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- 1. SEMINAR ON PRESENTED BY MR. Nitin P. Kanwale M.PHARM. 1ST
SEM. M.V.P. Samajs college of Pharmacy Nashik-2 GUIDED BY
DR.D.V.DERLE UV-VISIBLE SPECTROSCOPY NDMVP SAMAJS COLLEGE OF
PHARMACY NASHIK-2
- 2. Content Introduction Principle and Basic concept Some
important terms Instrumentation Applications Conclusion
References
- 3. INTRODUCTION Spectroscopy is a technique that measures the
interaction of molecules with electromagnetic radiation. Light in
the near-ultraviolet (UV) and visible (vis) range of the
electromagnetic spectrum has an energy of about 150 400 kJ mol. The
energy of the light is used to promote electrons from the ground
state to an excited state. A spectrum is obtained when the
absorption of light is measured as a function of its frequency or
wavelength. The absorbance of a solute depends linearly on its
concentration and therefore absorption spectroscopy is ideally
suited for quantitative measurements. Spectroscopic measurements
are very sensitive and nondestructive, and require only small
amounts of material for analysis Application of derivative
technique of spectrophotometry offers a powerful tool for
quantitative analysis of multi-component mixtures.
- 4. Principle and Basic concept Ultraviolet visible spectroscopy
( 200 - 800 nm) studies the changes in electronic energy levels
within the molecule arising due to transfer of electrons from - or
non- bonding orbital's. It commonly provides the knowledge about
-electron systems, conjugated unsaturations, aromatic compounds and
conjugated non-bonding electron systems etc UV- Visible is divided
into the ultraviolet (UV, 190-400 nm) and visible (VIS, 400-800 nm)
regions. Since the absorption of ultraviolet or visible radiation
by a molecule leads transition among electronic energy levels of
the molecule, it is also often called as electronic
spectroscopy.
- 5. Nature of Electronic Transitions The total energy of a
molecule is the sum of its electronic, its vibrational energy and
its rotational energy. Energy absorbed in the UV region produces
changes in the electronic energy of the molecule. As a molecule
absorbs energy, an electron is promoted from an occupied molecular
orbital (usually a non-bonding n or bonding orbital) to an
unoccupied molecular orbital (an anti-bonding or orbital) of
greater potential energy (figure1). Fig.1)Relative energies of
orbitals most commonly involved in electronic spectroscopy of
organic molecules increasing order of their energies viz. n* <
n* < * < * < *
- 6. Principles of Absorption Spectroscopy: Beers and Lamberts
Law The greater the number of molecules that absorb light of a
given wavelength, the greater the extent of light absorption and
higher the peak intensity in absorption spectrum . This makes the
basis of Beer-Lambert Law which states that the fraction of
incident radiation absorbed is proportional to the number of
absorbing molecules in its path . When the radiation passes through
a solution, the amount of light absorbed or transmitted is an
exponential function of the molecular concentration of the solute
and also a function of length of the path of radiation through the
sample. Therefore, Log Io / I = c l Where Io = Intensity of the
incident light (or the light intensity passing through a reference
cell) I = Intensity of light transmitted through the sample
solution c = concentration of the solute in mol l-1 l = path length
of the sample in cm = molar absorptivity or the molar extinction
coefficient , is numerically equal to the absorbance of a solution
of unit molar concentration (c = 1) in a cell of unit length ( l =
1) and its units are liters. moles-1. cm-1. The ratio I / Io is
known as transmittance T and the logarithm of the inverse ratio Io
/ I is known as the absorbance A. Therefore - Log I / Io = - log T
= c l and Log Io / I = A = c l or A = c
- 7. Spectral Measurements The UV-Vis spectra are usually
measured in very dilute solutions and the most important criterion
in the choice of solvent is that the solvent must be transparent
within the wavelength range being examined. Table lists some common
solvents with their lower wavelength cut off limits. Below these
limits, the solvents show excessive absorbance and should not be
used to determine UV spectrum of a sample. SR.NO. Solvent Cut off
wavelength(nm) 1 Acetonitrile 190 2 Water 191 3 Cyclohexane 195 4
Methanol 203 5 95% ethanol 304 Of the solvents listed in table ,
water, 95% ethanol and hexane are the most commonly used solvents.
For recording the spectrum 1 cm square quartz cell is commonly
used. These require approx. 3 ml of solution.
- 8. Solvent Effects Highly pure, non-polar solvents such as
saturated hydrocarbons do not interact with solute molecules either
in the ground or excited state and the absorption spectrum of a
compound in these solvents is similar to the one in a pure gaseous
state. However, polar solvents such as water, alcohols etc. may
stabilize or destabilize the molecular orbital's of a molecule
either in the ground state or in excited state and the spectrum of
a compound in these solvents may significantly vary from the one
recorded in a hydrocarbon solvent. Some important terms (i)
Chromophore: The energy of radiation being absorbed during
excitation of electrons from ground state to excited state
primarily depends on the nuclei that hold the electrons together in
a bond. The group of atoms containing electrons responsible for the
absorption is called chromophore. Most of the simple un-conjugated
chromophores give rise to high energy transitions of little
use.
- 9. (ii) Auxochrome: The substituents that themselves do not
absorb ultraviolet radiations but their presence shifts the
absorption maximum to longer wavelength are called auxochromes. The
substituents like methyl, hydroxyl, alkoxy, halogen, amino group
etc. are some examples of auxochromes. (iii) Bathochromic Shift or
Red shift: A shift of an absorption maximum towards longer
wavelength or lower energy. (iv) Hypsochromic Shift or Blue Shift:
A shift of an absorption maximum towards shorter wavelength or
higher energy. (v) Hypochromic Effect: An effect that results in
decreased absorption intensity. (vi) Hyper-chromic Effect: An
effect that results in increased absorption intensity.
- 10. INSTRUMENTATION
- 11. Simultaneous Estimation of multi-component formulation by
UV-Visible spectroscopy Simultaneous equation is applicable for the
estimation of those drugs where the spectra of drug overlap
properly whereas, DS method has been widely used to enhance the
signal and resolve the overlapped peak-signals due to its
advantages in differentiating closely adjacent peaks, and
identifying weak peaks obscured by sharp peaks. Following are the
method use 1. Derivative Spectroscopic Method 2. Simultaneous
Equation 3. The Absorption Ratio Method : iso-absorptive Point
Method 4. Multi-component Mode Method 5. Area Under Curve
Method
- 12. Conclusions UV-visible spectroscopy, a simple, rapid,
precise and highly accurate method for quantitative estimation is
in great use now a day. Derivative spectrophotometry is an
analytical technique of great utility for extracting both
qualitative and quantitative information from spectra composed of
unresolved bands by calculating and plotting one of the
mathematical derivatives of a spectral curve. Therefore the
derivative spectra (first to fourth-order) of the mixtures were
checked to select a suitable spectrum to be used for the
simultaneous determination of the components. Derivative techniques
in spectroscopy often offer a powerful tool for a resolution
enhancement, when signal overlaps or interference exists. Several
specific signals were singled out for the components in the spectra
of different derivative orders but the first-order derivative
spectra seemed to be generally the most suitable for analytical
aim. A derivative spectrum shows better resolution of overlapping
bands than the fundamental spectrum and may permit the accurate
determination of the max of the individual bands. Secondly, DS
discriminates in favor of substances of narrow spectral bandwidth
against broad bandwidth substances. All the amplitudes in the
derivative spectrum are proportional to the concentration of the
analyte provided that Beer's law is obeyed by the fundamental
spectrum.
- 13. References: 1) Gurdeep R. Chatwal, Sham K. Anand,
Instrumental Methods Of Chemical Analysis, Himalaya Publication,
Fifth Edition, Page No.2.149-2.184 2)Douglas A. Skoog, Holler,
Nieman, Principles Of Instrumental Analysis, Fifth Edition, Page
No.300-352 3)Frank A. Settle, Editor Handbook Of Instrumental
Techniques For Analytical Chemistry, Person Education, Page
No.481-499 4) C. Bosch Ojeda, F. Sanchez Rojas, Recent Applications
In Derivative Ultraviolet/Visible Absorption Spectrophotometry:
20092011, Microchemical Journal 106 (2013) 116, Contents Lists
Available At SciverseSciencedirect. Journal Homepage:
Www.Elsevier.Com/Locate/Microc. 5) Franz-XaverSchmid, Biological
Macromolecules: UV-Visible Spectrophotometry ENCYCLOPEDIA OF LIFE
SCIENCES / & 2001 Macmillan Publishers Ltd, Nature Publishing
Group / Www.Els.Net. 6)Jasmine Chaudhary, Akash Jain, Vipin Saini,
Simultaneous Estimation Of Multi- component Formulations By
UV-Visible Spectroscopy, International Research Journal Of
Pharmacy, Available Online Www.Irjponline.Com. 7) Www.Wikipedia.Com
Accessed On 2/11/2014; 06:30 PM