uv spectroscopy

1. Presented By
DISHA SHEE
ROLL.NO. - 19301920030
SUBJECT –INSTRUMENTAL METHODS OFANALYSIS
SUBJECT CODE - PT-711
B. Pharm, Year - 4rd , Semester - 7TH
BENGAL SCHOOL OF TECHNOLOGY
(A College of Pharmacy)
Sugandha, Hooghly, W.B- 712102
Electronic transition of UV spectroscopy &
Instrumentation

2. Spectroscopy
Spectroscopy is the study of the interaction between matter and electromagnetic radiation
as a function of the wavelength or frequency of the radiation.
OR
It is the measurement of electromagnetic radiation (EMR) absorbed or emitted when
molecule or ions or atoms of a sample move from one energy state to another energy
state.
Working principle of UV-visible spectroscopy:
When a chemical compound absorbs light, some excitation and de-excitation processes of
electrons occur in atoms which result in the production of the distinct spectrum.

3. Electronic Transitions
According to the molecular orbital theory, when a molecule is excited by the absorption of
energy (UV or visible light), its electrons are promoted from a bonding orbital to an anti
bonding orbital.
There are several types of electronic transitions available to a molecule including:
1. σ→ σ* transition:
➢ An electron in a bonding s-orbital is excited to the corresponding anti-bonding orbital and
observed with saturated compounds.
➢ The energy required is large.
➢ For example, methane (which has only C-H bonds, and can only undergo σ→ σ* transition
transitions) shows an absorbance maximum at 125 nm.
➢ Absorption maxima due to σ→ σ* transition are not seen in typical UV-VIS spectra (200 -
700 nm) but in UV-region (125- 135nm) .

4. 2. n → σ* transition:
➢ Saturated compounds containing atoms with lone pairs (non- bonding electrons) like
O, N, S and halogens are capable of n→ σ* transition.
➢ These transitions usually need less energy than n → σ* transition.
➢ They can initiated by light whose wavelength in range 150 -250 nm. Electronic
Transitions
➢ The number of organic functional groups with n → σ* peaks in the UV region is
small.
3. π→ π* transition:
➢ π electron in a bonding orbital is excited to corresponding anti- bonding orbital π*
and observed in conjugated compounds.
➢ Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles,
aromatic compounds, etc undergo π → π* transitions.
➢ e.g. Alkenes generally absorb in the region 170 to 205nm.

5. 4. n → π* transition:
➢ An electron from non-bonding orbital is promoted to anti- bonding π* orbital and
required lower energy.
➢ Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O)
undergo such transitions.
➢ n → π* transitions require minimum energy and show absorption at longer wavelength
around 300 nm.

6. INSTRUMENTATION COMPONENTS OF
SPECTROPHOTOMETER
➢ Source
➢ Wavelength selectors or Dispersing devices
➢ Sample compartment
➢ Detector
➢ Amplifier and Read-out devise
Figure 1. A basic block diagram of the elements in a single beam UV-Visible spectrometer.

7. Light Source:
Light sources that lie in the ultraviolet and visible region are used as UV-visible spectrometer
sources.
1.Hydrogen & deuterium lamps range 160-380nm
2.Xenon arc lamps range 250-600nm
3.Tungsten halogen lamps range 240-2500nm
Wavelength selector:
UV-Vis spectroscopy requires a single wavelength for proper functioning whereas the ideal output of
a single wavelength is not possible. This is so because no real wavelength selector is ideal. Although
a single wavelength is not possible, a band of radiation could be used. So an instrument with narrow
bandwidth would be better.
Types of the wavelength selectors
1.Filters
2.Monochromators

8. Filters:
Filters are used to permit a certain band of wavelength. The simplest type of filter is the
absorption filter. Most commonly colored glass filters are used. They absorb a broad portion of
the spectrum (complementary colors) and transmit other portions (its own color).
Monochromators:
A monochromator is an optical device that is used to select a narrow band of a wavelength of light.
It may be a quartz prism or grating.
Uses of monochromators:
•Monochromators are used for spectral scanning i.e. varying wavelength of radiation over a range.
•They can be used for the UV-visible region.
Components of a monochromator:
All monochromators are similar in mechanical construction. The essential components of a
monochromator are: Slit , Mirror , Lense , Grating/prism

9. SAMPLE HOLDER/ CUVETTES
➢ The cells or cuvettes are used for handling liquid samples.
➢ The cell may either be rectangular or cylindrical in nature.
➢ For study in UV region the cells are prepared from quartz or fused silica whereas
colour corrected fused glass is used for visible region. Glass is not suitable for the UV
region because it absorbs UV radiation i.e. it is not transparent in the UV region.
DETECTORS
➢ Device which converts light energy into electrical signals, that are displayed on
readout devices.
➢ The transmitted radiation falls on the detector which determines the intensity of
radiation absorbed by sample.

10. Types of UV-Vis spectrometers
There are two types of UV-vis spectrophotometers
1.Single beam UV-Vis spectrometer
2.Double beam UV-Vis spectrometer
Single beam UV-Visible spectrophotometer:
Single beam uv-vis spectrophotometer has a single beam as the name indicates. The incident light
coming from the source is passed through a monochromator then that incident monochromatic light
moves through a slit. Then it passes through the sample solution. Where some of the incident light is
absorbed by the sample while other is transmitted. That transmitted light is detected by the detector.
The detected light is then amplified, recorded, and then displayed on a suitable readout device.
Spectrum is plotted and the λmax is located.

11. Figure2 :Single beam spectrophotometer
Double beam UV-Visible spectrophotometer:
The basic difference from a single beam UV-Vis spectrophotometer is that the beam of incident
light is passing simultaneously from the sample and the reference cells.
The incident light splits and is directed towards both the reference and sample cuvette. The
refracted or transmitted beam is detected by the detectors. A double beam UV-vis
spectrophotometer needs two detectors that detect electron ratio to measure or calculate absorbance
in a test sample. It also requires a stabilized voltage supply.

12. Figure3 : double beam spectrophotometers

13. CONCLUSION
We can conclude that UV spectroscopy is best method which routinely used in analytical
chemistry for the quantitative determination of different analytes, such as transition metal
ions, highly conjugated organic compound and biological . UV spectrophotometer
instrumentation plays a crucial role in scientific research, enabling precise and rapid
measurements of UV light absorption and enhancing analytical capabilities in diverse
fields.

14. REFERENCE
1. Dr. Shankar ravi, “textbook of pharmaceutical analysis”, third edition, Rx
publication, page no.-2.2-2.5.
2. Harris DC. Quantitative Chemical Analysis. 7th ed, 3rd printing. W. H. Freeman;
2007.
3. Mårtensson N, Baltzer P, Brühwiler PA, Forsell JO, Nilsson A, Stenborg A,
Wannberg B. A very high resolution electron spectrometer. Journal of Electron
Spectroscopy and Related Phenomena. 1994 Dec 9;70(2):117-28.
4. Nitzan A. Electron transmission through molecules and molecular interfaces. Annual
review of physical chemistry. 2001 Oct;52(1):681-750.
5. Altemose IR. Evolution of instrumentation for UV-visible spectrophotometry Part I.
Journal of Chemical Education. 1986 Sep;63(9):A216.