Introduction to UV-Visible Spectroscopy

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2.  What is spectroscopy?
 Why UV spectroscopy?
 Theory
 Absorption laws
 Chromophore
 Instrumentation
 Application
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3. Spectrum + scopies
When a beam of light is allowed to pass through
a prism or grating, it will disperse into seven
colours from red to violet and the set of colours
produced is called spectrum.
Spectroscopy is the branch of science deals with
the study of interaction of EMR with matter.
Spectroscopy means examination of spectrum.
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4. From the type of radiation which is absorbed,
we can get idea about the nature of the
compound.
From the amount of radiation absorbed the
concentration of the substance can be found
Hence spectroscopy is used for both
quantitative and qualitative analysis
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5.  “The study of interaction of EM radiation with
molecules or Atoms.”
Types:
1.Absorption spectroscopy:
 The study of absorbed radiation by molecule, in
the form of spectra
 e.g.: UV, IR, NMR, colorimetry, atomic absorption
spectroscopy
2.Emission spectroscopy:
 The radiation emitted by molecules can also be
studied to reveal the structure of molecules.
e.g.: flame photometry, flourimetry
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6. 3.Atomic spectroscopy
 Interaction of EMR+ ATOMS
 Changes in energy take place at atomic level
 e.g.: atomic absorption spectroscopy
4. Molecular spectroscopy
 Interaction of EMR + MOLECULES
 Changes in energy take place at the
molecular level
 e.g.: UV,IR
 Results in transitions between vibrational &
rotational energy levels.
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8.  UV Visible spectrum can be ÷ into 3 regions
1. Far or vacuum UV region (10-200 nm)
2. Near or quartz UV region (200-380 nm)
3. Visible UV (380-780nm)
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9. 1. Detection of functional groups
2. Detection of impurities
3. Qualitative analysis
4. Quantitative analysis
5. Single compound without chromophore
6. Drugs with chromophore reagent
7. It helps to show the relationship between
different groups, it is useful to detect the
conjugation of the compounds.
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10.  When a beam of light falls on a solution or
homogeneous media, a portion of light is
reflected from the surface of media, a
portion is absorbed within the medium and
remaining is transmitted through the
medium.
 Thus if IO is the intensity of radiation falling on
the media
 IO = IR + IA + IT
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11.  The absorption of UV radiation brings about the
promotion of an electron from bonding to anti
bonding orbital.
 The wavelength of radiation is slowly changed from
min to max in the given region, and the absorbance
at every wavelength is recorded. Then a plot of
energy absorbed vs. wavelength is called
absorption spectrum.
 The significant features are:
 λ max (wavelength at max absorption)
 E max (The intensity of maximum absorption)
 The UV spectrum depends on
 Solvents
 Concentration of solution
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12. UV and Visible light cause only two kinds of electronic
transitions
• Only organic compounds with p electrons can absorb
energy in the UV/Visible region
• A visible spectrum is obtained if visible light is absorbed
• A UV spectrum is obtained if UV light is absorbed
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13.  Beer’s law
 Lambert’s law
 Beer- Lambert's law
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14.  “The intensity of a beam of monochromatic
light decrease exponentially with the
increase in concentration of the absorbing
substance”.
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15. light absorbed ∞ no: of absorbing molecules
(only true for dilute solutions)
A = log I0/I ∞ c
A - Absorbance or optical density
I0 - intensity of incident radiation
I - intensity of transmitted radiation
C - concentration of the absorbing species in
mol dm₃
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16.  The fraction of radiation absorbed is
independent of the intensity of the radiation
A = log I0/I ∞ l
l = Cell path length
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17. A = e c l
A = log(I / I0)
c = concentration of
substance in solution
l = length of the cell in cm
e = molar absorptivity
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18.  UV visible spectroscopy measure the
response of a sample to ultra violet and
visible range of EM radiation.
 molecules have either n,∏,σ electrons.
These electrons absorb UV radiation &
undergoes transitions from ground state to
excited state.
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19. The Visible Spectrum and Color
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20.  Defined as any isolated covalently bonded group
that shows a characteristic absorption in the UV
or Visible region.
 Chromophore in Greek means “color bearing.”
 only those molecules likely to absorb light in the
200 to 800 nm region which contain ∏-electrons
and may also have atoms with non bonding
electrons.
 such groups that absorb light in the UV- VIS
region are referred to as chromophore.
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21. INDEPENDENT DEPENDENT
When a single
chromophore is
sufficient to impart
color
e.g.:- NO2, NO ,O
and P quinoid
groups
When more than one
chromophore is required
to impart color
e.g.:-
CH3 CO CH3 (colorless)
CH3 CO CO CO CH3
(orange)
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22. SHIFTS IN UV VISIBLE SPECTRUM
BATHOCHROMIC SHIFT :
 Red shift
 A shift to lower energy
or longer wavelength.
HYPSOCHROMIC SHIFT :
 Blue shift
 A shift to higher energy
or shorter wavelength
HYPERCHROMIC SHIFT:
 An increase in intensity.
 HYPOCHROMIC SHIFT:
intensity of absorption
maximum decrease
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23.  Light source
 Deuterium and Tungsten lamps
 Monochromator
 Photodiodes
 Sample containers
 Cuvettes
 Plastic
 Glass
 Quartz
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24.  Two sources are required to scan the entire
UV-VIS band:
Deuterium lamp- covers the uv-200-330
Tungsten lamp-covers 330-700
 As with the dispersive IR, the lamps
illuminate the entire band of UV or visible
light; the monochromator ( grating/prism)
gradually changes the small bands of
radiation sent to the beam splitter
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25.  The beam splitter sends a separate band to a
cell containing the sample solution and a
reference solution.
 The detector measures the difference
between the transmitted light through the
sample (I) vs. the incident (Io) and sends this
information to the recorder.
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26. 1. Virtually all UV spectra are recorded
solution phase
2. Cells can be made of plastic, glass or
quartz
3. Only quartz is transparent in the full 200-
700 nm range; plastic and glass are only
suitable for visible spectra
4. Concentration can be empirically
determined
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31.  Measure the rates of a reaction
 Determine the pKa of a compound
 Estimate the nucleotide composition of
DNA
 Determination of structure of organic
compounds
e.g. element, functional group
 Determination of stereochemistry
e.g. cis or trans.
 Strength of hydrogen bond
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32.  Detection of impurities
determination of impurities in organic
molecules. Additional peaks can be observed due
to impurities in the sample and it can be
compared with that of standard raw material.
 Structure elucidation of organic compounds.
UV spectroscopy is useful in the structure
elucidation of organic molecules, the presence
of hetero atoms, and the presence of
unsaturated compounds. From the location of
peaks and combination of peaks, it can be
concluded that whether the compound is
saturated or unsaturated, hetero atoms are
present or not etc.
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33.  Quantitative analysis
UV absorption spectroscopy can be used for
the quantitative determination of compounds
that absorb UV radiation. This determination
is based on Beer’s law.
 Qualitative analysis
UV absorption spectroscopy can characterize
those types of compounds which absorbs UV
radiation. Identification is done by
comparing the absorption spectrum with the
spectra of known compounds.
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34.  Dissociation constants of acids and bases.
PH =pKa + log [A-] / [HA]
From the above equation, the PKa value can
be calculated from the graph plotted
between absorbance and wavelength at
different PH values.
 Quantitative analysis of pharmaceutical
substances
Many drugs are either in the form of raw
material form. They can be assayed by
making a suitable solution of the drug in a
solvent and measuring the absorbance at
specific wavelength
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35.  Molecular weight determination
Molecular weights of compounds can be
measured spectrophotometrically by preparing
the suitable derivatives of these compounds.
 Chemical kinetics
Kinetics of reaction can also be studied using UV
spectroscopy. The UV radiation is passed through the
reaction cell and the absorbance changes can be
observed.
 AS HPLC detector
A UV/Vis spectrophotometer may be used as a detector for
HPLC.
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