Photometry is the science of measuring light in terms of its brightness as perceived by the human eye. It only considers visible light since the eye can only see in this range. Spectrophotometry measures light intensity across the electromagnetic spectrum using instruments. Absorption spectrophotometry measures the absorption of light as it passes through a sample, relating absorption to characteristics like concentration through Beer's and Lambert's laws. A spectrophotometer directs light from a source through a sample and measures the intensity of transmitted light using a detector to analyze samples and identify substances.

1. Presented by –
Md. Ishtiyak
Jafar
PHOTOMETRY

3.  Light is the Electromagnetic radiation (EMR),
that is visible to human eye.
 composed of particles called photons
 ranges from 380 or 400 to about 760 or 780 nm.
 In Physics, the term light sometimes refers to
electromagnetic radiation of any type, whether
visible or not.

4. EMR SPECTRUM

5.  Energy associated with a given segment of the
spectrum is related to frequency and
wavelengths.

6. Photometry,
 Greek photo- ("light") and - metry ("measure")
 Science of measurement of light in terms of its
perceived brightness to the human eye.
 In photometry, the standard is the human eye.
 Since the human eye is only sensitive to visible
light, photometry only falls in that range.
 When intensity at each wavelength on the
whole range of electromagnetic spectrum is
measured, it is called Spectrophotometry.

7. Spectrophotometry
 Actually its a science that deals the use of
measurement of interaction between Matter and
electromagnetic radiations to quantize the
concentration of an analyte or for qualitative
purpose.
 Matter can be atoms, molecules or ions.
 The nature of interaction between the radiation
and matter may include –
Absorption
Emission or
Scattering

8. Spectrophotometry
 Three principal branches of Spectrophotometry
1. Absorption Spectrophotometry
2. Emission Spectrophotometry and
3. Scattering Spectrophotometry.

9. Absorption Spectrophotometry
 deals the measurement of radiation absorbed at
various wavelengths.
 When a beam of EMR passes through the
sample, some of the radiation’s intensity is
attenuated (decrease in number of photons)
 This process of attenuation is called absorption.

10. Major types of Absorption
Spectrophotometry
EMR Spectrophotometry Type
γ γ- ray Absorption Spectrophotometry
X - ray X – ray Absorption Spectrophotometry
UV/Visual UV/Visual Absorption Spectrophotometry
IR IR Absorption Spectrophotometry
Microwave Microwave Absorption Spectrophotometry
Radio Wave NMR Spectrophotometry

11. Emission Spectrophotometry
 Deals the measurement of Emitted Radiation
 Atoms or molecules that are excited to high energy
levels can decay to lower levels by emitting energy
as radiations.
 These emitted radiations are then passed through a
prism and measured directly.
 High temperature induced emission is called Atomic
emission (emission spectroscopy)
 EMR induced emission is called Atomic
Fluorescence (Fluorescence Spectroscopy).
 Used in Flame Spectrophotometry and
fluorometry

12. Scattering Spectrophotometry
 Measures certain physical properties by
measuring the amount of light that a substance
scatters at certain wavelengths.
 Most useful application is Raman
Spectrophotometry.

13. Principles of Absorption
Spectrophotometry
 When radiation falls on homogenous medium, a
portion of incident light is reflected, a portion is
absorbed and remainder is transmitted.
 The two laws governing the absorbance of the
radiation are known as Beer’s law and
Lambert’s law.
Incident Light (Io)
Transmitted
Light (I)

14. Beer’s Law
 Intensity of transmitted monochromatic light
decreases exponentially as the concentration of the
absorbing substance increases.
A α C

15. Lambert’s Law
 Intensity of transmitted monochromatic light
decreases exponentially as the thickness (path
length) of the absorbing material increases.
A α l

16. Mathematical expression of
Beer – Lamberts Law
A = log = εcl
T =
%T = × 100 or %T = T × 100 or T =
A = log10 or –log10 T or – log10
A = – log10 %T + log10100 or – log10 %T +
log10102
A = 2 – log10 %T
Io
I
I
Io
I
Io
%T
100
1
T
%T
100
Where,

17. Limitations
 Very elevated concentrations cann’t be measured
 If Incident radiation is not monochromatic
 If solvent absorption is not significant compared
to solute absorbance.
 If the sides of the cell are not parallel
 If Radiant energy is transmitted by other
mechanism (stray light)
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18. Spectrophotometer
 An instrument used to measure the absorbance
by measuring the amount of transmitted light of a
specific wavelength passes through a sample is
termed
Spectrophotometer

19. Components of
Spectrophotometer
Light sources
Enterance slit
Spectral isolator
Exit slit
Cuvettes
A photodetector
A read out device
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20. 1. Light Source
1. Tungsten filament lamp – continuous spectrum
2. Tungsten iodide lamp – Visible and UV
3. Hydrogen and Deuterium lamps – Continuous UV
4. Mercury vapour lamps – Discontinuous/line
spectrum
5. LEDs – two types of semi conductors
2. Entrance slit
• Focuses light on grating/prism, where it can be
dispersed with minimum stray lights
0

21. 3. Spectral isolator
 For the isolation of required wavelength/range of
wavelengths
Two types :
a) Filters
b) Monochromators
A. Filters
 Consists of only a material that selectively
transmits the desired wavelength and absorbs
the rest.
 2 types –
 Absorption filter and
 Interference filter Interference
filter
MgF
2

22. B. Monochromators
 A Grating/Prism disperses radiation energy from the
source lamp into a spectrum from which desired
wavelength is isolated by mechanical slits.
 Prism – non linear dispersion
 Grating – Linear dispersion
a. Prisms :
 Made up of fused silica
 Give only one order of emerging spectrum thus provide
higher optical efficiency.
 Wavelength of monochromatic light emerging from exit
slit can be change by rotating the prism.

23. b. Grating :
 Consists of highly polished
reflecting surfaces with many
equally placed parallel grooves with
sharp corners.
 Types –
 Transmittance grating (made up of
glass)
 Reflection grating (made up of
aluminium)
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4. Exit slit :
 Determine the band width of light that will be selected
from dispersed spectrum

24. 5. Cuvettes/Cell
 Receptacle for sample
 Optical property
depends on the Composition.
 Calibrated to path length 1 cm
 2 types : Silicate and Quartz or fused silica cell
6. Photodetectors
 A device that converts light into an electric signal that is
proportional to the no. Of photons striking its
photosensitive surface.
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25. 3 types :
a) Photomultiplier tube – highly sensitive
b) Photodiodes – high signal/noise
c) Charged couple device – constant response to
wavelengths and fast response time.
a. Photomultiplier tubes :
 An electron tube that is capable of significantly amplifying
a current.
 Extremely fast response time
 Slow to fatigue
b. Photodiodes :
 Semiconductors that change their charged voltage upon
being struck by light
 Change is converted to current and measured
 Higher response time
 Unlike PMT, it can be used for high intensity

26. C. Charged couple device :
 Solid phase devices that are made up of small silicon
cells.
 Electron released is captured and quantified
7. Read out devices
 Elecric energy from the detector is displayed on a meter
or display system
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27. Classes of Spectrophotometers
Single beam and double beam are the two major classes of
spectrophotometer.
 Single Beam: In this type, all the light passes through the
sample .To measure the intensity of the incident light the
sample must be removed so that all the light can pass through.
This type of spectrometer is usually less expensive and less
complicated.
 Double Beam: In this type, before reaches the sample the
light source is split into two separate beams. From these one
passes through the sample and second one is used for
reference. This gives the advantageous because at the same
time the reference reading and sample reading can take place.

28. 0
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0.72
Single beam Spectrophotometers
Double beam Spectrophotometers

29. Advantage of Double beam
Spectrophotometer
Compensate for variation in the source
intensity
Compensate for drift in the detector and
amplifier
Compensate for variation in intensity as
function of wavelength

30. COLORIMETER SPECTROPHOTOMET
ER
Light measures only in
visible region
UV, visible, IR, X-rays
Filters Prisms/gratings
Can choose only a
bandwidth of
wavelength
Can choose exact
wavelength
Only coloured solutions
measured
Colourless solutions
can also be measured
Absorbance – less Absorbance – More
Comparison

31. FLAME EMISSION
PHOTOMETRY
(More accurately
called Flame atomic
emission
Spectrophotometry)
Used for Quantitative
measurement of
certain metal ions
among them Na+, K+,
Ca2+, Li in Body

32. Principle
 Alkali metals when heated to high temperature,
energy is absorbed by orbital electron.
 Electron excited and move to a higher state.
 Being unstable there, return back to ground state
by reemitting the absorbed energy as radiation of
specific wavelength
 Isolated by optical filter
 Photo detector placed behind the filter converts it
in to an electric current which is measured

33.  The wavelength of the atomic spectral line gives
the identity of the element.
 The intensity of the emitted light is proportional
to the number of atoms of the element
 light intensity α no. of atoms α conc. of
substance
Na+ emits 589 nm YELLOW
K+ 766 nm VIOLET
Ca 622 nm Orange
Li 670 nm RED
Various metals emit a characteristic colour of light when
heated

41. ATOMIC ABSORPTION
SPECTROPHOTOMETRY
• This is an emission technique.
• Measures elements AL, Ca, Cu, Mg, Zn,
Li, Pb.

44. COMPONENTS
 Hollow cathode lamp
 Nebulizer
 Atomizer
 Monochromator
 Detector

54. Thank You