This document discusses the basic concepts and principles of spectrophotometry. It begins by defining photometry and spectrophotometry as the measurement of light intensity falling on a surface or at selected wavelengths, respectively. Spectrophotometers use prisms, gratings, or filters to isolate wavelengths. Beer's law states that absorbance is directly proportional to concentration and path length, allowing for quantitative analysis. Key relationships include transmittance, absorbance, and molar absorptivity. Conditions like monochromatic light and concentrations within Beer's law limits must be met for quantitative analysis.

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BASIC CONCEPTS,
INSTRUMENTATION
AND APPLICATION
By
Dr.Gurumeet C Wadhawa
Assistant Professor,
Department of Chemistry
Karmaveer Bhaurao Patil College,
Vashi,
Navi mumbai

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INTRODUCTION:
• Photometry is the measurement of the
amount of luminous light (Luminous
Intensity) falling on a surface from a source.
• Spectrophotometry is the measurement of
the intensity of light at selected
wavelengths.
• The method depends on the light absorbing
property of either the substance or a
derivative of the substance being analyzed
• Spectrophotometers use prisms and
gratings for isolating wavelengths while
devices that require filters for this purpose
are called Filter Photometers.

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NATURE OF LIGHT:
• Electromagnetic waves are characterized by
their frequency and wavelength.
• Light is a spectrum of different wavelengths which
the eye recognizes as “white” but can be isolated
into discrete portions and measured.
• Human eye responds to radiant energy btw 380
and 750nm, but modern instruments can
measure shorter wavelengths (UV) and longer
(IR) ones.
• Wavelength describes a position within a spectrum.
It is the distance btw 2peaks as the light travels in
a wave- like manner
• Light also is composed of discrete energy packs
called photons whose energy is inversely
proportional to the wavelength

4. BASIC CONCEPTS:
• When light passes through a solution, a certain fraction is
being absorbed.
• This fraction is detected, measured and used to relate the
light absorbed or transmitted to the concentration of the
substance.
• This enables both qualitative and quantitative analyses of
substances.
• The spectrophotometric technique is used to measure light
intensity as a function of wavelength. It does this by:
– Diffracting the light beam into a spectrum of wavelengths
– Direct it to an object
– Receiving the light reflected or returned from the object
– Detecting the intensities with a charge-coupled device
– Displaying the results as a graph on the detector and then
the display
device
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5. • The light absorption is directly related to the
concentration of the compound in the sample.
• As Concentration increases, light Absorption increases
linearly
and light Transmission decreases, exponentially
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6. Transmittance and
Absorbance:
• When a sample is illuminated, it absorbs some of the
light and transmits the rest.
• The transmitted light (Is) is of lower intensity than the
incident light (Io), and the transmitted light is defined
as:
T = Is / Io
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• Toensure accuracy (by eliminating effects of reflection by surface of
the cell, absorption by the cell wall and by solvent) an identical
reference cell without the compound of interest is also used.
• Thus, the amount of light absorbed (A) as the incident light
passes through the sample is equivalent to:
A = - log Is / IR = - logT
• In practice, the Reference cell is inserted and the instrument adjusted to
an arbitrary scale corresponding to 100% transmittance, after which the
percentage transmittance reading is made on the sample

8. (ἐ).
8
Beer’s Law:
• This states that the concentration of a substance if directly proportional to the amount of
light absorbed or inversely proportional to the logarithm of transmitted light.
A = abc
Where:
A = Absorbance
a = proportionality constant defined as absorptivity
b = light path in centimeters
c = concentration in g/L of the absorbing compound
NB: - Absorbance (A) has no units, so the unit for a = reciprocals those for b
and c.
• When b is 1cm and c is expressed in mol/L, ἐ (epsilon) is substituted for the constant,
a.
• The value of ἐ is a constant for a given compound at a given wavelength under prescribed
conditions of solvent, temperature, pH, etc., and is called the Molar absorptivity

9. Spectrometry
Nomenclature:
Application of Beer’s Law:
• In practice, a direct proportionality between absorbance
and concentration must be established for a given
instrument under specific conditions.
• A linear relationship exists up to a certain
concentration or absorbance beyond which the
solution is said to no longer obey Beer’s Law
• Within this limitation, a calibration constant (K) may be
derived and used to calculate the concentration of
unknown solutions by comparison
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Recall, a = A/bc
Thus, A1 / b1c1 = A2 / b2c2
Where 1 and 2 refers to the calibrating (c) and
unknown
(u) solutions respectively
• But because the Light path b remains constant,
b1 = b2
• Then, A1/c1 = A2 /c2 or Ac/cc = Au/cu
• Hence, concentration of the unknown:
cu = Au/Ac xcc
And,
Where
,
cu = Au x cc / Ac = Au x
K K = cc / Ac

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• The constant cannot be used once Beer’s
Law have been violated
• A non-linear calibration curve can be used if
a sufficient number if calibrators of varying
concentration is included to cover the entire
range encountered for reading on the
unknowns
• Published constants should be used only if
the method is followed in detail and readings
are made on a spectrophotometer capable
of providing light of high spectral purity at a
verified wavelength.
• Use of broader band light leads to some
decrease in absorbance

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• Beer’s Law is followed only if the
following conditions are met:
– Incident radiation on the substance of
interest is monochromatic
– Solvent absorption is insignificant
compared to the solute absorbance
– Solute concentration is within given limits
– Optical interferant is not present
– Chemical reaction does not occur between
the molecule of interest and another solute or
solvent molecule.

13. The Beer –Lambert Law
• When a monochromatic light of initial intensity Io passes
through a solution in a transparent vessel, some of the light
is absorbed so that the intensity of the transmitted light I
is less than Io .There is some loss of light intensity from
scattering by particles in the solution and reflection at the
interfaces, but mainly from absorption by the solution.
• Therelationship between I and Io depends on the path
length of theabsorbing medium, l, and the concentration of
the absorbing solution,c. These factors are related in the
laws of Lambert and Beer

15. Lambert’s law
• When a ray of monochromatic light
passes throughan absorbing medium
its intensity decreases exponentially
as the length of the absorbing
medium increases.

16. Beer’s law :
• When a monochromatic light passes
through an absorbing medium its
intensity decreases exponentially as
the concentration of the absorbing
medium increases.

18. Transmittance:
 The ratio of intensities is known as the
transmittance (T) and this is usually
expressed as percentage

19. Extinction
• If logarithms are taken of the
equation instead of a ratio then

20. • The expression log10 Io/I is known as the
extinction (E) or absorbance(A). The extinction is
some times referred as optical density.
• Therefore
• A (or) E = k cl
• where k is molar extinction co-efficient for the
absorbing material atwave length l, c = molar
concentration of the absorbing solution,l = path
length in the absorbing material in cm.

21. • If the Beer- Lambert law is obeyed
correctly and l is kept constant, then
a plot of extinction against
concentration gives a straight line
passing through the origin
• Extinction, E=log10 Io/I

22. Transmittance

23. Extinction