an atom, molecule or ion absorbs EMR and is
promoted to higher energy states or excited states
 The E.S. is a (T.S.) and the species soon looses the energy
it gained and returns to its (G.S.) by relaxation process
either as heat of collision or sometimes emits radiation of
specific wavelength

14. When molecule absorbs light
What happens to absorbed energy ?
S2, S1 = Singlet States

15. More Complex Electronic Processes
Fluorescence:
 absorption of radiation to an
excited state, followed by
emission of radiation to a
lower state of the same
multiplicity
Phosphorescence:
 absorption of radiation to an
excited state, followed by
emission of radiation to a
lower state of different
multiplicity
0 sec 1 sec
Example of Phosphorescence

16.  Singlet state:
 spins are paired, no net
angular momentum (and
no net magnetic field)
 Triplet state:
 spins are unpaired, net
angular momentum (and
net magnetic field)
More Complex Electronic Processes
Spins paired
No net magnetic field
Spins unpaired
net magnetic field
10-5 to 10-8 s
10-4 to 10 s

17.  E(ex)-E(g) = hn of the photon absorbed.
 UV / VIS - Electronic transition (along with vibrational/rotational)
 IR - Vibrational and rotational (No electronic transition)
 Microwave - Rotational transitions alone
 PMR/ NMR – Change in Nuclear Spin under magnetic
field
 Etotal = Eelectronic + Evibrational + Erotational
Total Energy of the System

18. Common Spectroscopic Methods Based on Electromagnetic Radiation
Type of Spectroscopy Usual Wavelength
Range
Type of Quantum Transition
Gamma-ray emission 0.005-1.4 Å Nuclear
X-ray absorption, emission,
fluorescence, and diffraction
0.1-100 Å Inner electron
Vacuum ultraviolet
absorption
10-180 nm Bonding electrons
Ultraviolet visible
absorption, emission,
fluorescence
180 -780 nm Bonding electrons
Infrared absorption and
Raman scattering
0.78-300 mm Rotation/vibration of molecules
Microwave absorption 0.75-3.75 mm Rotation of molecules
Electron spin resonance 3 cm Spin of electrons in a magnetic field
Nuclear magnetic
resonance
0.6-10 m Spin of nuclei in a magnetic field

19. Solvents used in Spectroscopic methods
Solvent λ of absorption
Water 191 nm
Ether 215 nm
Methanol 203 nm
Ethanol 204 nm
Chloroform 237 nm
Carbon tetrachloride 265 nm
Benzene 280 nm
Tetrahydrofuran 220 nm
Hexane 210
Dioxane 220
CH3CN 210
CH2Cl2 235
Acetone 300

20. Beers Lamberts law
When a light passes through absorbing medium at right
angle to the plane of surface or the medium or the solution,
the rate of decrease in the intensity of the transmitted light
decreases exponentially as the thickness of the medium
increases arithmetically
 Lambert’s law :
“When a beam of light is allowed to pass through a
transparent medium, the rate of decrease of intensity with the
thickness of medium is directly proportional to the intensity of
light”

21. Beers Lamberts law
Mathematically,
- dI / dt α I
-dI / dt = KI . . . . . . . . .(1)
Where,
I = intensity of incident light
t = thickness of the medium
K= proportionality constant
By integration of equation (1), and putting I=I0 when t=0,
I0/ It = kt or It= I0 e-kt

22. Beers Lamberts law
Where,
I0 = intensity of incident light
It = intensity of transmitted light
k = constant which depends upon wavelength and
absorbing medium used
By changing the above equation from natural log, we get,
It = I0 e-Kt . . . . . . . . . .(2)
Where,
K = k/ 2.303
So, It = I0 e-0.4343 kt
It = I010-Kt . . . . . . . . . .(3)

23. Beers Lamberts law
 Beer’s law :
“Intensity of incident light decreases exponentially as the
concentration of absorbing medium increases arithmetically.”
The above sentence is very similar to Lambert’s law.
So, It = I0 e-k' c
It = I0 10-0.4343 k' c
It = I0 10 K' c . . . . . . . . . .(4)
Where, k' and K'= proportionality constants
c = concentration

24. Beers Lamberts law
 Beer’s law :
By combining equation (3) and (4), we get,
It = I0 10 -act
I0 / It = 10 act
Where, K and K' = a or ε
c = concentration
t or b = thickness of the medium
log I0 / It = εbc . . . . . . . . . .(5)
Where, ε = absorptivity, a constant dependent
upon the λ of the incident radiation and
nature of absorbing material

25. Beers Lamberts law
 Beer’s law :
The value of ε will depend upon the method of expression of
concentration.
The ratio I0 / It is termed as transmittance T, and
The ratio log I0 / It is termed as absorbance A
Formerly, absorbance was termed as optical density D or
extinction coefficient E, and
The ratio I0 / It is termed as opacity.
Thus,
A = log I0 / It . . . . . . . . . .(6)

26. Beers Lamberts law
 Beer’s law :
From equation (5) and (6),
A = εbc . . . . . . . . . .(7)
Thus,
“absorbance is the product of absorptivity, optical path length
and the concentration of the solution”
According to equation (7),
A = log I0 / It

27. Beers Lamberts law
 Beer’s law :
Transmittance T is a ratio of intensity of transmitted light to
that of the incident light.
T = I0 / It
The more general equation can be written as follows:
A = log I0 / It
A = log 1/ T
A = – log T
A = abc
A = εbc

28. Instrumentation

29. 1. Sources
2. Wavelength selectors (filters, monochromators)
3. Sample containers
4. Detectors
5. Readout devices
Components of Spectrophotometer

30. Excitation sources
Deuterium Lamp UV
Tungsten Lamp UV-vis
Laser X-ray, UV, vis, IR
X-ray tube X-ray
Mercury lamp UV-vis
Xenon lamp UV-vis
Silicon carbide globar IR
Flame
Furnaces
Plasmas
Hollow-cathode lamp
1. Sources (Excitation Sources)

31. 1. Sources
 A Light Source should have following properties
 Continuous : Emit radiation of all wavelengths within
the spectral region for which they are to be used
 Stable
 Brightness (high intensity)
 Narrow Line Width
 Background
 Lifetime
Visible and Near IR = 390-700nm UV = 400 –10nm

32. Types of Lamps Used as Sources
1. Tungsten lamp
 Excellent source
 Operates at 3000 oK
 Produces l from 320 to 2500 nm
2. Deuterium Arc lamp
 Common lamp
 electric discharge causes D2 to dissociate and emit UV
radiation (160 nm – 325 nm)
 Used for UV spectroscopic studies
1. Sources

33. Types of Lamps Used as Sources
3. Lasers
 High power
 Narrow line width
 Very good for studying reactions
 Coherence : Frequency and waveform are identical and
their phase difference is constant
 can fine-tune the desired wavelength (but choice of
wavelength is limited)
 Too much expensive
 Other good sources are: Xe (250 – 1000 nm),
Hg (280 – 1400 nm)
1. Sources

34. 1. Sources
Continuous
Sources
Tungsten
Lamp
320-2500 nm
Visible and
near IR
radiation
Deuterium
Lamp
200-400 nm
Ultraviolet
radiation

35.  They are used for spectral scanning (varying the wavelength of radiation
over a considerable range )
 They can be used for UV/Vis region
 All monochromators are similar in mechanical construction
 All monochromators employ slits, mirrors, lenses, gratings or prisms
2. Wavelength Selectors (Monochromators)

36. 2.Wavelength Selectors (Monochromators)
a. Optical/Interference Filters
b. Diffraction Gratings
c. Prism

37. i. Absorption Filters
 Simplest kind of filter
 This type of filters is colored glass filters
 Absorbs a broad portion of the spectrum (complementary color) and transmits
other portions (its color)
 Used in the visible region
 It permits certain bands of wavelength (bandwidth of ~ 50 nm) to pass through
a. Optical / Interference Filters

38. a. Optical / Interference Filters
ii. Interference Filters

39.  Disadvantages
 They are not very good wavelength selectors and can’t be used in instruments
utilized in research
 This is because they allow the passage of a broad bandwidth which gives a
chance for deviations from Beer’s law
 They absorb a significant fraction of the desired radiation
a. Optical / Interference Filters

40. b. Diffraction Gratings
 The reflection grating is ruled with a series of
closely spaced, parallel grooves with repeated
distance d
 The grating is covered with Al to make it
reflective
 When polychromatic light is reflected from the
grating, each groove behaves as a new point
source of radiation.

41. b. Diffraction Gratings
 Polychromatic radiation from the entrance
slit is collimated (made into beam of
parallel rays) by a concave mirrors
 These rays fall on a reflection grating,
whereupon different wavelengths are
reflected at different angles
 The orientation of the reflection grating
directs only one narrow band wavelength,
to the exit slit of the monochromator
 Rotation of the grating allows different
wavelengths, to pass through the exit slit

42. b. Diffraction Gratings
 When adjacent light rays are in phase,
they reinforce one another
(constructive interference)
 When adjacent light rays are not in
phase, they partially or completely
canceled one another (destructive
interference)

43. b. Diffraction Gratings
r
 Bragg’s Law (Sir W.H. Bragg and his son Sir W.L. Bragg) : “When the x-ray is incident
onto a crystal surface, its angle of incidence, θ, will reflect back with a same angle of
scattering, θ. And, when the path difference, d is equal to a whole number, n, of wavelength,
a constructive interference will occur”.
nλ=2d (sin θi + sin θr)
Where,
• λ is the wavelength of the x-ray,
• d is the spacing of the crystal layers (path difference),
• θi is the incident angle and θr is angle of reflection
• n is an integer
Since incident angle i = Constant;
Therefore, l  r
Reflection Grating
i
d

44. b. Prism Monochromators
 Dispersion by prism depends on refraction
of light which is wavelength dependent
 Violet color with higher energy (shorter
wavelength) are diffracted or bent most
 While red light with lower energy (longer
wavelength are diffracted or bent least
 As a result, the poly-chromatic white light
is dispersed into its individual colors

45. Advantages and disadvantages of decreasing monochromator slit width
 Exit slit determines the width of radiation (bandwidth) emitted from the
Monochromator
 A wider slit width gives higher sensitivity because higher radiation intensity
passes to the sample but on the other hand, narrow slit width gives better
resolution for the spectrum
 In general, the choice of slit width to use in an experiment must be made by
compromising these factors. Still, we can overcome the problem of low
sensitivity of the small slit by increasing the sensitivity of the detector

46.  Glass – visible
 Quartz -UV ,visible both
3. Sample Containers (Cuvettes)
Long
pathlength
Short pathlength (b)
1 cm pathlength
cuvette
1 cm 1 cm
Opaque
Face
Transparent
Face
Square Cylindrical Rectangular

47.  Convert radiant energy (photons) into an electrical signal
 Ideal Detector :-
 High sensitivity - A detector should be sensitive
 Fast response time- Fast response over a considerable range of wavelengths
 High signal/noise
 Constant response for λmax
 linear response - Electrical signal produced by the detector must be directly
proportional to the transmitted intensity
4. Detectors

48.  Types of Detectors :-
 PMT-Photo Multiplier Tube
 CCD-Charge-Coupled Device
 CID- Charge Injection Device
 MCT-Mercury Cadmium Telluride
 Photodiode
 Thermocouple
 Pyroelectric detector
4. Detectors

49.  Phototube emits electrons from a photosensitive, negatively charged cathode
when struck by visible or UV radiation
 The electrons flow through vacuum to an anode to produce current which is
proportional to radiation intensity
4. Detectors
a. Phototube
hn
e-
-Ve
Photosensitive cathode
amplifier
anode

50.  Single channel, but very high sensitivity
 Electrons emitted from the photosensitive cathode alloy (Cs3Sb, K2CsSb,
Na2KSb) strike a second surface called dynode which is positive with respect to
the original cathode
 Electrons are thus accelerated and can knock out more than one electrons from
the dynode
4. Detectors
b. Photomultiplier Tube
photochathode
anode
high voltage
voltage divider network
dynodes
light
electrons
e-

51.  For 9 stages giving 64 electrons for 1
photon, the amplification is 49 or 2.6 x 105)
 The output is fed to an amplifier which
generates a signal
 To minimise noise it is necessary to operate
at the lowest possible voltage
4. Detectors
b. Photomultiplier Tube

52.  An integrated-circuit chip that contains an array of capacitors that store charge
when light creates e-hole pairs
 The charge accumulates and is read in a fixed time interval
 CCDs are used in similar applications to other detectors, although the CCD is
much more sensitive for measurement of low light levels
4. Detectors
c. Charge-Coupled Devices (CCD)

53.  Single Beam Spectrophotometer : Light beam follows a single path from the
source, to the monochromator, to the sample cell and finally to the detector
Types of Spectrophotometers
 Double Beam Spectrophotometer
• P = Intensity of transmitted Light
• P0 = Intensity of Incident Light

54.  Single beam spectrophotometer is inconvenient because :
 The sample and blank must be placed alternately in the light path
 For measurements at multiple wavelengths, the blank must be run at each
wavelength
 Advantages of double beam instruments are :
 The absorption in the sample is automatically corrected for the absorption
occurring in the blank, since the readout of the instrument is log the difference
between the sample beam and the blank beam
 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
 Automatic scanning and continuous recording of spectrum (absorbance versus
wavelength)
Advantages of double beam instruments over single beam instruments

55.  Deviations from Beer’s
law appear when :
 The absorbing species
undergoes association,
dissociation, hydrogen
bonding, complex formation,
hydrolysis, ionization,
polymerization or reaction
with the solvent to give
products that absorb differently
from the analyte
Deviation From Beer’s Law
1. Chemical Deviations

56.  The extent of such deviations can be predicted from the molar absorptivities of
the absorbing species and the equilibrium constants for the equilibria involved
 Typical equilibria that give rise to this effect include monomer dimer equilibria,
metal complexation equilibria where more than one complex is present,
acid/base equilibria, and solvent-analyte association equilibria
 A common example of this behavior is found with acid/base indicators.
Deviations arising from chemical factors can only be observed when
concentrations are changed
Deviation From Beer’s Law
1. Chemical Deviations

57.  Since HA is weak acid it exists in equilibrium with its conjugated base A-
 Deviations arising from chemical deviations observed only when concentrations
are changed eg. Phenolphthalein:
Deviation From Beer’s Law
1. Chemical Deviations
l = 600 nm

58.  Beer’s law strictly applies only when measurements are made with
monochromatic source of radiation
 If the band selected corresponds to a region in which the absorptivity of the
analyte is essentially constant, deviation from Beer’s law will be minimum
 To avoid deviation, it is advisable to select a wavelength band near the
wavelength of maximum absorption where the analyte absorptivity changes little
with wavelength
 The deviations due to stray light are most significant at high absorbance values
Deviation From Beer’s Law
2. Instrumental Deviations

59.  Stray light always causes the apparent absorbance to be lower than the true
absorbance
 Because stray radiation levels can be as high as 0.5% in modern instruments,
absorbance levels above 2.0 are rarely measured
 Another deviation is caused by mismatched cells (unequal pathlengths and
optical characteristics)
 This error can be avoided either by using matched cells or by using a linear
regression procedure to calculate both the slope and intercept of the calibration
curve
Deviation From Beer’s Law
2. Instrumental Deviations

60.  Wide applicability to both organic and inorganic systems
 High sensitivity of 10-6-10-4 M
 Moderate to high selectivity
 Good accuracy the relative error encountered in concentration lie in the range
from 1% - 3%
 Ease and convenience of data acquisition
The important characteristics of Spectrophotometric methods

61.  UV-Vis absorption spectrophotometry is
employed primarily for quantitative
analysis
 UV-Vis - Used for identification and
estimation of inorganic, organic and
biomedical species
 UV/Vis - More widely used in chemical
and clinical laboratories throughout the
world than any other single method
Important Applications of Spectrometric Methods