Applications OF UV-Visible Spectroscopy.pptx

1. Application OF UV-
Visible Spectroscopy

2. Applications of UV Visible Spectroscopy
• Used for qualitative and quantitative analysis of all molecules that absorb
ultraviolet and visible electromagnetic radiation.
QUALITATIVE ANALYSIS:-
1. Structure elucidation of organic compound
2. Determination of impurities
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Basic Spectroscopy, Principle & Applications By ILMI

3. Structure elucidation of organic compound
• U.V spectroscopy is useful for the structure elucidation of organic
compound.
• The presence and absence of a particular absorption band at particular
wavelength may be taken as evidence for presence and absence of
particular chromophores in compound.
EXAMPLE:-
Vitamin A lambda max=325 nm
Vitamin B lambda max=287 nm and 351 nm
• 351 nm is due to presence of extra ethylenic bond.
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Basic Spectroscopy, Principle & Applications By ILMI

4. Detection of impurities
• Best method for determination of impurities in organic compound.
Additional peaks are observed due to impurities in a sample.
FOR EXAMPLE:-
Benzene appear as common impurity in cyclohexane. Its presence
can be detected by its absorption at 255 nm.
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Basic Spectroscopy, Principle & Applications By ILMI

5. TLC is used for qualitative analysis of reaction
mixture
Sample is applied on TLC plate.
Allowed to run in appropriate solvent.
Different compounds travel different distance which then visualized under UV
lamp.
Extra spots are due to impurities.
UV visible spectroscopy can be used as detector for HPLC.
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6. Quantitative Analysis
Determination of concentration of compound
Chemical Kinetics; study of chemical reaction
Dissociation constant of acid and bases
Quantification and thermal denaturation of DNA
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7. Determination of concentration of the
compound
• Principle of absorption in UV-Spectroscopy based on Beer Lambert
Law
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A = log lo /I =log 1/T=-log T=€CL
Where, €=molar extinction coefficient
C = concentration
L = path length
lo = intensity of incident radiation
I= the intensity of transmitted radiation
 Absorbance is directly related to concentration
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8. Example:
 The estimation of anthracene in a mixture of anthracene and
naphthalene.
 UV spectrum shows lamda max at 375nm, where naphthalene does
not absorb in this region.
 Thus by using Beer Lambert Law we can calculate the concentration
of pure anthracene.
 Similar procedure for estimation of anthracene in benzene,
ergosterol in fat, chlorophyll in plant material,
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9. UV-Absorption spectrum of Benzene,
Naphthalene & Anthracene
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10. Chemical Kinetics
UV visible spectroscopy can also be used to “ REACTION
RATES .”
The UV radiation is passed through the reaction cell and
absorbance changes can be observed .
In UV visible spectroscopy “ Rate of a reaction can be
determined provided one of the Reactant OR Product
absorbs UV at a wavelength where other reactants or
products have little or low absorbance .”
https://youtu.be/bR86HHClmYY
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11. Continued . . .
For example in Nitroethane ,
Reaction :
To measure the rate at which hydroxide removes a Proton from
nitro-ethane , UV spectrophotometer is adjusted to measure the
Absorbance as a function of Time . Anion of nitro-ethane has max
absorbance at 240nm , but neither other product show significant
absorption at this wavelength .
https://youtu.be/bR86HHClmYY
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12. Other Examples
Enzymes cannot be measured directly but their catalytic
properties allow their estimation from the Speed of
reactions they catalyze .
The relative proportion of different enzymes can be used to
diagnose the disease say of Liver , Pancreas or any other
organ .
In clinical diagnostic , it is used as an indicator of tissue
damage .
https://youtu.be/bR86HHClmYY
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Dissociation constant of acid and base
• The pKa of a compound cab be determined by UV/Vis spectroscopy.
• If either the acidic or basic form of the compound absorbs UV or visible light.
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Dissociation constant of acid and base
For example, the phenoxide ion has a λmax at 287 nm. If the absorbance at 287 nm
is determined as a function of pH, the pKa of phenol can be calculated by using
Henderson-Hasselbalch equation
pH = pKa + log [A-] / [HA]
By plotting graph between absorbance and wavelength at different pH values, the
ratio [A-]/[HA] can be determined and hence the pKa value of a compound can be
calculated.
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Quantification and thermal denaturation of
DNA
• UV Spectroscopy is used to estimate the nucleotide composition of
DNA.
• In DNA
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Quantification and thermal denaturation of
DNA
• When we heat DNA
Less Absorbance at 260 nm More Absorbance at 260 nm
Hydrogen Bond
Breaks
Hydrogen Bond
Present
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Quantification and thermal denaturation of
DNA
• Melting Temperature Of DNA (Tm)
The temperature at which half of the DNA
molecules are denatured.
Tm increases with GC pairs.
%G-C= 2.44*(Tm – 81.5 – 16.6 [log(M)] + 500/K)
Where, M is the molarity of the solution in mol/L
K is the DNA base pair length
UV/Visible Spectroscopy by Ian Robertson PerkinElmer, Inc.
Seer Green, UK
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