Spectrophotometry

Advanced Pharmaceutical Analysis

Lab. 1
Course 2 …..5th year
Teacher Rawa M. M. Taqi

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Spectrophotometry:-
Is the measurement of the interaction of light with matter.
Principles of Spectroscopy
If matter is exposed to electromagnetic radiation, e.g. UV light, the radiation can
be absorbed, transmitted or reflected.

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 Spectrophotometer
• The use of Spectrophotometer for the analysis of compounds
which is divided into
• Quantitative analysis (how much is there?)
• Qualitative analysis ( what are they?)

• Atoms and molecules show the ability for absorbtion of energy in

the form of electromagnetic radiation .
• The type and quantity of the absorbed energy depends on :
1. The chemical structure of these molecules .
2. The quantity of absorbed energy depends on the number of the
molecules absorbing this solution. 4
Electromagnetic radiation
• Electromagnetic radiation : is a term applied to the energy
diffused in the form of waves.
• Electromagnetic spectrum:- is composed of wide range of
radiation frequencies. It is possible to divide the spectrum into
the following regions according to
 the wave length
 energy
 frequency
 and the nature of interaction between the molecules and
radiation ( ionization ,exititation, or vibration).
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Electromagnetic radiation

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Electromagnetic spectrum
• The types of electromagnetic radiation are broadly classified into the
following classes depends on the increasing order of wavelength:-
• Cosmic rays( higher energy, higher frequency, and shorter wave length)
• γ- rays
• X- rays
• UV- rays
• Visible rays
• IR- rays
• Microwaves
• Radio waves ( lower energy, lower frequency, and longer wave length)
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Electromagnetic spectrum

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Electromagnetic radiation

Wavelength is expressed in nanometers (10 -9 meters). Other

units which may be encountered, but whose use is now
discouraged, are the Angstrom (Å) and the millimicron (mμ).
1mμ = 10Å = 10 -7 cm.
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Electromagnetic radiation

The higher the

frequency, the greater
the energy:
E = hn
The higher the
frequency, the shorter
the wavelength:
Where l =c/n
E = Energy of light
h = plank's constant
This is why UV
n = frequency of light
λ = wave length radiation from the sun
C = velocity of light (3 x 10 10 cm s-1 ). burns you.
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 The UV radiation
region extends
from
200 nm to 380 nm
and the visible
radiation region
extends from
380 nm to 780 nm.
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PRINCIPLES OF UV - VISIBLE SPECTROSCOPY
In order for absorption to occur within this region of spectrum
the compounds should be conjugated systems or aromatics
compounds
While saturated hydrocarbons show no absorption in this region
so they can be used as solvents e.g. cyclohexane , n-hexane.
Some inorganic salts are colored and show absorption within the
visible region e.g. chromic nitrate.
Absorption spectra in the ultraviolet and visible regions are
due to energy transitions of both bonding and nonbonding
outer electrons of the molecule.
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PRINCIPLES OF UV - VISIBLE SPECTROSCOPY
• An electron is exited from a full orbital (low energy ground state
orbital) into an empty anti-bonding orbital (high energy exited
state orbital). anti-bonding
orbital (empty)
• Each jump takes energy from the
light ,and a big jump needs more
energy than a small one.
• Each wavelength of light has Full orbital
(Bonding)
a particular energy associated
with it.
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 Electrons in σ bonds (a single covalent bond) are tightly bound and
radiation of high energy (short wavelength) is needed to excite
them.
 Certain atoms like N,O ,and halogens have non bonding electrons
( lone pair) that are less tightly bound than the previous and can be
exited at a lower energy (longer wavelength) radiation.
 Electrons in double or triple bonds ( π electrons) are easy to be
exited ( loosely bound) and in compounds that contain series of
alternating double bonds (conjugated systems) , the electrons are
delocalized due to resonance and require less energy for excitation
( longer wave length). 14
The possible electronic transitions

These are
normally
empty

These contain
lone pairs

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Visible light
White light is a combination of
light of different wave length
in the visible spectrum .
passing white light through a prism
splits it up into the several colors
of light observed in the visible
spectrum between 400 nm and 780 nm.
A common feature of colored compounds is a system of
extensively conjugated pi – electrons. 16
Absorption spectrum
It is a spectrum of Absorbance versus wave length (λ) or
Transmittance(T) versus wave length (λ).
An example of simple UV – Visible absorption spectrum for Isoprene
is shown below.
Absorbance (on the vertical y-axis)
is just a measure of the amount
of light absorbed .
The higher the value,
the more of a particular
wavelength is being absorbed
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 Lambda max (λ max): is the wave length at which maximum
absorption occurs (%T is minimum).
 While Lambda min (λ min): is the wave length at which minimum
absorption occurs (%T is maximum).

Some compounds show more than one λ max.

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Instrument ( spectrophotometer) parts
1. Light source: deuterium lamp (UV light), or tungsten lamp (visible).
2. Monochromator: allow the passage of light in certain selected wave length and
neglecting the other unwanted wavelengths (using wave control knob).
3. Sample compartment: accommodate the sample (inside cuvette) to be exposed to
the monochromatic light.
4. Detector: responsible for converting light signals (transmitted) to electrical
signals.
5. Microprocessor: translate the electrical signals to digital signals.
6. Displayer: display the digital signals on screen (A or T %).

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Notes
Cuvette: either quartz (UV) or glass (visible).
The use of scratched or contaminated cuvettes
should be avoided since they reflect and /or absorb
radiation that will give you inaccurate measurements.
Also, bubbles, turbidity, fingerprints, should be avoided since they will diminish
the accuracy of readings.
The cuvettes commonly used for accurate work have an optical path length of 1 cm
and require 2.5 – 3 ml of sample for all accurate reading.
Blank : the medium in which the substance being measured is located may itself
absorb light of certain wavelength so in order to measure the absorbance due to only
a particular species in solution , zeroing is needed , in which the blank is added in
the light path and the light control knob is rotated until read 100% T (A=0). 21
Operating procedure
[Link] the spectrometer and allow standing for 20 minutes (for
warming).
[Link] the wavelength dial on the spectrometer to lambda required nm.
With the sample chamber empty, until reads zero %T (which is the
case when no cuvette is placed in sample holder.
[Link] your reference blank into the sample compartments until
reads 100%T (A=0).
[Link] sample is added now and the A or %T is recorded.
[Link] a change in wavelength is made the 0 % T and 100%T
must then be reset since the amount of compensation varies with
the wave length.
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The purpose
Qualitative
[Link] the absorption spectrum of
unknown compounds (qualitative analysis).
2. Determine the λ max of compound.
Quantitative
3. Determine the concentration of a compound in
solution using Beer- Lambert's Law.
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Determination of the absorption spectrum of
POTASSIUM PERMANGANATE solution KMnO4
(Qualitative analysis).
By gradual scanning along wide range of wave lengths
then reading A and % T.
Plot your data on graph paper ( plot absorbance (A) on
the y-axis as a function of wavelength (λ) on the x-
axis).
Plot Transmittance (%T ) on the y-axis as a function of
wavelength (λ) on the x-axis. 24
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