Beer's law and Lambert's law describe the relationship between light absorption and the properties of the material that the light passes through. Specifically, Beer's law states that absorption increases exponentially with increasing concentration or path length, while Lambert's law states it increases exponentially with path length alone. Together they form Beer-Lambert law. A spectrophotometer uses these principles to measure the absorption of light by a sample to identify unknown substances or determine concentrations. It has components like a light source, monochromator, and detector to measure transmittance or absorbance. Single beam spectrometers measure one path at a time, while double beam measures a sample and reference simultaneously for better accuracy.

1. PRINCIPLE (BEER’S LAMBERT’S LAW)
VISIBLE SPECTROPHOTOMETER
(SINGLE BEAM & DOUBLE BEAM)
PAPER 4 UNIT 4
_PRASHANT SINGH

2. BEER’S LAW = when a ray of monochromatic light passes through an
absorbance medium, its intensity decreases exponentially as the
concentration of the absorbing medium increases, provided the length of the
absorbing medium is constant.
Equation :
Where as, I = Intensity of transmitted ray
Iо = Intensity of the incident ray .
k1 = constant
c = concentration of he absorbing medium

3. LAMBERT’S LAW = when a ray of monochromatic light passes through an
absorbing medium, its intensity decreases exponentially as the light of the
absorbing medium increases, provided the concentration of the absorbing
medium is constant.
Equation =
Where as, I = Intensity of transmitted ray
Iо = Intensity of the incident ray .
k2 = constant
l = path lenghtof he absorbing medium

4. BEER’S-LAMBERT’S LAW
 When a ray of monochromatic light of initial intensity Iо passes through a
solution or a transparent vessel, some of the light is absorb so that the
intensity of the transmitted light I is less than Iо.
 The relationship between I and Iо depends on the path length l of the
absorbing medium and the concentration C of the absorbing solution
 Equation

5. Transmittance (T)
Transmittance is the ratio of intensities and usually expressed as a percentage
(%)
Equation: T= I/Iо % T= I/Iо × 100
The absorbance A or extinction E is expressed as :
E=log I/Iо
According to the beer’s lambert’s law the extinction or absorbance is
propotional to the concentration of the absorbing solution (C) and to the
thickness of the absorbing medium (L) i.e. A∞C & A∞L
Therefore, A=ἐCL
Where, ἐ= molar extinction co-efficient for absorbing medium.

6. Transmittance
I0 I
b
303.2
log)log(
)log(303.2)ln(
0
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0
0
00
0
k
bcAT
I
I
I
I
kbc
I
I
dbkc
I
dI
kcdb
I
dI
I
I
T
I
I
b





 



8. Electronic Spectroscopy
 Ultraviolet (UV) and visible (VIS) spectroscopy
 This is the earliest method of molecular
spectroscopy.
 A phenomenon of interaction of molecules with
ultraviolet and visible lights.
 Absorption of photon results in electronic
transition of a molecule, and electrons are
promoted from ground state to higher
electronic states.

9. UV and Visible Spectroscopy
 In structure determination : UV-VIS
spectroscopy is used to detect the presence of
chromophores like dienes, aromatics,
polyenes, and conjugated ketones, etc.

10. Instrumentation

11. Components of a Spectrophotometer
Light Source
 Deuterium Lamps－a truly continuous
spectrum in the ultraviolet region is
produced by electrical excitation of
deuterium at low pressure.
(160nm~375nm)
 Tungsten Filament Lamps－the most
common source of visible and near
infrared radiation.

12. Components of a Spectrophotometer
Monochromator
 Used as a filter: the monochromator will
select a narrow portion of the spectrum
(the bandpass) of a given source
 Used in analysis: the monochromator will
sequentially select for the detector to
record the different components
(spectrum) of any source or sample
emitting light.

13. Monochromator

14. Grating

15. Detector
Barrier Layer/Photovoltaic

16. Principle of Barrier
Layer/Photovoltaic Detector
 This device measures the intensity of photons
by means of the voltage developed across the
semiconductor layer.
 Electrons, ejected by photons from the
semiconductor, are collected by the silver
layer.
 The potential depends on the number of
photons hitting the detector.

17. Principle of Phototube Detector
 This detector is a vacuum tube with a cesium-
coated photocathode.
 Photons of sufficiently high energy hitting the
cathode can dislodge electrons, which are
collected at the anode.
 Photon flux is measured by the current flow in
the system.

18. Phototube detector

19. Detector
Photomultiplier

20. Principle of Photomultiplier
Detector
 The type is commonly used.
 The detector consists of a photoemissive
cathode coupled with a series of electron-
multiplying dynode stages, and usually called
a photomultiplier.
 The primary electrons ejected from the photo-
cathode are accelerated by an electric field so
as to strike a small area on the first dynode.

21. Principle of Photomultiplier
Detector
 The impinging electrons strike with enough
energy to eject two to five secondary electrons,
which are accelerated to the second dynode to
eject still more electrons.
 A photomultiplier may have 9 to 16 stages,
and overall gain of 106~109 electrons per
incident photon.

22. Single and Double Beam
Spectrometer
 Single-Beam: There is only one light beam or
optical path from the source through to the
detector.
 Double-Beam: The light from the source, after
passing through the monochromator, is split
into two separate beams-one for the sample
and the other for the reference.

24. advantage
 Less expensive
 High sensitivity
disadavntage
 Instability due to lack of
compensation for
disturbance like voltage
fluctuation, mechanical
components instability or
drift in energy of light
source.
 Such drifts result in
abnormal fluctuation in
the result
Single beam
spectrophotometer

25. Double beam
spectrophotometer
advantage
 No warm up time
 Better level of detection
 Instability factor do not
affect the measurement
in real time result
disadvantage
 expensive

26. Application
 UV/Vis spectroscopy is routinely used in analytical
chemistry for the quantitative determination of different
analytes, such as transition metal ions, highly conjugated
organic compounds, and biological macromolecules.
 Spectroscopic analysis is commonly carried out in
solutions but solids and gases may also be studied.

27. Quantitative Analysis
Beer’s Law
A=bc
: the molar absorptivity (L mol-1 cm-1)
b: the path length of the sample
c :the concentration of the compound in
solution, expressed in mol L-1