The document provides a comprehensive overview of UV/Visible spectroscopy, detailing its types, principles, instrumentation, and applications. It explains emission and absorption spectroscopy, emphasizing their roles in analyzing the interaction of light with matter and various electronic transitions involved. Additionally, it discusses practical applications such as detection of impurities, structure elucidation, and quantitative analysis in pharmaceuticals.

1. UV / VISIBLE
SPECTROSCOPY
MASH’HOOD MAHMODD KHAN SHAHID
MUHAMMAD UZAIR AMIR JUTT
MUHAMMAD SAIFULLAH KHAN

2. SPECTROSCOPY
 Study of spectrum formed by interaction of matter with
radiant energy.
 Spectroscopy is the branch of science that involves the
study of interaction of electromagnetic radiations with
atoms or molecules.
It is of two types;
 Emission spectroscopy
 Absorption spectroscopy

3. EmISSIOn SPECTROSCOPY
 It is the measure of emitted light and spectroscopic
analysis of this emitted light gives the emission spectrum.
 An emission spectrum thus obtained gives the information
about the light source under study.
 This phenomenon is primarily caused by excitation of
atoms by the thermal or electrical means, absorbed energy
causes electron in the ground state to be promoted to the
state of higher energy where they are short lived and will
return to the ground state by emitting the radiations.
 But there may also be phosphorescence in some instances.
Example:
 Flame photometry
 Flourimetry

4. ABSORPTIOn SPECTROSCOPY
 It is the measure of energy absorbed by an excited atom
or molecule and the spectroscopic analysis of the
energy gives the absorption spectrum.
 It also represents the energy absorbed relative to the
energy of given frequency of electromagnetic radiation.
Example:
 IR
 UV/VISIBLE
 NMR

5. UV/VISIBLE
SPECTROSCOPY:-
 Uv-vis spectroscopy is also known as
electronic spectroscopy. In which the
amount of light absorbed at each
wavelength of Uv and visible regions
of electromagnetic spectrum is
measured. This absorption of
electromagnetic radiations by the
molecules leads to molecular
excitation.

6. ELECTROmAgnETIC
RAdIATIOnS

7. PRInCIPLE
 The absorption of energy by ground state atom in the
gaseous state forms the basis of atomic absorption
spectroscopy.
 Spectrum is a graph of intensity of absorbed or emitted
radiation by sample verses frequency (ν) or wavelength
(λ).
 A phenomenon of interaction of molecules with
ultraviolet and visible lights.
 Absorption of photon results in electronic transition of a
molecule, and electrons are promoted from ground state
to higher electronic states.

8. Principle is based upon the electronic
transitions:
 σσ →→ σσ* transition* transition
ππ →→ ππ* transition* transition
n →n → σσ* transition* transition
n →n → ππ* transition* transition
σσ →→ ππ* transition* transition
ππ →→ σσ* transition* transition

9. ElEctronic transition

10. σ →→ σ* transition* transition
 σ electron from orbital is excited to corresponding anti-
bonding orbital σ*.
 Methane (CH4) has C-H bond only

11. n →n → σσ* transition* transition
 Saturated compounds containing atoms with lone pair of
electrons like O, N, S and halogens are capable of n →
σ* transition..

12. ππ →→ ππ* transition* transition
 π electron in a bonding orbital is excited to
corresponding anti-bonding orbital π*.
 Compounds containing multiple bonds like alkenes,
alkynes, carbonyl, nitriles, aromatic compounds, etc
undergo π π* transitions.→

13. n →n → ππ* transition* transition
 An electron from non-bonding orbital is promoted to
anti-bonding π* orbital.
 Compounds containing double bond involving hetero
atoms (C=O, C≡N, N=O) undergo such transitions.

14. instrumEntation
Following are the general components of atomic absorption
spectrometer:
Sources of Radiation (UV and Visible)
Wavelength selector (Monochromator)
Cuvette (sample and refrence container)
Detectors
Signal processor (amplifier) and read-out device.

15. radiation sourcE
UV:
Deutrium and hydrogen lamps.
The electrical excitation of deuterium or hydrogen at low
pressure produces a continuous UV spectrum.
Range: 160-375nm
Precaution:
Quartz windows and quartz cuvette must be used b/c
glass absorb radiation of wavelength less then 350nm.

16. radiation sourcE
Visible source:
Tungston filament lamp
Range: 350-2500nm
The lifetime of a tungsten/halogen lamp is approximately
double that of an ordinary tungsten filament lamp.

17. Wavelength selector
There are following components in every monochromators
namely:
Entrance slit
Collimating lens
Dispersing device (prism of grating)
Focusing lens
Exit slit

19. cuvette
 The containers for the sample and reference
solution must be transparent to the radiation
which will pass through them. Quartz or fused
silica cuvettes are required for spectroscopy in
the UV region. These cells are also transparent in
the visible region. Silicate glasses can be used for
the manufacture of cuvettes for use between 350
and 2000 nm.

20. Detector
 Photomutiplier tube
 Linear photodiode array
 Charged coupled devices

22. applications
 Detection of impurities
 Structure elucidation of organic compound
 Quantitative analysis
 Qualitative analysis
 Dissociation constants of acids and base
 Chemical kinetics
 Quantitative analysis of pharmaceutical analysis
 Molecular weight determination.
 As HPLC detector.

23. Detection of
impurities UV absorption spectroscopy is one of the best methods for
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. By also measuring the absorbance
at specific wavelength, the impurities can be detected.
Benzene appears as a common impurity in cyclohexane. Its presence
can be easily detected by its absorption at 255 nm.
 The bands due to impurities are very intense.
 Saturated compounds show little absorption while unsaturated show
strong absorption bands.

24. structure
eluciDation of
organic compounDs UV spectroscopy is useful in the structure elucidation of organic
molecules, the presence or absence of unsaturation, the presence of
hetero atoms.
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.

25. Quantitative analysis
 UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation.
 This determination is generally based on Beer’s Law;
which states that Absorption is directly proportional to Conc. of
sample in the solution.
 For this, we produce calibration curve for different standard
concentrations of the sample solution. Then unknown sample
concentration can be determined by comparing the values in standard
graph.

26. Qualitative analysis
 UVabsorption 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.
UV absorption spectroscopy is generally used for characterizing
aromatic compounds and aromatic olefins.

27. Dissociation
constants of aciDs
anD bases.
 PH = PKa + log [A-
] / [HA]
From the above equation, the PKa value can be calculated if the ratio
of [A-
] / [HA] is known at a particular PH. and the ratio of [A-
] / [HA]
can be determined spectrophotometrically from the graph plotted
between absorbance and wavelength at different PH values.

28. 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.

29. Quantitative
analysis of
pharmaceutical
substances Many drugs are either in the form of raw material or in the form of
formulation. They can be assayed by making a suitable solution of the
drug in a solvent and measuring the absorbance at specific wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the
wavelength 284 nm.

30. molecular weight
Determination
 Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable derivatives of these
compounds.
For example, if we want to determine the molecular weight of amine
then it is converted in to amine picrate. Then known concentration of
amine picrate is dissolved in a litre of solution and its optical density is
measured at λmax 380 nm. After this the concentration of the solution
in gm moles per litre can be calculated by using the following formula.
 "c" can be calculated using above equation, the weight "w" of amine
picrate is known. From "c"and "w", molecular weight of amine picrate
can be calculated. And the molecular weight of picrate can be
calculated using the molecular weight of amine picrate.

31. SHEEEEE!
REVOLUTIONARY KHANS
KYA COOL HAIN HUM THREE?