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الفصل الثالث-الجزء الثاني

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29 views16 pages

الفصل الثالث-الجزء الثاني

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bifusk481
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Al-Esra’a University College

Department of Pharmacy

Spectrophotometric Analysis

Prof. Dr.
A. M. AL. Haideri
First Stage – Term 1 part 2
Electromagnetic Spectrum
The electromagnetic radiation covers a vast spectrum of
frequencies and wavelengths. This includes the very energetic
gamma-rays radiation with a wavelength range from 0.005-1.4
A° to radio waves in the wavelength range up to meters
(exceedingly low energy). However, the region of interest to us
in this course is rather a very limited range from 180-780 nm.
This limited range covers both ultraviolet and visible radiation. It
is of interest to indicate at this point that each region of
electromagnetic spectrum requires a special set of instrumental
components. This can therefore make it clear for us that the type
of instrumental components we will deal with will be extremely
limited but, at the same time, will be representative enough.
Fundamentals of Spectrophotometry
Spectroscopy is any procedure that uses the
interaction of Electromagnetic Radiation (EMR)
with matter to identify and/or to estimate an analyte.

Molecules
Ions Solid Qualitative Quantitative
Atoms Liquid Analysis Analysis
mixtures Gas
solutions
➢Electromagnetic radiation consists of discrete packets of energy, which
we call photons.
➢ Photons are the particles of light or the quanta of light.
➢ Each photon carries the energy, E (Joule).
𝑬 = 𝒉𝝂
where h is the Planck’s constant (=6.626x10-34 J.s)
➢The all characteristics of light can be related as follows:
𝒄
𝑬 = 𝒉𝝂 = 𝒉 = 𝒉𝒄 𝝂
𝝀
The greater the energy, the higher the frequency and
wavenumber and the shorter the wavelength.
*The particle nature can explain phenomena like absorption and emission
of light.
Absorption of Light
A molecule that absorbs light photons will end up with increased energy. The
molecule will be promoted to an excited state. Microwave energy will cause
rotation of compounds. IR energy is high enough to promote bond stretching,
UV/Vis energy promotes electrons into higher orbitals. Short-𝝺 UV and X-rays
can ionize molecules or even break bonds.

Most excited molecules relax again to the ground state emitting the excess energy
in the form of heat.
Ultraviolet-Visible Spectrophotometry
What happens when a molecule absorbs UV-Visible radiations?
❖When a molecule absorbs light having sufficient energy (e.g. UV-Vis radiation) to
cause an electronic transitions, additional vibration and rotation transitions also
occur.
❖Molecule can absorb one photon of just the right energy to cause the following
simultaneous changes:
1. A transition from the ground electronic state E0 to the E1 excited electronic state.
2. A change in the vibrational energy from the ground vibrational state of E0 to an
excited vibrational state of E1.
3. A transition from one rotational state of E0 to a different rotational state of E1.
4. All the above transitions are quantized which means that they required certain
exact amount of energy.
5. Thus, total energy absorbed = Eelec + Evib + Erot

As a result, a large number of photons of


certain wavelengths are absorbed by a
molecule. These individual wavelengths
are too numerous and too close to each
other and a spectrum of broad band of
absorbed wavelengths are obtained.
Transmittance and Absorbance
There are two quantities that relate the change in the intensity or radiant
power of EMR before P0, and after P, interaction with matter.
1. Transmittance T is simply defined as “the fraction of light that reaches
a detector after passing through a sample”
𝑷
𝑻= 0≤T≤1
𝑷
The percent transmittance %T, is simply 100 T
𝑷
%𝑻 = × 𝟏𝟎𝟎 0 < %T < 100
𝑷
2. absorbance define as:
𝑷 𝑷
A=-log T → A=-log → A=log ( )
𝑷 𝑷
For purpose of chemical analysis
Absorbance is directly proportional to:
1. concentration c of absorbing species in the sample (A α c)
2. path length of light b through the sample (A α b)

Beer's law 𝑨 = 𝜺𝒃𝒄

The previous equation is the heart of spectrophotometry as


applied to analytical chemistry, it is called Beer-Lambert law
or simply Beer‘s law.
❑ concentration of the analyte is given in unit mol/L (M)
❑ The path length b in cm
❑ ε is called the molar absorptivity or molar absorption
coefficient “Absorbance of 1 M solution measured in a cell of
1 cm pathlength”
❑ ε is characteristic for each substance at a particular
wavelength λ and particular solvent.
Beer-Lambert’s law proves a direct correlation between the absorbance (A) of a molecule to
the concentration (c) and the path length (b).

Derivation of Beer Lambert Law


This relationship is a linear for the most part. However, under certain circumstances the
Beer relationship gives a non-linear relationship. These deviations from the Beer Lambert
law can be classified into three
Categories:
Real Deviations These are fundamental deviations due to the limitations of the law itself.
Chemical Deviations These are deviations observed due to specific chemical species of the
sample which is being analyzed.
Instrument Deviations These are deviations which occur due to how the
absorbance measurements are made.

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