1. Prepared by-
Mr.Akshay V. Patil
M.pharm 1st sem
Dr, Rajendra gode college
of pharmacy Malkapur.
Guide by-
Mr. P .V. Burukle sir
Associate professor
Dept. Pharamaceutical Analysis.
Dr,Rajendra Gode college
of pharmacy Malkapur.
UV- visible Spectroscopy.
2. Introduction.
Ultraviolet and visible (UV-Vis) absorption spectroscopy
is the measurement of the attenuation of a beam of light
after it passes through a sample or after reflection from a
sample surface.
Absorption measurements can be at a single wavelength or
over an extended spectral range.
The UV radiation region extends from 10 nm to 400 nm.
3. Near UV Region: 200 nm to 400 nm
Far UV Region: below 200 nm
Far UV spectroscopy is studied under vacuum condition.
the visible radiation region extends from 400 nm to 800 nm.
Ultraviolet absorption spectra arise from transition of
electron with in a molecule from a lower level to a higher
level.
A molecule absorb ultraviolet radiation of frequency (𝜗),
the electron in that molecule undergo transition from
4. lower to higher energy level. The energy can be calculated
by the equation, E=h𝜗 erg
Absorption spectrum
When a sample is exposed to light energy that matches the energy
difference between a possible electronic transition within the
molecule, a fraction of the light energy would be absorbed by the
molecule and the electrons would be promoted to the higher energy
state orbital. A spectrometer records the degree of absorption by a
sample at different wavelengths and the resulting plot of absorbance
(A) .
5. versus wavelength (λ) is known as a spectrum.
The significant features:
λmax (wavelength at which there is a maximum absorption)
єmax (The intensity of maximum absorption)
6.
7. Every time a molecule has a bond, the atoms in a bond have their
atomic orbitals merged to form molecular orbitals which can be
occupied by electrons of different energy levels.
Ground state molecular orbitals can be excited to anti-bonding
molecular orbitals.
These electrons when imparted with energy in the form of light
radiation get excited from the highest occupied molecular orbital
(HOMO) to the lowest unoccupied molecular orbital (LUMO) and the
resulting species is known as the excited state or anti-bonding state.
8. TYPES OF TRANSITIONS:
In U.V spectroscopy molecule undergo electronic
transition involving σ, π and n electrons.
Four types of electronic transition are possible.
i. σ ⇾ σ* transition
ii. n ⇾ σ* transition
iii. n ⇾ π* transition
iv. π ⇾ π* transition
9. I. σ ⇾ σ* transition
An electron in a bonding σ orbital of a molecule is excited to the
corresponding anti-bonding orbital by the absorption of radiation.
To induce a σ ⇾ σ* transition it required LARGE ENERGY.
Ex: Methane
Methane contain only single C-H bonds.
it undergo only σ ⇾ σ* transition only, it gives absorption
maximum at 125nm.
10. II. n ⇾ σ* transition:
In this type saturated compounds containing atoms with unshared
electron pairs are undergo n ⇾ σ* transition.
It require less energy than the σ ⇾ σ* type. Most of the
absorption peaks appearing below 200nm.
In the presence of polar solvents the absorption maximum tend to
shift shorter wavelength
Ex: Water , ethanol.
11. III. n ⇾ π* & π ⇾ π* transition
Most organic compounds are undergo transitions for n ⇾ π*
and π ⇾ π* transition.
Because energies required for processes bring the absorption peaks
into spectral region.
Both transition require the presence of an unsaturated functional
group to the ´∏´ orbitals.
Ex: For π ⇾ π* ⧐ Alkenes compounds, alkynes carbonyl
For n ⇾ π* ⧐ carbonyl compounds.
12. Beer´s law
Beer´s law states that The intensity of a beam of
monochromatic light decreases exponentially with
increase in the concentration of absorbing species
arithmetically.
Accordingly.
- dI /dc α I
[The decrease in the intensity of incident light with conc. is proportional to
intensity of incident light]
- dI /dc = k I
... [Removing and introducing constant of proportionality k]
13. -dI / I = k d c .... [rearranging terms]
-InI = kc + b ....... Eq 1
[When conc =0 there is no absorbance hence I = I0 sub.in Eq 1.]
-In I0 = k×0+b
-In I0= b ...substituting the value in Eq 1
-InI = kc – In I0
In I0 -InI= kc
In I0 /I=kc ....since (logA-log B = log A/B)
14. I0 /I= ekc ... [ removing natural logarithm]
I/ I0 = e-kc ...[making inverse on both side]
I = I0 e-kc ...[ Eq 2 For Beer´s Law ]
15. Lambert s law
The rate of decrease of intensity (monochromatic light) with the
thickness of the medium is directly proportional to the intensity of
light.
-dI /dt α I
[This eq. Can be simplified similar to eq. 2 to get the following eq.
By replacing c with t]
I = I0 e-kt .... Eq.3
Eq .2 & Eq.3 get combined
I = I0 e-kct
16. I = I0 10-kct ... ( convert. Nat. Log.to base 10 & k k×0.4343)
I/ I0 = 10-kct ... (rearranging terms)
I0/I = 10kct .....(inverse on both side)
log I0/I = kct ....( taking log on both side) Eq .4
These Eq. Infer that,
A =kct ....since log I0/I =A
Insted of k we can use Є
A = Єct (Mathematically Eq.for Beer Lambert Law)
Where A = Absorbance
Є= molecular extinction coefficint
18. Instrumentation
Instruments for measuring the absorption of U.V. or visible radiation
are made up of the following components;
1. Sources (UV and visible)
2. filter or monochromator
3. Sample containers or sample cells
4. Detector
20. 1. Radiation source
It is important that the power of the radiation source does not change
abruptly over its wavelength range.
The electrical excitation of deuterium or hydrogen at low pressure
produces a continuous UV spectrum. The mechanism for this
involves formation of an excited molecular species, which breaks up
to give two atomic species and an ultraviolet photon
Both Deuterium and Hydrogen lamps emit radiation in the range 160
- 375 nm. Quartz windows must be used in these lamps, and quartz
cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.
21. Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
Various Visible radiation sources are as follows
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbonone lamp
22. 2. filters or monochromators
All monochromators contain the following component parts;
• An entrance slit
• A collimating lens
• A dispersing device (a prism or a grating)
• A focusing lens
• An exit slit
Polychromatic radiation (radiation of more than one
wavelength) enters the monochromator through the entrance
slit. The beam is collimated, and then strikes the dispersing
element at an angle.
23. The beam is split into its component wavelengths by the grating or
prism. By moving the dispersing element or the exit slit, radiation of
only a particular wavelength leaves the monochromator through the
exit slit.
3. sample containers or sample cells
A variety of sample cells available for UV region. The choice of
sample cell is based on
a) the path length, shape, size
b) the transmission characteristics at the desired wavelength
c) the relative expense
24. The cell holding the sample should be transparent to the
wavelength region to be recorded. Quartz or fused silica cuvettes
are required for spectroscopy in the UV region. Silicate glasses can
be used for the manufacture of cuvettes for use between 350 and
2000 nm.The thickness of the cell is generally 1 cm. cells may be
rectangular in shape or cylindrical with flat ends.
4. Detectors
In order to detect radiation, three types of photosensitive devices
are
a. photovoltaic cells or barrier- layer cell
25. b. phototubes or photoemissive tubes
c. photomultiplier tubes
Phototubes are also known as photoemissive cells. A phototube
consists of an evacuated glass bulb. There is light sensitive cathode
inside it. The inner surface of cathode is coated with light sensitive
layer such as potassium oxide and silver oxide.
When radiation is incident upon a cathode, photoelectrons are
emitted. These are collected by an anode. Then these are returned
via external circuit. And by this process current is amplified and
recorded.
26. CHOICE OF SOLVENTS
It should not itself absorb radiations in the region under
investigations.
It should be less polar so that it has minimum interaction with the
solute molecule.
Most commonly: 95%ethanol
Cheap, good dissolving power, does not absorb radiation above
210nm.
Typical examples
27.
28. SOLVENT EFFECT
The position and intensity of an absorption band may shift when the
spectrum is recorded in different solvents.
A dilute sample solution is prefered for analysis.
Most commonly used solvent is 95% ethanol. It is best solvent as
it is cheap,transparent down to 210nm.
Position as well as intensity of absorption maxima get shifted for
a particular chromophore by changing the polarity of solvent.
By increasing polarity of solvent→dienes.
