Colorimeter and Spectrophotometer Electromagnetic Radiation EMR THE ELECTROMAGNETIC SPECTRUM Interaction of e.m.r. with Matter Molecular Spectra Spectrophotometry Principles OF Spectrophotometry

1. SPECTROMETRY
Colorimeter and
Spectrophotometer
1
EMR & SPECTRUM
PRESENTED BY:
PRASHANT V C
DEPT OF ZOOLOGY
GUK

2. Electromagnetic Radiation
 Characteristics of wave
◦ Frequency, v - number of oscillations per unit time, unit: hertz (Hz) - cycle per
second
◦ velocity, c - the speed of propagation, for e.m.r c=2.9979 x 108 m×s-1 (in vacuum)
◦ wave-length, l - the distance between adjacent crests of the wave
wave number, v’, - the number of waves per unit distance v’ =l-1
 The energy carried by an e.m.r. or a photon is directly proportional to the
frequency, i.e. where h is Planck’s constant h=6.626x10-34J×s
2
c
'
v
c
v 


c
'
hv
hc
hv
E 




3. 3
EMR
Electromagnetic waves travel at the speed of light (2.998 x 108 m s-1) in a vacuum.
.

4. 4
THE ELECTROMAGNETIC SPECTRUM
Atkins & de Paula 2002
.

5. Interaction of e.m.r. with Matter
 Interaction of electromagnetic radiant with matter
◦ Types of interactions
 Absorption
 Reflection
 Transmission
 Scattering
 Refraction
◦ Each interaction can disclose certain properties of the matte
5
refraction
transmission
absorption
reflection scattering

6. Molecular Spectra
 Energy change of excited molecules
An excited molecules can lose its excess
energy via several processes
◦ B - Releasing Energy {E} as heat when changing
from a sub-state [v] to the singlet state
◦ The remaining energy can be release by one
of following:
 C - Transfer its remaining E to other chemical
species by collision
 D - Emitting photons when falling back to the
ground state - Fluorescence
 F - Radiating E from triplet to ground
state (triplet quenching) -
Phosphorescence 6
S0
T1
S2
S1
v1
v2
v3
v4
v1
v2
v3
v4
v1
v2
v3
v4
v1
v2
v3
v4
Inter- system
crossing
Internal
transition
B
B
E1
E2
C
F
A
B
Fluorescence
D
Fluorescence
Jablonsky diagram
.

7. Spectrophotometry
 Theory of light absorption
Quantitative observation
◦ Higher concentration the liquid
- the less the emergent light intensity
These observations are summarised by Beer’s Law:
When a ray of monochromatic light of initial intensity Io passes through an absorbing medium its
intensity decreases exponentially as the concentration of the absorbing medium increases.
7
Incident light
I0
Emergent light
I
C
b
Thus
I = Io e - K2 C
Where I= emergent light
Io=Incident Light
C=concentration

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Colorimeter
Scale
Absorbance
Transmission
100
0
0
2
Transmission [%]
Absorbance [Log Scale]
0
100 0

40
0.4
80
0.1
60
0.2 0.6 0.8 1
20
0.3

9. Spectrophotometry: Principles
 Theory of light absorption
Quantitative observation
◦ The thicker the cuvette
- more diminishing of light in intensity
These observations are summarised by Lambert Law:
When a ray of monochromatic light of initial intensity Io passes through an absorbing medium its
intensity decreases exponentially as the length of the absorbing medium increases.
9
Incident light
I0
Emergent light
I
C
b
Thus
I = Io e - K1 l
Where I= emergent light
Io=Incident Light
l=Path length
Thus combining both the laws
I = Io e - K3 C l
Where I= emergent light
Io=Incident Light
C=concentration
l=Path length

10. Spectrophotometry: Principles
 Transmittance & Absorbance
The ratio of intensities is known as the transmittance i.e. intensity of
transmittted and incident light
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Thus
T= I/ Io
Where T=Tansmittance [0-100]
I= emergent light
Io=Incident Light
% Transmission = (I/Io) x 100
Where as absorbance is equal to:
A= E = Log 10 (1 /T)
Or E=Log 10 (Io / I) or Log 10 Io / I= Kcl
Where A= Absorbance
E= Extinction coefficient
I= emergent light
Io=Incident Light
C=concentration
l=Path length

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A  CL = KCl by definition and
it is called the Beer Lambert Law.
K = Specific Extinction Coefficient
- 1 g of solute per liter of solution
A = ECL
E = Molar Extinction Coefficient
- Extinction Coefficient of a solution containing
1g molecule of solute per 1 liter of solution
Beer Lambert Law

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Colour Filter
Blue
Green
Yellow
Red
COLORIMETER

13. Spectrophotometry
 Instrumentation
UV visible
Light source Hydrogen discharge lamp Tungsten-halogen lamp
Cuvette QUARTZ glass
Detectors photomultiplier photomultiplier
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Type of Monochromators
.
* Filters
-Coloured
-Interference
*Dispersing Elements
-Prism
-Diffraction Grating
Most modern spectrometers use Diffraction Gratings

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Coloured Filters

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Monochromators: Interference Filters

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Monochromators: Diffraction Grating
shown

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Sample Compartment

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Detectors

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Photomultipliers Tube

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Types of Spectrophotometers
Single Beam UV-Vis Spectrophotometer-1
Light
Source
Sample
Cuvette
Detector
Aperture
Diffraction
Grating

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Types of Spectrophotometers
Single Beam UV-Vis Spectrophotometer-2
Light
Source
Detector
Mono
chromator
Detector
Cuvette
Half-
Mirror
Collecting
Transmitted Light
Slits

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Types of Spectrophotometers
Single Beam UV-Vis Spectrophotometer-2
Light
Source
Detector
Mono
chromator
Detector
Cuvette
Half-
Mirror
Collecting
Transmitted Light
Slits
Beam
Selector
Reference
Cuvette
Half-
Mirror
Mirror
Designed for detecting more than one absorbance at a time

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Double Beam Spectrophotometer
Mono
chromator
Reference Detector
Ratio
Sample Detector
Lamp

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Array Detector Spectrophotometer:
Advantage
1-Allows rapid recording of absorption spectra.
2-Light source is dispersed after it passes through a sample
which allows the use of array detector to simultaneously
record the transmitted light power at multiple λ.
3-More precision, more sensitivity and more reproducible
results.
4-The spectrophotometer can be controlled from a computer
equipped with compatible software.
5-Besides allowing rapid spectral recording, these instruments
are relatively small and robust.

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-A beam of light from a visible and/or UV light source (colored red)
is separated into its component wavelengths by a prism or
diffraction grating.
-Each monochromatic (single wavelength) beam in turn is split into
two equal intensity beams by a half-mirrored device.
-One beam, the sample beam (colored magenta), passes through a
small transparent container (cuvette) containing a solution of the
compound being studied in a transparent solvent.
-The other beam, the reference (colored blue), passes through an
identical cuvette containing only the solvent.

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-The intensities of these light beams are then measured
by electronic detectors and compared.
-Over a short period of time, the spectrometer
automatically scans all the component wavelengths in
the manner described.
-The ultraviolet (UV) region scanned is normally from 200
to 400 nm, and the visible portion is from 400 to 800 nm.

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-Absorption may be presented as transmittance (T = I/I0) or
absorbance (A= log I0/I). If no absorption has occurred, T = 1.0 and
A= 0.
-Most spectrometers display absorbance on the vertical axis, and
the commonly observed range is from 0 (100% transmittance) to 2
(1% transmittance).
The wavelength of maximum absorbance is a characteristic value,
designated as λmax.

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-Different compounds may have very different absorption
maxima and absorbances.
-Intensely absorbing compounds must be examined in
dilute solution, so that significant light energy is
received by the detector, and this requires the use of
completely transparent (non-absorbing) solvents.

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THANKS